ARTICLE-01
Synthesis and crystal structures of 5'-phenylspiro[indoline-3,
2'-pyrrolidin]-2-one derivatives
1,3-Dipolar
cycloaddition of azomethineylides to exocyclic olefins constitutes a versatile
protocol for the construction of poly functionalized spiro-heterocycles viz.
pyrrolidines [1] and pyrrolizines [2], which widely occur
in natural products and biologically active compounds. The spiro-
indole-pyrrolidine ring system is a frequently encountered structural motif in
many biologically important and pharmacologically relevant alkaloids. Compounds
with an indole/oxindole framework are promising pharmacophore which exhibit
interesting applications in the biological and pharmacological arena [3]. The derivatives of
spirooxindole ring systems are used as antimicrobial, antitumour agents and as
inhibitors of the human NKI receptor besides being found in a number of
alkaloids like horsifiline, spirotryprostatin and (+) elacomine [4]. The recently
discovered small-molecule MDM2 inhibitor MI-219 and its analogues are in advanced
preclinical development as cancer therapeutics [5]. Our interest in
preparing pharmacologically active pyrrolidines led us to the compounds,
4'-Nitro-3',5'-diphenylspiro[indoline-3,2'-pyrrolidin]-2-one (I) and 3'-(4-Methoxyphenyl)- 4'-nitro
-5'-phenylspiro[indoline-3, 2'-pyrrolidin]-2-one (II), and we have undertaken the X-ray
crystal structure determination of these compounds in order to establish their
conformations.
The spiro
compounds reported in the present work were prepared (Scheme 1) by following our
earlier literatures method [6-8]. A mixture of (E)-(2-nitrovinyl) benzene or (E)-1-methoxy-4-(2-nitrovinyl) benzene (1
mmol), isatin (1 mmol) and phenylglycine (1 mmol) was heated to reflux in
methanol on a water-bath for 40 min. The progress of the reaction was monitored
by thin layer chromatography (TLC). The starting materials vanished in the TLC
indicating the completion of the reaction i.e, the azomethineylide (dipole)
reacts with the substituted vinyl benzene (dipolarophile). Then, the reaction
mixture was poured into crushed ice, the resulting solid filtered and washed
with water to afford pure regio and stereoselective
3'-Phenyl-4'-nitro-5'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one or
3'-(4-Methoxyphenyl)-4'-nitro-5'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one in
good yields. The synthesis scheme of
3'-(aryl)-4'-nitro-5'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one is shown
below. For compound (I):
Yield 80%; M.p. 239°C. For compound (II): Yield 78%; M.p. 231°C.
Scheme 1
Synthesis scheme of the compounds.
Scheme 1
Synthesis scheme of the compounds.
In both the molecules,
the 2-oxyindole ring is planar (r.m.s deviation: 0.031 Å and 0.018 Å for I and II, respectively), which is common in spiro complexes [9,10]. The spiro rings
of both molecules have the twisted envelope structure with the N atom at the
flap position. The distance to the flap position from the mean plane of spiro
carbon atoms, are 0.531(3) Å and 0.503(2) Å in compounds (I) and (II), respectively. The phenyl ring and
methoxyphenyl rings are inclined by an angle of 31.45 (2)° in compound (II) which is similar to the inclination of
the two phenyl rings in compound (I) (31.60(2)°). In compound (II), H9 and H8 have trans conformation with the torsion angle of 152.45(2)°
(H9/C9/C8/H8) and H8 and H7 have cis conformation with the torsion angle of -5.43(2)°
(H8/C8/C7/H7). In compound (I)
also, similar conformation is found. The hydrogen conformation torsion angles
in compound (I) are
152.81(3)° and 7.14(3)° for H9 & H8 and H8 & H7, respectively. Even
though these conformations are similar, the directions in which the hydrogens
are attached, are reciprocal in both the compounds. Figure 1, a superimposition
of the planar 2-oxyindole rings, drawn using Mercury [11], clearly shows the
reciprocal conformations of both the compounds. In both molecules, N-H···O
hydrogen bonds make the R22 (8) ring motifs (Figure 2 and Figure 3). Further, the
structures are stabilized by intermolecular hydrogen bonds.
Figure
1. Reciprocal conformations of both compounds, as seen from
the superimposition of the planar 2-oxyindole rings.
Figure 2. Figure showing the
intramolecular hydrogen bonds resulting in R22(8) motif
in compound (I).
Figure 3. Figure showing the
intramolecular hydrogen bonds resulting in R22(8) motif
in compound (II).
Figure
1. Reciprocal conformations of both compounds, as seen from
the superimposition of the planar 2-oxyindole rings.Figure 2. Figure showing the
intramolecular hydrogen bonds resulting in R22(8) motif
in compound (I).Figure 3. Figure showing the
intramolecular hydrogen bonds resulting in R22(8) motif
in compound (II).
X-ray Crystallography
Single crystal
X-ray intensity data for the compounds (Scheme 2) and (Scheme 3) were collected
using a Nonius CAD-4 MACH 3 diffractometer with MoKα (0.71073 Å) radiation at room temperature (293 K). The data
reduction was carried out using XCAD4 [12]. The absorption
corrections were applied using the ψ-scan method [13]. The structures of
both the compounds were solved by direct methods using SHELXS97 [14] and all the
non-hydrogen atoms were refined anisotropically by full-matrix least-squares on
F2 taking all the unique reflections using SHELXL97 [14]. The hydrogen
atoms attached with carbon atoms were placed in their calculated positions and
included in the isotropic refinement using the riding model with C-H = 0.93Å
(-CH) or 0.97Å (-CH2) Å or 0.96Å (-CH3) Å with Uiso(H) =
1.2 Ueq (parent C atom) and amino bound hydrogen atoms were located from the
difference Fourier map and include in the refinement isotropically. The crystal
data, experimental conditions and structure refinement parameters for the
compounds (I) and (II) are presented in Table 1. The
re-crystallization of the compound (I) and repeated data collection with different crystal samples did
not improve the R value and other statistical parameters. Crystals of better quality could not be obtained
for the compound (I).
Table 2 gives the geometry
of the hydrogen bonds present in I and II.
The molecular structures of compounds (I) and (II)
showing the atom numbering scheme using ORTEP-3 [15] are given in
Figures 4 and 5, respectively.
Scheme 2
Scheme showing the structural formula of compound (I).
Scheme 3
Scheme showing the structural formula of compound (II).
Table 1. The crystal data, experimental conditions and structure refinement parameters for the compounds (I) and (II)
Table 2. The geometry of the hydrogen bonds (Å, °)
Figure 4. The molecular structure of
compound (I) showing the atom numbering scheme. Displacement ellipsoids are
drawn at the 40% probability level, using ORTEP-3. Hydrogen atoms are drawn as
spheres of arbitrary size.
Figure 5. The molecular structure of
compound (II) showing the atom numbering scheme. Displacement ellipsoids
are drawn at the 40% probability level, using ORTEP-3. Hydrogen atoms are drawn
as spheres of arbitrary size.
Scheme 2
Scheme showing the structural formula of compound (I).
Scheme 3
Scheme showing the structural formula of compound (II).
Table 1. The crystal data, experimental conditions and structure refinement parameters for the compounds (I) and (II)
Table 2. The geometry of the hydrogen bonds (Å, °)
Figure 4. The molecular structure of
compound (I) showing the atom numbering scheme. Displacement ellipsoids are
drawn at the 40% probability level, using ORTEP-3. Hydrogen atoms are drawn as
spheres of arbitrary size.Figure 5. The molecular structure of
compound (II) showing the atom numbering scheme. Displacement ellipsoids
are drawn at the 40% probability level, using ORTEP-3. Hydrogen atoms are drawn
as spheres of arbitrary size.
The title
compounds were synthesized and the corresponding molecular crystal structures
have been determined by single-crystal X-ray diffraction. In both the
compounds, the R22(8) motif is present. Even though most
of the conformational features are similar when seen separately, by super
positioning the two structures it is found that the entire configuration is
inverted with respect to the 2-oxyindole ring. This is due to the substitution
of methoxyphenyl instead of phenyl ring in compound (I).
Crystallographic
data (excluding structure factors) for the structures of compounds (I) and (II) reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre as supplementary
publication numbers, CCDC 802309 and CCDC 802308, respectively. Copies of the
data can be obtained free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1 EZ, UK. (fax: +44-(0)1223-336033 or email: deposit@ccdc.cam.ac.uk).
The authors
declare that they have no competing interests.
JKS collected the
X-ray data and solved the crystal structures under the guidance of SN. SMR and
JS synthesized the title compounds under the guidance of SP. All the authors
read and approved the final manuscript.
One of the authors
(JK) thanks the UGC for the RFSMS fellowship. SN thanks the CSIR for the
funding provided under the Emeritus Scientist Scheme.
1. Watson AA, Fleet GWJ, Asano N, Molyneux RJ,
Nash RJ: Polyhydroxylated alkaloids - natural occurrence and therapeutic
application.
Org Lett 2000,
17:2639-2641. 
5. Ding K, Lu Y, Nikolovska-Coleska Z, Wang G,
Qiu S, Shangary S, Gao W, Qin D, Stuckey J, Krajewski K, Roller PP, Wang S: Structure-based
design of spiro-oxindoles as potent, specific small-molecule inhibitors of the
MDM2-p53 interaction.
6. Ranjith Kumar R, Perumal S, Senthilkumar P,
Yogeeswari P, Sriram D: A Facile Synthesis and Antimycobacterial Evaluation of
Novel Spiro-pyrido-pyrrolizines and Pyrrolidines.
7. Karthikeyan SV, Devi Bala B, Alex Raja VP,
Perumal S, Yogeeswari P, Sriram D: A Highly Atom Economic, Chemo-, Regio-
and Stereoselective Synthesis and Evaluation of Spiro-Pyrrolothiazoles as
Antitubercular Agents.
8. Prasanna P, Balamurugan K, Perumal S,
Yogeeswari P, Sriram D: A Regio- and Stereoselective 1,3-Dipolar Cycloaddition for
the Synthesis of Novel Spiro-Pyrrolothiazolyloxindoles and Their Antitubercular
Evaluation.
9. Suresh J, Suresh Kumar R, Rajapriya A,
Perumal S, Nilantha Lakshman PL: 1-Benzyl-4',5'-diphenylpiperidine-3-spiro-3'-pyrrolidine-2'-spiro-3''-indoline-4,2''-dione.
Acta Cryst
2009, E65:o147-o148.
10. Nagamuthu S, Sribala R, Ranjithkumar R,
Krishnakumar RV, Srinivasan N: 4'
(2,4-Dichlorophenyl)-1,1'-dimethylpiperidine-3-spiro-3'-pyrrolidine-2'-spiro-3''-indoline-4,2''-dione.
Acta Cryst
2010, E66:o717.
Acta Cryst
1968, A24:351-359.
Acta Cryst
2008, A64:112-122.
15. Farrugia LJ: ORTEP-3 for Windows - a version of ORTEP-III with a
Graphical User Interface (GUI).
J Appl Cryst 1997, 30:565.
THE END
ARTICLE-02
Background
The use of cocoa
has been documented for almost 4,000 years. The first population thought to
consume the material was the Mesoamericans [1,2]. In the past decade
there has been increasing interest and numerous publications on the putative
health effects associated with the moderate consumption of cocoa and chocolate
products [3-5]. In a parallel
fashion, several groups initiated studies into the potential agents responsible
for cardiovascular effects with the flavanols, (±)-catechin and (±)-epicatechin
being candidate compounds [6].
The growing interest in these compounds resulted in a plethora of methods for quantification in various foodstuffs including tea, wine, grapes and chocolate. While other analytical methods have been used, HPLC was the predominant method developed [7-13]. A thorough literature search using Google and Pubmed resulted in thousands of citations on polyphenol analysis and almost 900 citations on flavanol analysis by HPLC in chocolate, indicating the recent explosive growth in methods for these analytes. Considering the increased interest in the cocoa flavanols' potential cardiovascular effects, a standard quantification method would be pertinent for accurate determination of dose-response effects in clinical trials. There is both an ISO and an Institute for Nutraceutical Advancement (INA) method for flavanols in tea, but not yet a standard method for flavanol quantification in chocolate and cocoa [14,15].
With this as background, the National Confectioners Association (NCA) convened an analytical chemistry working group to develop a consensus HPLC method. This group conducted a collaborative study using samples provided by NCA to establish a method to quantify (±)-catechin and (±)-epicatechin in cocoa and chocolate and make it available to the industry.
The growing interest in these compounds resulted in a plethora of methods for quantification in various foodstuffs including tea, wine, grapes and chocolate. While other analytical methods have been used, HPLC was the predominant method developed [7-13]. A thorough literature search using Google and Pubmed resulted in thousands of citations on polyphenol analysis and almost 900 citations on flavanol analysis by HPLC in chocolate, indicating the recent explosive growth in methods for these analytes. Considering the increased interest in the cocoa flavanols' potential cardiovascular effects, a standard quantification method would be pertinent for accurate determination of dose-response effects in clinical trials. There is both an ISO and an Institute for Nutraceutical Advancement (INA) method for flavanols in tea, but not yet a standard method for flavanol quantification in chocolate and cocoa [14,15].
With this as background, the National Confectioners Association (NCA) convened an analytical chemistry working group to develop a consensus HPLC method. This group conducted a collaborative study using samples provided by NCA to establish a method to quantify (±)-catechin and (±)-epicatechin in cocoa and chocolate and make it available to the industry.
Scope and Applicability
This method is
applicable for the analysis of (±)-epicatechin and (±)-catechin in cocoa
powder, chocolate liquor and formulated chocolate products. This ring trial
only included pure chocolate; any products containing inclusions (such as fruit
or nuts) may not be appropriate for this method due to potential interference.
A. Principle
This method
determines the (±)-catechin and (±)-epicatechin content of cocoa and chocolate
products. Fat is removed from the sample in order to prevent potential
interference and protect the column by using multiple hexane extractions.
Defatted samples are then dried for subsequent extraction of analytes.
Defatted, dried samples are extracted, with sonication, at 40°C for 15 minutes
using an acetone: water: acetic acid (70: 29.5: 0.5) solvent mixture. Extracted
samples are then centrifuged to remove insoluble materials and brought up to a
defined volume. The extracts are filtered into HPLC vials for chromatographic
analysis. (±)-Catechin and (±)-epicatechin are separated by a reverse phase
mechanism on a C18 column with an acidic acetonitrile-water mobile phase
gradient. Analytes are detected and quantified by their fluorescence, with
excitation at 280 nm and emission at 315 nm.
B. Apparatus
(a) HPLC system: With solvent degasser, binary gradient pumping, gradient mixer,
injector capable of 10 μL
injection (either autosampler or manual), column oven, fluorescence detector
and data analysis system
(b) Chromatography column: Reversed phase HPLC column octadecylsilane (ODS; C18)
derivatized silica reversed phase HPLC column, pore size from 100 - 125 A, are
recommended. Recommended column: Phenomenex Luna, 5 μm, C18(2), 100A, 250
× 3.0 mm (alternate
columns may be used if they provide acceptable resolution)
(c) Analytical balance: Readability 0.1 mg or lower
(d) Pipettes: Capable of accurately delivering 20-1000 μL; 1-5 mL
(e) Vials: 2
mL, amber glass, screw cap, for storing Stock Standard solutions and for
holding filtered HPLC sample prior to injection
(f) Test Tubes: Screw capped, with caps, capable of holding at least 10 mL
(g) Volumetric flasks: 10 mL, 20 mL, 50 mL and 100 mL, Class A, glass
(h) Centrifuge tubes: Plastic, for single use, 50 mL, screw cap (air tight)
(i) Vortex Mixer
(j) Ultrasonic Bath
(k) Flame-Proof Centrifuge: For centrifuging 50 mL tubes at 2500 × g
(l) Syringe filters: For filtering HPLC samples, 0.45 μm PVDF, PTFE or hydrophilic polypropylene, 13 or 25 mm
diameter (Nylon filters are not recommended due to potential adsorption of
metabolites)
(m) Syringe: All plastic, 1 mL to 5 mL as appropriate
(n) Glass beads: (approx. diameter 5 mm)
C. Reagents
(a) Water: High
purity deionized water, filtered through a 0.45 μm or smaller pore filter
(b) Acetonitrile: HPLC grade
(c) Hexane: HPLC
grade
(d) Acetone: HPLC grade
(e) Acetic Acid: Glacial
(f) Extraction Solvent: Mix 700 mL Acetone, 295 mL Water and 5 mL Acetic Acid
(g) Mobile Phase A: 0.2% Acetic Acid in Water. Add 2 mL Acetic Acid to 1 L Water.
(h) Mobile Phase B: 0.2% Acetic Acid in Acetonitrile. Add 2 mL Acetic Acid to 1 L
Acetonitrile.
(i) Standards: (±)-Catechin hydrate, purity ≥ 98%, Sigma-Aldrich C1251-5G or
equivalent; (±)-epicatechin, purity ≥ 98%, Sigma-Aldrich E4018-1G or
equivalent. Certificate of analysis from supplier is required for purity
correction of each new lot number.
D. Standards and Reagent
Blank Preparation
(a) (i.) Stock standard
solution A (approx 1000 μg/mL): Into a 50 mL volumetric flask, accurately weigh approximately 50
mg (±)-catechin hydrate and 50 mg (±)-epicatechin and record the weights. Add
extraction solvent and mix or sonicate to dissolve. Bring to 50.00 mL with
extraction solvent and mix. Label as Stock standard A.
(ii.) Stock standard
solution B (approx 100 μg/mL): Pipette, using a Class A volumetric pipette, 5 mL of Stock
standard A into a 50 mL volumetric flask and dilute to volume with extraction
solvent. Label as Stock standard B.
(b) Obtain loss on drying (important for (±)-catechin hydrate): Crystal water is not stoichiometrically
distributed; for (±)-epicatechin loss on drying normally equals 0) and HPLC
purity of analyte from supplier's certificate of analysis for each new lot
number to calculate purity: Purity (%) = [100 (%) - loss on drying (%)] * HPLC purity
(%)/100 (%)
Calculate exact
concentrations of each component in stock standard solution as shown below:
Stock standard A use concentration as is.
Stock standard B concentrations will require multiplication by an additional 1/10 factor.
Stock standard B concentrations will require multiplication by an additional 1/10 factor.
(c) Fluorescence Detector Sensitivity Assessment: Stock
Standard Selection (Stock Standard Solutions A, B): When running this method for the first time
inject the appropriate injection volume, 10 μl, of Stock Standard A and B onto the HPLC system under the
conditions provided in Section G. Examine the detector response for the two
concentrations provided. Choose the most concentrated stock standard solution
that does not saturate the detector as the Stock Standard with which to
proceed. Discard the other stock standard solution. If proceeding with stock
standard B, for future analysis note that the stock standard preparation
procedure can be modified by preparing a stock standard solution of 0.1 mg/mL
to save one dilution step.
(d) Reagent Blank: Use extraction solvent for the blank.
(e) Working standards: Prepare Working Standard Solutions from the chosen stock standard.
Add indicated amounts of appropriate working standard (100 or 1000 μg/mL) to a 10 mL volumetric flask; bring to
volume with the extraction solvent. Transfers should always be made with Class
A volumetric pipettes. Alternatively test tubes can be used and the remaining
extraction solvent for dilution to 10 mL can be added with Class A volumetric
pipettes. See an example of the working standard dilution scheme in Table 1 below.
Table 1. Example of Working Standard Dilution Scheme(s)
(f) Calculate
the exact concentration of each component of the working standards as follows:
E. Lipid Removal from
Cocoa and Chocolate Samples
(a) Accurately
weigh approximately 2 grams of
each finely divided/grated milk chocolate sample or 1 gram for cocoa powders/baking chocolate/dark
chocolate samples into a labeled, tared 50 mL disposable centrifuge tube.
Record the weight of the sample W SAMPLE.
(b) Add
approx. 40 mL hexane (dispenser) and cap tightly.
(c) Mix until
the sample is completely dispersed (check visually).
(d) Centrifuge
for 5 minutes at 2500 × g.
(e) Carefully
decant and dispose of the hexane phase immediately.
(f) Repeat
defatting steps (b) to (e) one additional time.
(g) Remove
the cap and allow the residual solvent to evaporate in an appropriate fume hood
until remaining hexane has evaporated (e.g. over night). Alternately, a stream
of nitrogen may be used to accelerate the drying process.
F. Preparation of Test
Solutions
Continue with
whole sample remaining in the centrifugation tube.
(a) Add 2
glass beads to the centrifuge tube containing the dried, defatted sample.
(b) Add 9 mL
of extraction solvent (dispenser) and vigorously shake the sample to break
centrifugation pellet. Sample does not need to be completely suspended yet.
Shake headlong, if necessary gently tap several times.
(c) Place in
an ultrasonic bath at 40°C for 15 minutes in total. After 5-10 minutes of
sonication, remove sample from bath and handshake again until sample is
completely suspended (check visually). Alternately, vortex sample.
(d) Remove
the sample from the ultrasonic bath, centrifuge at 2500 × g for 5 minutes.
(e) Carefully
and slowly decant the liquid portion into a 20 mL Class A volumetric flask
(wide neck, if possible).
(f) Repeat
the extraction steps (b) to
(d)
one additional time.
Decant the liquid from the second extraction into the same 20 mL volumetric
flask.
(g) Bring to
volume with extraction solvent.
(h) Assemble
a Syringe and Syringe Filter. Filter approximately 1 mL of sample into a HPLC
Vial.
(i) Analyze
by HPLC as described in Section G.
G. Chromatography
(a) Injection volume: 10 μL
(b) Flow rate: 0.65 mL/min for 3 mm i.d. column; Alter flow rate to maintain
linear flow for other column dimensions.
(c) Detection: Fluorescence with excitation at 280 nm and emission at 315 nm
(d) Column temperature: 40°C
(e) Gradient Elution: See Table 2 for example
gradient conditions. HPLC
columns differ in their selectivity for these compounds. Gradient conditions
should be altered as needed to achieve resolution of (±)-catechin and
(±)-epicatechin from interfering peaks.
Table 2. HPLC Gradient Example
(f) Concentration of analytes in sample extract: Check if concentration of analytes in
sample extract lie within their calibration ranges. If necessary, dilute and
re-run extraction solution.
(g) Check sample: Check sample by re-runing mid point calibration curve in middle
and at end of sample sequence. Calculate the mean, standard deviation and
coefficient of variation (%CV) of the peak areas.
H. Calculations
Integrate peak
area for quantitation. If peak areas of analytes in sample extracts are above
calibration curve, dilute sample extract solution with extraction solvent
accordingly. If peak areas of analytes in sample extracts are below calibration
curve, repeat sections E and F and increase sample weight accordingly.
Construct standard curves, plotting calibration standard concentration of each standard against the area of the standard peak, using linear regression. Calculate the analytes (±)-catechin and (±)-epicatechin in the original sample as follows:
Analyte in sample [μg/g] = assay concentration of analyte [μg/mL] * × [mL]/Wsample[g]
Construct standard curves, plotting calibration standard concentration of each standard against the area of the standard peak, using linear regression. Calculate the analytes (±)-catechin and (±)-epicatechin in the original sample as follows:
Analyte in sample [μg/g] = assay concentration of analyte [μg/mL] * × [mL]/Wsample[g]
WSample [g]
= initial sample weight from section E (a)
× [mL] = 20 mL
(volume of extraction solution in volumetric flask; section F)
I. HPLC System and Column
Performance Criteria Qualification
An HPLC column
which fully resolves the analytes of interest may be used for the method.
Gradient slope, flow rates and injection volumes may be altered as appropriate
to accommodate columns of differing dimensions.
J. System Suitability
System suitability
is a required procedure to ensure the HPLC system is working correctly. The
following suitability tests are recommended to ensure correct system operation
prior to initial use:
Repeatability and carry-over: Before running any test solutions, demonstrate the repeatability and lack of carryover of the HPLC system as follows:
Repeatability and carry-over: Before running any test solutions, demonstrate the repeatability and lack of carryover of the HPLC system as follows:
(a) System Artifacts: As the first two injections of the day, analyze the blank standard
twice in succession. Inspect the two chromatograms for artifact peaks from the
HPLC system. Artifacts in the first chromatogram, absent in the second,
indicate a buildup of impurities on the system. Artifacts present in both runs
indicate impurities expected in every run. If the first chromatogram shows
artifact peaks but the second chromatogram does not, inject a blank solution as
the first sample in every analytical set. The presence of artifact peaks
indicates impurities in the HPLC solvents, the needle wash system, or carryover
in the injection system. These problems, if present, should be corrected.
(b) Carryover: Inject Standard 5 and then the blank. Carefully examine the blank
injection for carryover peaks. Calculate the carryover of any peaks seen in the
blanks as a percentage of the concentration found in standard 5. Carryover of
standard 5 to the blank injection should be less than 0.1%.
(c) Linearity of the standard curve: Analyze each of the 5 standards, and
construct a standard curve. The R2 of each standard curve should be
greater than 0.9990. If this linearity is not achieved, prepare fresh
standards.
(d) System precision: Analyze five replicate analyses
of standard number 3. Calculate the concentration of each analyte and calculate
the mean, standard deviation and percent coefficient of variation (%CV) of the
results. The %CV for all peaks should be ideally less than 2%.
K. Samples
Samples for
analysis were prepared by the NCA Study Director and submitted as blind samples
to five participating laboratories. Samples consisted of cocoa, dark chocolate,
milk chocolate and NIST SRM [2384] Baking Chocolate having certified values for
(±)-catechin and (±)-epicatechin. One dark chocolate samples was used as a
blind duplicate to assess method repeatability.
L. Quantification
Quantification was
performed using the external calibration method as described in the method with
all laboratories reporting regression coefficients in excess of 0.99 with the
labs equally divided whether calibration was forced through zero.
All results were
submitted to the Study Director using the form that was provided with the
samples and with all data statistically evaluated. Samples were run in
duplicate or triplicate. Furthermore each data set was evaluated using the
Q-test to test for outliers with some data being eliminated. The results can be
seen in Table 3.
.
Figure 1. Chromatogram of Dark Chocolate
Extract Analyzed Under Method Parameters. Peak at 11.964 is (±)-catechin while peak at 14.422 is
(±)-epicatechin.
Each laboratory also provided information about LOD (Limit of Detection) which was in pure solvent in the 40-50 ng/mL. The LOQ (Limit of Quantitation) ranged from 1- 2 μg/g Repeat injections of standards were also accomplished with %CVs reported in the 1-4% range.
The NIST (National Institute of Standard Technology) reference values for (±)-catechin and (±)-epicatechin in SRM (Standard Reference Material) 2384 are 245 +/- 51 (μg/g) and 1220 +/- 22 (μg/g) respectively with the data from this study indicating value of 254 +/- 23.5 (μg/kg) and 1137 +/- 68 (μg/kg) which are within the acceptable range of determinations established by NIST. While recovery studies have become a default method to assess method accuracy according to Swartz and Krull, the analysis of an established SRM is by itself a generally accepted method of validation [17]. Additionally, guidance from AOAC on methods validation indicates that spiking is not a desirable method to assess method accuracy as spiking solutions tend to be easily extractable hence the choice of the NIST standard to evaluate the method.
The sample labeled Milk Chocolate 1 is an example for a product containing very low amounts of the target analytes. With the analyte concentration in the sample extract at their lower limit of quantification and the chromatographic performance negatively affected by co-extracted matrix compounds the applied method operates at its limit. Hence the sample was not included in the statistical evaluation of the method. That being said, no issues related to complexation of polyphenols with milk reported by some researchers were seen [18].
The %CV ranged from 7-15% in this study. Laboratories used a column that satisfied the requirements of U.S. Pharmacopeia, previously described in methodology section [19]. The method was reviewed and compared with recommendations of Swartz and Krull. Finally, while the data in Table 3 may seem excessive to the casual observer, it is well within the parameters established by AOAC for another complex analyte [20].
The literature reports on the use of numerous solvents for the extraction of flavan-3-ols including mixtures of methanol, acetone, water and acid therefore the solvent combination used is within established parameters [21,22]. Furthermore, a variety of HPLC detector types have been used including UV, Diode Array, Mass Spec and fluorescence [23-27]. The choice of fluorescence detection is within established analytical parameters for this determination as it offers selectivity and sensitivity for these compounds with the identity of the peaks being established by the use of authentic standards.
.
Figure 1. Chromatogram of Dark Chocolate
Extract Analyzed Under Method Parameters. Peak at 11.964 is (±)-catechin while peak at 14.422 is
(±)-epicatechin.Each laboratory also provided information about LOD (Limit of Detection) which was in pure solvent in the 40-50 ng/mL. The LOQ (Limit of Quantitation) ranged from 1- 2 μg/g Repeat injections of standards were also accomplished with %CVs reported in the 1-4% range.
The NIST (National Institute of Standard Technology) reference values for (±)-catechin and (±)-epicatechin in SRM (Standard Reference Material) 2384 are 245 +/- 51 (μg/g) and 1220 +/- 22 (μg/g) respectively with the data from this study indicating value of 254 +/- 23.5 (μg/kg) and 1137 +/- 68 (μg/kg) which are within the acceptable range of determinations established by NIST. While recovery studies have become a default method to assess method accuracy according to Swartz and Krull, the analysis of an established SRM is by itself a generally accepted method of validation [17]. Additionally, guidance from AOAC on methods validation indicates that spiking is not a desirable method to assess method accuracy as spiking solutions tend to be easily extractable hence the choice of the NIST standard to evaluate the method.
The sample labeled Milk Chocolate 1 is an example for a product containing very low amounts of the target analytes. With the analyte concentration in the sample extract at their lower limit of quantification and the chromatographic performance negatively affected by co-extracted matrix compounds the applied method operates at its limit. Hence the sample was not included in the statistical evaluation of the method. That being said, no issues related to complexation of polyphenols with milk reported by some researchers were seen [18].
The %CV ranged from 7-15% in this study. Laboratories used a column that satisfied the requirements of U.S. Pharmacopeia, previously described in methodology section [19]. The method was reviewed and compared with recommendations of Swartz and Krull. Finally, while the data in Table 3 may seem excessive to the casual observer, it is well within the parameters established by AOAC for another complex analyte [20].
The literature reports on the use of numerous solvents for the extraction of flavan-3-ols including mixtures of methanol, acetone, water and acid therefore the solvent combination used is within established parameters [21,22]. Furthermore, a variety of HPLC detector types have been used including UV, Diode Array, Mass Spec and fluorescence [23-27]. The choice of fluorescence detection is within established analytical parameters for this determination as it offers selectivity and sensitivity for these compounds with the identity of the peaks being established by the use of authentic standards.
The data from
these studies indicate the proposed chocolate and cocoa method is suitable as
an HPLC method for the determination of flavanol monomers, (±)-catechin and
(±)-epicatechin in chocolate and cocoa. The method is the first such method
developed by an industry group such as NCA for this purpose.
HPLC:
High-Performance Liquid Chromatography; ISO: International Organization for
Standardization; mL: Milliliter;°C: Degrees Celsius; nm: Nanometer; mm:
Millimeter; μm: micrometer;
PVDF: Polyvinylidene Fluoride; PTFE: Polytertrafluoroethylene; L: Liter; mg:
Milligram; g: Gram; Min: Minutes; μg: Microgram; %: Percent; NIST: National Institute of Standards
and Technology; SRM: Standard Reference Method; LOD: Limit of Detection; LOQ:
Limit of Quantification; AOAC: Association of Official Analytical Chemists; UV
-Ultraviolet
The authors
declare that they have no competing interests.
AB selected
standard and sample preparation methods, chromatography parameters and suitable
commercial samples. LS distributed samples to participating laboratories,
collected and analyzed data and prepared the manuscript. All authors read and
approved the final manuscript.
The authors would
like to thank the following company's laboratories for their participation in
validating the method by testing commercial samples: The Hershey Company,
Archer Daniels Midland Company, Kraft Europe, Kraft-Cadbury and Barry
Callebaut. NCA is especially grateful to the following individuals for their
commitment throughout this process: Jeff Hurst, Mark Payne, Lindo Groff, Mark
Collison, Eva-Maria Berndt, Verena Jendreizik, Alison Branch and Olivier
Nuytten.
References
1. powis TG, Hurst WJ, Rodriguez MC, Ortiz CP,
Blake M, Cheetham D, Coe MD, Hodgson JG: The Origins of Cacao Use in Mesoamerica.
Mexicon 2008,
30(2):35-38.
2. Crown PL, Hurst WJ: Evidence of Cacao
Use in the Prehispanic American Southwest.
Proc Natl Acad Sci USA 2009, 106(7):2110-3. PubMed Abstract |
Publisher Full Text
| PubMed Central Full Text
3. Farouque H, Leung M, Hope S, Baldi M,
Schechter C, Cameron J, Meredith I: Acute and Chronic Effects of
Flavanol-Rich Cocoa on Vascular Function in Subjects With Coronary Artery
Disease: a Randomized Double-Blind Placebo-Controlled Study.
4. Flammer AJ, Hermann F, Sudano I, Spieker L,
Hermann M, Cooper KA, Serafini M, Luscher TF, Ruschitzka F, Noll G, Corti R: Dark Chocolate
Improves Coronary Vasomotion and Reduces Platelet Reactivity.
5. Corti R, Flammer AJ, Hollenberg NK, Luscher
TF: Cocoa and Cardiovascular Health.
6. Heiss C, Dejam A, Kleinbongard P, Schewe T,
Sies H, Kelm M: Vascular Effects of Cocoa Rich in Flavan-3-Ols.
7. Harborne JB: Phytochemical
methods: a guide to modern techniques of plant analysis. Springer: New Delhi, India; 1998.
8. Merken HM, Beecher GR: Liquid
Chromatographic Method for the Separation and Quantification of Prominent
Flavonoid Aglycones.
9. Moon JK, Shibamoto T: Antioxidant
Assays for Plant and Food Components.
10. Beekwilder J, Jonker H, Meesters P, Hall RD, van der Meer IM, De
Vos CHR: Antioxidants in Raspberry: on-Line Analysis Links Antioxidant
Activity to a Diversity of Individual Metabolites.
THE END
ARTICLE-03
Salicylic acid functionalized silica-coated magnetite nanoparticles for
solid phase extraction and preconcentration of some heavy metal ions from various
real samples
1.
Background
Heavy metals are
released into the environment from industrial applications, including mining,
refining and production of textiles, paints and dyes. These pollutants greatly
threaten the health of human populations and the natural ecosystems even at low
concentration. As they do not degrade biologically like organic pollutants,
their presence in drinking water or industrial effluents is a public health
problem due to their absorption and therefore possible accumulation in organisms
[1-5]. The toxicities of
heavy metals may be caused by the inhibition and reduction of various enzymes,
complexation with certain ligands of amino acids and substitution of essential
metal ions from enzymes [4-6]. Hence, their
determination in industrial effluents, various water resources, environmental
and biological samples is important, especially in the environment monitoring
and assessment of occupational and environmental exposure to toxic metals.
However, the direct determination of heavy metal ions at trace levels in real samples remains a challenging problem because of their low concentration and matrix effects even with frequently used sophisticated instrumental techniques such as inductively coupled plasma atomic emission spectrometry (ICP-AES), electrothermal atomic absorption spectrometry (ET-AAS) etc. without sample preconcentration and separation [7-10].
Flame atomic absorption spectrometry (F-AAS) is among the most widely used methods for the determination of the heavy metals at trace levels, but the sensitivity and selectivity of F-AAS is usually insufficient for the determination of heavy metals at trace concentrations in complex matrix environmental samples [11-13]. In trace analysis, therefore, preconcentration or separation of trace elements from the matrix is frequently necessary in order to improve their detection and selectivity by F-AAS [13-16]. Different techniques are used for the separation and preconcentration of metals in the solution. These include liquid-liquid extraction, precipitation, cation-exchange resins, cloud point extraction and solid phase extraction [2,17-20]. However, disadvantages such as significant chemical additives, solvent losses, complex equipments, large secondary wastes, prefiltration problems and time consuming procedures, limit the application of most of these techniques.
Solid phase extraction (SPE) addresses these problems. It can extend the detection limits and remove interfering constituents thereby improving the precision and accuracy of the analytical results. Activated carbon, polymeric fibers, Ambersorb, inorganic ion-exchanger, alumina and silica gel have been used to preconcentrate trace metal ions. However, they suffer from lack of selectivity, which leads to high interference of other existing species with the analyte metal ion and chemical stability [2,17].
Recently, using nanometer-sized materials in SPE as metals ions extractors has turned out to be an active area of research in the field of separation science because of their special properties. Magnetic nanoparticles, a new kind of nanometer-sized material, are widely used in the fields of biotechnology, biomedicine and as an efficient adsorbent with large specific surface area and small diffusion resistance [2,4,6,21-24]. The use of synthetic iron oxides is much more economical than commercial highly efficient activated carbon, in a 30:1 relative ratio depending on the particular kind of activated carbon [25]. The magnetic separation provides suitable route for online separation, where particles with affinity to target species are mixed with the heterogeneous solution. Upon mixing with the solution, the particles tag the target species. External magnetic fields are then applied to separate the tagged particles from the solution.
However, it should be pointed out that pure inorganic nanoparticles (such as Fe3O4 and Fe2O3) can easily form large aggregates, which may alter their magnetic properties [6,25]. Moreover, these nanometer-sized metal oxides are not target-selective and are unsuitable for samples with complicated matrices. Therefore, a suitable coating is essential to overcome such limitations. To overcome latter problem, chemical or physical modification of the sorbent surface with some organic compounds, especially chelating ones, is usually used to load the surface with some donor atoms such as oxygen, nitrogen, sulfur and phosphorus [2,6]. These donor atoms are capable of selective binding with certain metal ions. Salicylic acid (SA) is a commercial ligand with a carboxylic and a phenolic function site which can act as electron pair donors reacting with most of hard and intermediate cations. It has already been used, for example, as the modifier in chelating resins like Amberlite XAD-2-SA, Amberlite XAD-4-SA and silica gel-SA and it have shown good sorption capacity [26-28]. This may be due to the small size of ligand molecules that has facilitated extensive functionalization of the solid support matrices.
In this study, silica-coated magnetic nanoparticles modified with SA were synthesized by a sol-gel method. These magnetic nanoparticles were employed as an SPE adsorbent for separating and concentrating trace amounts of Cu(II), Cd(II), Ni(II) and Cr(III) ions from environmental and various other real matrices prior to their determination by F-AAS and was found to have superior preconcentration and metal loading ability compared to other adsorbents prepared using salicylic acid as the functional group. The propose method was validated by analyzing certified reference materials (both environmental and biological) and by performing recovery studies on water and food samples by F-AAS.
However, the direct determination of heavy metal ions at trace levels in real samples remains a challenging problem because of their low concentration and matrix effects even with frequently used sophisticated instrumental techniques such as inductively coupled plasma atomic emission spectrometry (ICP-AES), electrothermal atomic absorption spectrometry (ET-AAS) etc. without sample preconcentration and separation [7-10].
Flame atomic absorption spectrometry (F-AAS) is among the most widely used methods for the determination of the heavy metals at trace levels, but the sensitivity and selectivity of F-AAS is usually insufficient for the determination of heavy metals at trace concentrations in complex matrix environmental samples [11-13]. In trace analysis, therefore, preconcentration or separation of trace elements from the matrix is frequently necessary in order to improve their detection and selectivity by F-AAS [13-16]. Different techniques are used for the separation and preconcentration of metals in the solution. These include liquid-liquid extraction, precipitation, cation-exchange resins, cloud point extraction and solid phase extraction [2,17-20]. However, disadvantages such as significant chemical additives, solvent losses, complex equipments, large secondary wastes, prefiltration problems and time consuming procedures, limit the application of most of these techniques.
Solid phase extraction (SPE) addresses these problems. It can extend the detection limits and remove interfering constituents thereby improving the precision and accuracy of the analytical results. Activated carbon, polymeric fibers, Ambersorb, inorganic ion-exchanger, alumina and silica gel have been used to preconcentrate trace metal ions. However, they suffer from lack of selectivity, which leads to high interference of other existing species with the analyte metal ion and chemical stability [2,17].
Recently, using nanometer-sized materials in SPE as metals ions extractors has turned out to be an active area of research in the field of separation science because of their special properties. Magnetic nanoparticles, a new kind of nanometer-sized material, are widely used in the fields of biotechnology, biomedicine and as an efficient adsorbent with large specific surface area and small diffusion resistance [2,4,6,21-24]. The use of synthetic iron oxides is much more economical than commercial highly efficient activated carbon, in a 30:1 relative ratio depending on the particular kind of activated carbon [25]. The magnetic separation provides suitable route for online separation, where particles with affinity to target species are mixed with the heterogeneous solution. Upon mixing with the solution, the particles tag the target species. External magnetic fields are then applied to separate the tagged particles from the solution.
However, it should be pointed out that pure inorganic nanoparticles (such as Fe3O4 and Fe2O3) can easily form large aggregates, which may alter their magnetic properties [6,25]. Moreover, these nanometer-sized metal oxides are not target-selective and are unsuitable for samples with complicated matrices. Therefore, a suitable coating is essential to overcome such limitations. To overcome latter problem, chemical or physical modification of the sorbent surface with some organic compounds, especially chelating ones, is usually used to load the surface with some donor atoms such as oxygen, nitrogen, sulfur and phosphorus [2,6]. These donor atoms are capable of selective binding with certain metal ions. Salicylic acid (SA) is a commercial ligand with a carboxylic and a phenolic function site which can act as electron pair donors reacting with most of hard and intermediate cations. It has already been used, for example, as the modifier in chelating resins like Amberlite XAD-2-SA, Amberlite XAD-4-SA and silica gel-SA and it have shown good sorption capacity [26-28]. This may be due to the small size of ligand molecules that has facilitated extensive functionalization of the solid support matrices.
In this study, silica-coated magnetic nanoparticles modified with SA were synthesized by a sol-gel method. These magnetic nanoparticles were employed as an SPE adsorbent for separating and concentrating trace amounts of Cu(II), Cd(II), Ni(II) and Cr(III) ions from environmental and various other real matrices prior to their determination by F-AAS and was found to have superior preconcentration and metal loading ability compared to other adsorbents prepared using salicylic acid as the functional group. The propose method was validated by analyzing certified reference materials (both environmental and biological) and by performing recovery studies on water and food samples by F-AAS.
2.1. Reagents and Apparatus
Chemicals used for
experiments were all in analytical reagent grade. Aqueous solutions of
chemicals were prepared with deionized water. The glass equipments kept in HNO3
10% (v/v) solution overnight and washed with deionized water several
times, oven dried and kept in closed bags before use. Standard solutions of
Cu(II), Cd(II), Ni(II) and Cr(III) ions were prepared from the nitrates of
these elements each as 1000.0 mg L-1. Working solutions, as per the
experimental requirements, were freshly prepared from the stock solution for
each experimental run. pH adjustments were performed with 0.01-1.0 mol L-1
HCl and NaOH solutions.
Certified reference materials such as vehicle exhaust particulates (NIES-8), human hair (NIES-5), tea leaves (NIES-7) and pepperbush (NIES-1) were obtained from the National Institute for Environment Studies (NIES). Zinc base die-casting alloy C (NBS-627) were provided by the Iron and Steel Institute of Japan (Tokyo, Japan) and the National Bureau of Standards, U.S. Department of Commerce, (Washington D.C., USA), respectively. A multivitamin capsule (bearing the commercial name Maxirich) was procured from Arjang pharmacy (Hamedan, Iran) and infant milk substitute, IMS, (commercially available as Lactogen 1), spinach, tomato and hydrogenated oil were obtained from the local market, Hamedan.
The concentration of metals was determined by atomic absorption spectrometry using a Varian model Spect AA 220 apparatus. The instrumental settings of the manufacturer were followed. Infrared spectra were recorded with a Fourier transform infrared spectrometer (FT-IR, Perkin Elmer, spectrum 100). Samples were gently ground and diluted in nonabsorbent KBr matrices to identify the functional groups and chemical bonding of the coated materials. Scanning electron microscopy (SEM) was performed to measure the particle size and shape (SEM-EDX, XL30 and Philips Netherland). The crystal structure of synthesized materials was determined by an X-ray diffractometer (XRD) (38066 Riva, d/G.Via M. Misone, 11/D (TN) Italy) at ambient temperature. Surface area and porosity were defined by N2 adsorption-desorption porosimetry (77 K) using a porosimeter (Bel Japan, Inc.). A Metrohm model 713 (Herisau, Switzerland) pH-meter with a combined glass electrode was used for pH measurements.
Certified reference materials such as vehicle exhaust particulates (NIES-8), human hair (NIES-5), tea leaves (NIES-7) and pepperbush (NIES-1) were obtained from the National Institute for Environment Studies (NIES). Zinc base die-casting alloy C (NBS-627) were provided by the Iron and Steel Institute of Japan (Tokyo, Japan) and the National Bureau of Standards, U.S. Department of Commerce, (Washington D.C., USA), respectively. A multivitamin capsule (bearing the commercial name Maxirich) was procured from Arjang pharmacy (Hamedan, Iran) and infant milk substitute, IMS, (commercially available as Lactogen 1), spinach, tomato and hydrogenated oil were obtained from the local market, Hamedan.
The concentration of metals was determined by atomic absorption spectrometry using a Varian model Spect AA 220 apparatus. The instrumental settings of the manufacturer were followed. Infrared spectra were recorded with a Fourier transform infrared spectrometer (FT-IR, Perkin Elmer, spectrum 100). Samples were gently ground and diluted in nonabsorbent KBr matrices to identify the functional groups and chemical bonding of the coated materials. Scanning electron microscopy (SEM) was performed to measure the particle size and shape (SEM-EDX, XL30 and Philips Netherland). The crystal structure of synthesized materials was determined by an X-ray diffractometer (XRD) (38066 Riva, d/G.Via M. Misone, 11/D (TN) Italy) at ambient temperature. Surface area and porosity were defined by N2 adsorption-desorption porosimetry (77 K) using a porosimeter (Bel Japan, Inc.). A Metrohm model 713 (Herisau, Switzerland) pH-meter with a combined glass electrode was used for pH measurements.
2.2. Preparation of samples
2.2.1. Natural and sewage
water samples
The water samples,
river water (collected from Alvand, Hamedan, Iran), canal water (collected from
Yazd, Iran), sewage water (collected from area in the vicinity of local nickel
electroplating industry, Hamedan) and tap water (collected from our faculty)
were immediately filtered through Millipore cellulose membrane filter (0.45 μm pore size), acidified to pH 2 ± 0.01 with
HNO3, and stored in precleaned polyethylene bottles. After then, pH
of the sample was adjusted to 6.0 and the procedure described in section 2.5
has been carried out.
2.2.2. Digestion of standard
environmental, biological and metal alloy samples
Two certified
reference materials (CRMs); vehicle exhaust particulates (NIES-8), pepperbush
(NIES-1) and tea leaves (NIES-7), were analyzed. Approximately 0.50 g of this
material, were weighed accurately into a Teflon cup, and dissolved in
concentrated nitric acid (~10 mL), with heating in a water bath. The solution
was cooled, diluted and filtered. The filtrate was made to 100.0 mL, with
deionized water in a calibrated flask. An aliquot of the sample solution was
taken, and the target metals ions were determined by the given procedure.
The sample solutions of biological CRMs such as human hair (NIES-5) was prepared as proposed by International Atomic Energy Agency [29]. A 50.0 mg of each of the samples was agitated with 25 mL of acetone, and then washed three times with deionized water and with 25 mL of acetone. The washed samples were placed in a glass beaker individually and allowed to dry at room temperature. Decomposition of organic matter is an important part for determination of heavy metals in these samples. Therefore, each of the samples was dissolved in 10 mL of concentrated nitric acid. After adding 2.5 mL of 30% H2O2 the solution was boiled to dryness. The residue obtained was dissolved in minimum amount of 2% HCl and made up to a 50 mL volume in a calibrated flask. Then the procedure given in Section 2.5 was performed.
To dissolve the standard reference alloy, zinc based die-casting alloy C (NBS-627), 25 mg of the sample was taken into a beaker and dissolved in 10-50 mL of HCl:HNO3 mixture (3:1). The solution was boiled to near dryness. Finally, the residue was dissolved in minimum volume of 2% HCl and filtered through Whatman filter paper No. 1. The residue was washed with two 5 mL portions of hot 2% HCl. The solution was evaporated to dryness. The residue was dissolved in 5 mL of 2% HCl and make up to 50 mL with deionized water after its pH was adjusted to desired value.
The sample solutions of biological CRMs such as human hair (NIES-5) was prepared as proposed by International Atomic Energy Agency [29]. A 50.0 mg of each of the samples was agitated with 25 mL of acetone, and then washed three times with deionized water and with 25 mL of acetone. The washed samples were placed in a glass beaker individually and allowed to dry at room temperature. Decomposition of organic matter is an important part for determination of heavy metals in these samples. Therefore, each of the samples was dissolved in 10 mL of concentrated nitric acid. After adding 2.5 mL of 30% H2O2 the solution was boiled to dryness. The residue obtained was dissolved in minimum amount of 2% HCl and made up to a 50 mL volume in a calibrated flask. Then the procedure given in Section 2.5 was performed.
To dissolve the standard reference alloy, zinc based die-casting alloy C (NBS-627), 25 mg of the sample was taken into a beaker and dissolved in 10-50 mL of HCl:HNO3 mixture (3:1). The solution was boiled to near dryness. Finally, the residue was dissolved in minimum volume of 2% HCl and filtered through Whatman filter paper No. 1. The residue was washed with two 5 mL portions of hot 2% HCl. The solution was evaporated to dryness. The residue was dissolved in 5 mL of 2% HCl and make up to 50 mL with deionized water after its pH was adjusted to desired value.
2.2.3. Preparation of
multivitamin capsule and food samples
Five multivitamin
capsules (5.83 g) were taken in a beaker containing 25 mL of concentrated HNO3
and digested by slowly increasing the temperature of the mixture to 40 ±
0.2°C. The solution was gently evaporated on a steam bath until a residue was
left. It was subsequently mixed with 50 mL of deionized water and HNO3 was
then added drop wise until a clear solution was obtained on gentle heating.
Powdered IMS food sample (200.0 mg) was heated in a beaker containing mixture of concentrated H2SO4 (20 mL) and HNO3 (10 mL) till a clear solution was obtained. It was allowed to cool and most of the acid was neutralized with NaOH. The total volume was made up to 50 mL with deionized water and kept as stock.
Hydrogenated oil (2.00 g) was taken in a beaker and dissolved in 15 mL of concentrated nitric acid with heating. The solution was cooled, diluted and filtered. The filtrate was made up to 50 mL with deionized water after adjusting its pH to the optimum value.
A 10 g sample of tomato sample was heated in silica crucible for 3 h on a hot plate and the charred material was transferred to a furnace for overnight heating at 650°C. The residue was cooled, treated with 10.0 mL concentrated nitric acid and 3 mL 30% H2O2 and again kept in a furnace for 2 h. The final residue was treated with 3 mL concentrated hydrochloric acid and 2-4 mL of 70% perchloric acid and evaporated to fumes. The solid residue was dissolved in water, filtered and the pH was adjusted to 6.0 by the addition of NaOH and HCl solutions. The preconcentration procedure given above in section 2.5 was then applied to these solutions.
0.1 g of vegetable sample was placed in a 100 mL beaker and 10 mL of concentrated HNO3 was added. The mixture was evaporated near to dryness on a hot plate at about 150°C. After cooling to room temperature, 3 mL of concentrated hydrogen peroxide was added. The mixture was again evaporated to dryness and the residue dissolved with 0.5 mol L-1 HNO3. It was filtered through a filter paper. The preconcentration procedure was applied to this sample solution.
Powdered IMS food sample (200.0 mg) was heated in a beaker containing mixture of concentrated H2SO4 (20 mL) and HNO3 (10 mL) till a clear solution was obtained. It was allowed to cool and most of the acid was neutralized with NaOH. The total volume was made up to 50 mL with deionized water and kept as stock.
Hydrogenated oil (2.00 g) was taken in a beaker and dissolved in 15 mL of concentrated nitric acid with heating. The solution was cooled, diluted and filtered. The filtrate was made up to 50 mL with deionized water after adjusting its pH to the optimum value.
A 10 g sample of tomato sample was heated in silica crucible for 3 h on a hot plate and the charred material was transferred to a furnace for overnight heating at 650°C. The residue was cooled, treated with 10.0 mL concentrated nitric acid and 3 mL 30% H2O2 and again kept in a furnace for 2 h. The final residue was treated with 3 mL concentrated hydrochloric acid and 2-4 mL of 70% perchloric acid and evaporated to fumes. The solid residue was dissolved in water, filtered and the pH was adjusted to 6.0 by the addition of NaOH and HCl solutions. The preconcentration procedure given above in section 2.5 was then applied to these solutions.
0.1 g of vegetable sample was placed in a 100 mL beaker and 10 mL of concentrated HNO3 was added. The mixture was evaporated near to dryness on a hot plate at about 150°C. After cooling to room temperature, 3 mL of concentrated hydrogen peroxide was added. The mixture was again evaporated to dryness and the residue dissolved with 0.5 mol L-1 HNO3. It was filtered through a filter paper. The preconcentration procedure was applied to this sample solution.
2.3. Preparation of
silica-coated magnetite nanoparticles
The magnetite
nanoparticles (MNPs) were prepared by the conventional co-precipitation method
with minor modifications [30]. In this method,
ultrasonic vibration by an ultrasonic bath was used instead of magnetic
stirring. FeCl3.6H2O (11.68 g) and FeCl2.4H2O
(4.30 g) were dissolved in 200 mL deionized water under nitrogen gas in an
ultrasonic bath at 85°C for a few minutes leading to smaller and more
homogenized particles. Then, 20 mL of 30% NH3 2O, which is different
from the 15 mL of 20% NH3 2O used in Ref. [30], were added to the
solution. The color of bulk solution changed from orange to black immediately.
The magnetite precipitates were washed twice with deionized water and once with
0.02 mol L-1 sodium chloride. The washed magnetite was stored in
deionized water at a concentration of 40.0 g L-1.
Then, the magnetite suspension prepared above (20 mL) was placed in a 250 mL round-bottom flask and allowed to settle. The supernatant was removed, and an aqueous solution of tetraethoxysilane [TEOS, 10% (v/v), 80 mL] was added, followed by glycerol (60 mL). The pH of the suspension was adjusted to 4.6 using glacial acetic acid, and the mixture was then stirred and heated at 90°C for 2 h under a nitrogen atmosphere. After cooling to room temperature, the suspension was washed sequentially with deionized water (3 × 500 mL), methanol (3 × 500 mL), and deionized water (5 × 500 mL). The silica magnetite composite was stored in deionized water at a concentration of 40.0 g L-1.
Then, the magnetite suspension prepared above (20 mL) was placed in a 250 mL round-bottom flask and allowed to settle. The supernatant was removed, and an aqueous solution of tetraethoxysilane [TEOS, 10% (v/v), 80 mL] was added, followed by glycerol (60 mL). The pH of the suspension was adjusted to 4.6 using glacial acetic acid, and the mixture was then stirred and heated at 90°C for 2 h under a nitrogen atmosphere. After cooling to room temperature, the suspension was washed sequentially with deionized water (3 × 500 mL), methanol (3 × 500 mL), and deionized water (5 × 500 mL). The silica magnetite composite was stored in deionized water at a concentration of 40.0 g L-1.
2.4. Preparation of
silica-coated magnetite nanoparticles modified with salicylic acid
25 mL of
silica-coated magnetite prepared as described above was washed with ethanol (2
× 100 mL) and then diluted to 150 mL with 3.3% SA solution and 16 mmol L-1
acetic acid solution (pH 4.5). The solution was transferred to a 500 mL
3- necked round-bottom flask and then stirred and heated at 60°C for 2 h under
a nitrogen atmosphere. After that, the resulting nanospheres were washed with
deionized water three times and twice with methanol, then dried into powders at
room temperature under vacuum (Figure 1).
Figure 1. Schematic of the preparation of
adsorbent (a) and solid phase extraction of the analytes (b).
Figure 1. Schematic of the preparation of
adsorbent (a) and solid phase extraction of the analytes (b).
2.5. Recommended procedure
for sorption and desorption of heavy metal ions
A series of sample
solutions containing heavy metal ions were transferred into a 1 L beaker. The
pH of the solution was adjusted to 6.0 using 0.01-0.1 mol L-1 HCl
and/or NaOH solutions. After that, 0.11 g of the sorbent was added to the
solution and the mixtures were dispersed by ultrasonication for 10 min at room
temperature to attain equilibrium, and then magnetically separated (Figure 1). Then, the sorbent
was washed with deionized water and afterwards, the metal ions retained on
sorbent were eluted with the solution of the mixture of 6.0 mL of 1.0 mol L-1
HNO3. The analytes in the eluate were then determined by
F-AAS.
3.1.
Characteristics of modified magnetite nanoparticles
The surface and
textural morphology of silica coated magnetite nanoparticles by SEM image is
illustrated in Figure 2. As shown in Figure 2, the naked magnetite
nanoparticles had a mean diameter of 29 nm. Using the ultrasonic vibrations
caused the prepared nanoparticles were smaller and more homogenized particles [30]. After
modification process, the modified nanoparticles prepared are in the range of
58-73 nm in diameter. This shows that the magnetite nanoparticles have been
completely coated by the silica and SA. Also, this could be attributed to the
reaction occurring only on the particle surface, and thus our attempt to
prepare SA-silica coated magnetite nanoparticles in this work has been
achieved.
Figure 2. SEM images of synthesized
magnetite nanoparticles (left) and modified magnetite nanoparticles (right).
The typical X-ray diffraction (XRD) profile of silica-coated magnetite nanoparticles is shown in Figure 3. The broad peak at around 2θ = 20° in the XRD pattern is due to the amorphous silica shell on the surface of the magnetite nanoparticles. The characteristic peaks of the magnetite nanoparticles were also clearly identified in the XRD pattern for a standard magnetite pattern (Joint Committee on Powder Diffraction Standards (JCPDS) file no. 19-0629) [30,31]. Also, with comparison of peaks of silica-coated magnetite nanoparticles and silica-coated magnetite nanoparticles modified with SA concluded although the magnetic particle surfaces were coated with SA, the very distinguishable FCC peaks of magnetite crystal were observed, which means that these particles have the phase stability. The different functional groups such as hydroxide and carboxylylic did not affect on crystallinity and morphology in this study [32].
Figure 3. X-ray diffraction pattern of
silica coated magnetite nanoparticles.
The specific surface area was determined using the Brunauer-Emmett-Teller (BET) equation applied to the adsorption data on nitrogen adsorption/desorption experiments. The results of the BET method showed that the average specific surface area of silica-coated magnetite nanoparticles modified with SA was 41.62 m2 g-1. It can be concluded from these values that this type adsorbent is nanoparticles with relatively large specific surface area.
The adsorbent was subsequently characterized by FT-IR spectral data. The FT-IR spectrum of silica-coated magnetite nanoparticles modified with SA has prominent bands at 1680 cm-1, 1486 cm-1, and 1387 cm-1 due to carboxylate, OH (bending) and phenolic group vibrations, respectively (Figure 4). This supports the immobilizing of SA onto silica-coated magnetite nanoparticles. The red shifts of the two peaks namely hydroxyl and carboxylic by 20-25 cm-1 for metal loaded adsorbent further suggest that chelation with salicylic acid functionality is responsible for the sorption of metal ions by adsorbent. Furthermore, the adsorbent shows good chemical stability with no less of capacity up to 4.0 mol L-1 of HCl/HNO3/H2SO4 used for stripping of metal ions. It can withstand alkaline medium up to 3.0 mol L-1 of NaOH.
Figure 4. FT-IR spectra (a) salicylic
acid (b) Fe3O4 nanoparticles (c) SiO2 coated
Fe3O4 nanoparticles (d) SiO2 coated Fe3O4
nanoparticles modified with salicylic acid.
Figure 2. SEM images of synthesized
magnetite nanoparticles (left) and modified magnetite nanoparticles (right).The typical X-ray diffraction (XRD) profile of silica-coated magnetite nanoparticles is shown in Figure 3. The broad peak at around 2θ = 20° in the XRD pattern is due to the amorphous silica shell on the surface of the magnetite nanoparticles. The characteristic peaks of the magnetite nanoparticles were also clearly identified in the XRD pattern for a standard magnetite pattern (Joint Committee on Powder Diffraction Standards (JCPDS) file no. 19-0629) [30,31]. Also, with comparison of peaks of silica-coated magnetite nanoparticles and silica-coated magnetite nanoparticles modified with SA concluded although the magnetic particle surfaces were coated with SA, the very distinguishable FCC peaks of magnetite crystal were observed, which means that these particles have the phase stability. The different functional groups such as hydroxide and carboxylylic did not affect on crystallinity and morphology in this study [32].
Figure 3. X-ray diffraction pattern of
silica coated magnetite nanoparticles.The specific surface area was determined using the Brunauer-Emmett-Teller (BET) equation applied to the adsorption data on nitrogen adsorption/desorption experiments. The results of the BET method showed that the average specific surface area of silica-coated magnetite nanoparticles modified with SA was 41.62 m2 g-1. It can be concluded from these values that this type adsorbent is nanoparticles with relatively large specific surface area.
The adsorbent was subsequently characterized by FT-IR spectral data. The FT-IR spectrum of silica-coated magnetite nanoparticles modified with SA has prominent bands at 1680 cm-1, 1486 cm-1, and 1387 cm-1 due to carboxylate, OH (bending) and phenolic group vibrations, respectively (Figure 4). This supports the immobilizing of SA onto silica-coated magnetite nanoparticles. The red shifts of the two peaks namely hydroxyl and carboxylic by 20-25 cm-1 for metal loaded adsorbent further suggest that chelation with salicylic acid functionality is responsible for the sorption of metal ions by adsorbent. Furthermore, the adsorbent shows good chemical stability with no less of capacity up to 4.0 mol L-1 of HCl/HNO3/H2SO4 used for stripping of metal ions. It can withstand alkaline medium up to 3.0 mol L-1 of NaOH.
Figure 4. FT-IR spectra (a) salicylic
acid (b) Fe3O4 nanoparticles (c) SiO2 coated
Fe3O4 nanoparticles (d) SiO2 coated Fe3O4
nanoparticles modified with salicylic acid.
3.2. Variables Optimization
of preconcentration process
3.2.1. Effect of pH for metal
ions uptake
Solution acidity
can show either of two different effects on metal adsorption: protonation of
binding sites of the chelating molecules, and complexation or precipitation
many metal ions by hydroxide ion. Therefore, since the solution pH is an
important parameter to obtain quantitative recovery for heavy metal ions, this
was the first parameter that was optimized. The influence of pH on the
preconcentration of target metal ions over the pH range from 2.0 to 10.0 was
studied (keeping the other parameters constant). As could be seen from Figure 5, quantitative
recovery was obtained for Cr(III), Cu(II), Ni(II) and Cd(II) within the pH
range 5.0-7.0. This may be attributed to the presence of free lone pair of
electrons on oxygen atoms, which are suitable functional sites for coordination
with the metal ions. The decrease in recovery at pH values lower than 5.0 may
be due to the competition of proton with cations in binding to donor atoms.
Considering these facts and in order to avoid an abrupt change in adsorption (which
may occur due to minor changes in the pH), and also to preconcentrate of these
ions simultaneously, pH 6.0 was selected as the optimal pH for all subsequent
experiments.
Figure 5. Relation between pH and
recoveries of analytes (N = 3).
Figure 5. Relation between pH and
recoveries of analytes (N = 3).
3.2.2. Effect of contact time
The efficiencies
of the analytes deposition depend on the contact time of sample with the solid
phase. It is necessary to require the preconcentrate of metal ions in short
time. In this regard, replicate sets of analytes and adsorbents were prepared
and investigating at different time intervals 2, 5, 8, 10, 15, 20, 30 min.
Results showed that the rate of uptake of the analytes was quite high (Figure 6). Adsorption of
Cd(II), Cu(II), Ni(II) and Cr(III) from the solution reached more than 95% at
about 8 min. Therefore, ultrasonication time of 10 min was selected for further
works.
Figure 6. Effect of contact time on
recovery percentage of 20 μg L-1 heavy metal ions; pH 6.0; adsorbent
0.1 g.
Figure 6. Effect of contact time on
recovery percentage of 20 μg L-1 heavy metal ions; pH 6.0; adsorbent
0.1 g.
3.2.3. Effect of the adsorbent
amount
Figure 7. Recovery percentage of metal
ions at different adsorbent dosage.
3.2.4. Effect of sediment
time
Conventional SPE
usually requires filtration or centrifugation to separate the adsorbent from
aqueous solutions, which makes the method time-consuming. In this study, the
adsorbent could be separated rapidly from the sample solution using an external
magnetic field, due to the superparamagnetism of these nanoparticles. The
effect of sediment time on the recovery of metal ions was investigated, and no
significant effect was seen when the sedimentation time was greater than 60 s.
A sediment time of 1 min was therefore selected in subsequent experiments.
3.2.5. Effect of the type,
concentration and volume of the eluent
The selection of
suitable eluent was a difficult problem. As could be seen from Figure 4, the uptake of these
metal ions was negligible at low pH; therefore, the acidic eluent is the best
solution to obtain efficient extraction. The optimal eluent was a difficult
problem because of the limitation of FAAS to tolerate organic solvents while
the eluent should not destroy the solid phase. Various acids were used to
identify the best eluent for the adsorbed metal ions on the adsorbent. The
results are given in Table 1. Deionized water was
found to be unsuitable for the purpose of elution as <0.5% recovery was
achieved indicating that the metal ions were retained by the adsorbent by some
strong bonding forces. Among of different eluents used, 1.0 mol L-1 of
HNO3 provided higher recovery and reproducibility. Therefore, this
solution was chosen as an eluent for the metal ions from nano-sized adsorbent.
Table 1. Effect of type and concentration eluting solution (4 mL) on analytes recovery (%)
Subsequent experiments showed that even 2.0 mL of the eluent solution was enough for elution of metal ions, however, in all experiments metal ions were eluted by 4.0 mL of 1.0 mol L-1 of HNO3 solution because this volume was necessary for reading absorption signal of analytes by F-AAS.
Table 1. Effect of type and concentration eluting solution (4 mL) on analytes recovery (%)
Subsequent experiments showed that even 2.0 mL of the eluent solution was enough for elution of metal ions, however, in all experiments metal ions were eluted by 4.0 mL of 1.0 mol L-1 of HNO3 solution because this volume was necessary for reading absorption signal of analytes by F-AAS.
3.2.6. Effect of the sample
volume
Due to the low
concentrations of trace metals in real samples, these analytes should be taken
into smaller volumes for high preconcentration factor by using sample solutions
with large volumes. Therefore the maximum applicable sample volume was
determined by increasing the dilution of metal ion solution, while keeping the
total amount of loaded metal ion fixed for analytes. Different feed volumes
varied between 50 and 1000 mL. The recoveries were found to be stable until 800
mL and were chosen as the largest sample volume to work. In this study, the
final solution volume to be measured by F-AAS was 4.0 mL; therefore the
preconcentration factors were 200 for all metal ions. At volumes higher than
800 mL probably the analyte ions are not sorbed effectively because of low
amount of adsorbent in those volumes. As stated previously, the final solution
volume, after eluting the metal ions, was 4.0 mL, therefore the preconcentration
factors of 200 was obtained for all analytes.
3.3. Stability and
reusability of adsorbent
The reusability and stability of
the adsorbent was investigated. The capacity of the modified adsorbent was
found to be apparently constant (less than 3%) after the repeated use of more
than 9 cycles of sorption and desorption of the target analytes.
3.4. Effect of potentially
interfering ions
In view of the
fact that flame atomic absorption spectrometry provides high selectivity, the
only interference may be attributed to the preconcentration step. For
application of recommended solid phase extraction to real samples, effects of
some interfering species on the recovery of metal ions were investigated with
the optimized procedure. Various species, which are inevitably associated with
heavy metals, may interfere in the final determination through precipitate
formation, redox reactions or competing complexation reactions. In order to
assess the analytical applicability of the adsorbent to real samples, common chemical
species such as sodium citrate, sodium tartrate, sodium oxalate, humic acid,
fulvic acid, NO3-, CO32-, NH4+,
SO42-, PO43-, Cl-, K+
and Na+ were checked for any interference in the sorption of
these metals. The tolerance limit is defined here as the species concentration
causing a relative error smaller than ± 5% related to the preconcentration and
determination of the analytes. The tolerable limits of interfering ions are
given in Table 2.
Table 2. Effects of the matrix ions on the recoveries of the examined metal ions.
The allowable amount of Fe(III) ions as an interfering species was lower than the other investigated species in preconcentration of cadmium. The usage of masking agent such as NH4F for this interfering species in the present method resulted in suppressing the effect of Fe(III) interference and improving the selectivity. However, the tolerance ratio for Fe(III) ion could be raised to 700 times when 2 mL of 0.25 mol L-1 NH4F solution is also added when necessary.
Table 2. Effects of the matrix ions on the recoveries of the examined metal ions.
The allowable amount of Fe(III) ions as an interfering species was lower than the other investigated species in preconcentration of cadmium. The usage of masking agent such as NH4F for this interfering species in the present method resulted in suppressing the effect of Fe(III) interference and improving the selectivity. However, the tolerance ratio for Fe(III) ion could be raised to 700 times when 2 mL of 0.25 mol L-1 NH4F solution is also added when necessary.
3.5. Adsorption capacities
The capacity of
the adsorbent is an important factor because it determines how much adsorbent
is required to quantitatively remove a specific amount of metal ions from the
solutions. The adsorption capacity was tested following the batch procedure.
110 mg of sorbent was equilibrated with 800 mL of various concentrations of
Cu(II), Ni(II), Cd(II) and Cr(III) for 1 h. In order to reach the
"saturation", the initial metal ions, concentrations were increased
till the plateau values (adsorption capacity values) obtained. The results
showed that adsorption capacity of various metal ions probably differ due to
their size, degree of hydration and the value of their binding constant with
the adsorbent. The maximum adsorption capacity has been found to be 39.9, 39.8,
27.8 and 17.3 mg g-1 for Cu(II), Cr(III), Cd(II) and Ni(II),
respectively.
3.6. Analytical precision and
detection limits
Under the selected
conditions, eight portions of standard solutions were enriched and analyzed
simultaneously following the general procedure. Linearity was maintained
between 0.73 μg L-1 to
0.40 mg L-1 for copper; 0.91 μg L-1 to 0.35 mg L-1 for nickel; 0.44 μg L-1 to 0.64 mg L-1 for
chromium and 0.37 μg L-1 to
0.45 mg L-1 for cadmium, in initial solution. The detection limit
(DL) of the present work was calculated under optimal conditions after the
application of preconcentration and separation procedure of blank solutions
analyzed by F-AAS. The DL was calculated as DL = kSb/m, where k is equal to 3 according to the desired
confidence level (95%), Sb
is the standard
deviation of the blank signal and m is the slope of the analytical curve (n = 8). The detection
limits were found to be 0.15, 0.22, 0.27 and 0.11 μg L-1 for Cr(III), Cu(II),
Ni(II) and Cd(II), respectively. The relative standard deviation (RSD) of the
eight replicate determinations was lower than 4.0% (Cr(III): 3.1%; Cu(II):
2.2%; Ni(II): 3.3%; Cd(II): 2.6%), which indicated that the method had good
precision for the analysis of trace target ions in solution samples.
3.7. Application to real
samples
Certified
reference materials were analyzed by developed method. The results are given in
Table 3. The results show
that the results are good agreement with the certified values for the
investigated analyte ions. The proposed method was applied to a various water
samples. The results were given Table 4. Proposed solid
phase extraction method for determination of these metals in some water samples
were applied successfully. Recovery values can be quantitatively except in all
samples. Multivitamin capsule, IMS and hydrogenated oil samples were
investigated as samples with complex matrices. These results indicate the
applicability of the developed procedure; for selective preconcentration of
target analytes; and that it is free of interference (Table 5). Also, the proposed
procedure has been applied to the determination of copper, nickel, chromium and
cadmium content; in tomato and spinach samples. The results are given in Table 6. As can be seen from
the results in Table 6 the metal ions were
quantitatively recovered from the food samples; by the proposed procedure.
These results demonstrate, the applicability of the procedure for target ions
determination in water samples.
Table 3. Results for metal ions determination in certified reference samples obtained using the optimum conditions.
Table 4. Results for metal ions determination in various water samples obtained using the optimum conditions.
Table 5. Results for metal ions determination in various samples obtained using the optimum conditions.
Table 6. Results for metal ions determination in food samples obtained using the optimum conditions.
Table 3. Results for metal ions determination in certified reference samples obtained using the optimum conditions.
Table 4. Results for metal ions determination in various water samples obtained using the optimum conditions.
Table 5. Results for metal ions determination in various samples obtained using the optimum conditions.
Table 6. Results for metal ions determination in food samples obtained using the optimum conditions.
A simple,
sensitive and selective method was developed for the preconcentration of
cadmium, copper, nickel and chromium in various real samples. In summery,
silica coated Fe3O4 nanoparticles modified with SA with
well defined diameter prepared by such a simple, time-saving and low cost route
using sol-gel method combined ultrasonic stirring. These nanoparticles have
relatively high adsorption as compared to the similar materials because of
their smaller size. The size of the produced modified maghemite nanoparticles
was determined by X-ray diffraction (XRD) analysis and scanning electron
microscopy (SEM). The present method has following advantages over reported
methods. Synthesized adsorbent is distinct in terms of sensitivity, selectivity
towards investigated metal ions. Also, these magnetic nanoparticles carrying
the target metals could be easily separated from the aqueous solution simply by
applying an external magnetic field; no filtration or centrifugation was
necessary. Furthermore, the proposed method gives an efficient and cost
effective method with very low detection limits and good relative standard
deviation and can be applied to the determination of traces of these ions in
various real samples.
The authors
declare that they have no competing interests.
HB carried out the
synthesis of adsorbent and performed the some of experimental AA carried out
the survey of prepared adsorbent, participated in the design of the study.
sections. MRS performed the experimental sections, participated in the sequence
alignment and drafted the manuscript. All authors read and approved the final
manuscript.
The authors
gratefully acknowledge the financial and technical support provided by the
Research Council of Islamic Azad University, Yazd Branch.
References
1. Tu ZF, He Q, Chang XJ, Hu Z, Gao R, Zhang
L, Li ZH: 1-(2-Formamidoethyl)-3-phenylurea functionalized activated carbon
for selective solid-phase extraction and preconcentration of metal ions.
2. Afkhami A, Saber-Tehrani M, Bagheri H,
Madrakian T: Flame atomic absorption spectrometric determination of trace
amounts of Pb(II) and Cr(III) in biological, food and environmental samples
after preconcentration by modified nano-alumina.
3. Li ZH, Chang XJ, Zou XJ, Zhu XB, Nie R, Hu
Z, Li RJ: Chemically-modified activated carbon with ethylenediamine for
selective solid-phase extraction and preconcentration of metal ions.
4. Afkhami A, Saber-Tehrani M, Bagheri H: Simultaneous
removal of heavy-metal ions in wastewater samples using nano-alumina modified
with 2,4-dinitrophenylhydrazine.
5. Marahel F, Ghaedi M, Montazerozohori M,
Nejati Biyareh M, Nasiri Kokhdan S, Soylak M: Solid-phase extraction and
determination of trace amount of some metal ions on Duolite XAD 761 modified
with a new Schiff base as chelating agent in some food samples.
6. Huang CZ, Hu B: Silica-coated magnetic
nanoparticles modified with γ-mercaptopropyltrimethoxysilane for fast and
selective solid phase extraction of trace amounts of Cd, Cu, Hg, and Pb in
environmental and biological samples prior to their determination by
inductively coupled plasma mass spectrometry.
7. Dadfarnia S, Haji Shabani AM, Dehgan Shirie
H: Determination
of lead in different sSamples by atomic absorption spectrometry after
preconcentration with dithizone immobilized on surfactant-coated alumina.
Bull Korean Chem Soc 2002, 23:545-548.
8. Haji Shabani AM, Dadfarnia S, Dehghani Z: On-line solid
phase extraction system using 1,10-phenanthroline immobilized on surfactant
coated alumina for the flame atomic absorption spectrometric determination of
copper and cadmium.
Talanta 2009,
76:1066-1070.
9. Soylak M, Kars A, Narin I: Coprecipitation
of Ni2+, Cd2+ and Pb2+ for preconcentration in
environmental samples prior to flame atomic absorption spectrometric
determinations.
10. Narin I, Surme Y, Bercin E, Soylak M: SP70-α-benzoin
oxime chelating resin for preconcentration-separation of Pb(II), Cd(II), Co(II)
and Cr(III) in environmental samples.
11. Sun ZM, Liang P: Determination of Cr(III) and total
chromium in water samples by cloud point extraction and flame atomic absorption
spectrometry.
12. Ghaedi M, Tavallali H, Shokrollahi A, Zahedi M, Montazerozohori M,
Soylak M: Flame atomic absorption spectrometric determination of zinc,
nickel, iron and lead in different matrixes after solid phase extraction on
sodium dodecyl sulfate (SDS)-coated alumina as their bis
(2-hydroxyacetophenone)-1,3-propanediimine chelates.
13. Mohammadi SZ, Afzali D, Pourtalebi D: Flame atomic
absorption spectrometric determination of trace amounts of lead, cadmium and
nickel in different matrixes after solid phase extraction on modified
multiwalled carbon nanotubes.
14. Divrikli U, Kartal AA, Soylak M, Elci L: Preconcentration
of Pb(II), Cr(III), Cu(II), Ni(II) and Cd(II) ions in environmental samples by
membrane filtration prior to their flame atomic absorption spectrometric
determinations.
15. Mikuła B, Puzio B, Feist B: Application of 1,10-phenanthroline for
preconcentration of selected heavy metals on silica gel.
THE END
ARTICLE-04
Amorphous calcium phosphate and its application in dentistry
1. Review
Amorphous calcium
phosphate (ACP) is the initial solid phase that precipitates from a highly
supersaturated calcium phosphate solution, and can convert readily to stable
crystalline phases such as octacalcium phosphate or apatitic products. Its
morphological form, structural model and X-ray diffraction patterns are typical
for noncrystalline substances with short-range periodic regularity. ACP has
been demonstrated to have better in vivo osteoconductivity than hydroxyapatite
(HAP), better biodegradability than tricalcium phosphate, good bioactivity but
no cytotoxicity [1]. These excellent
biological properties make ACP widely used in dentistry, orthopaedics and
medicine. This review provides an overview of the development, structure,
composition and morphology characterization, phase transformation and
biomedical application of ACP in dentistry.
Generally, it is
believed that ACP was firstly described by Aaron S. Posner [1] in the mid 1960s.
It was obtained as an amorphous precipitate by accident when mixing high
concentrations (30 mM) of calcium chloride and sodium acid phosphate (20 mM) in
buffer [2]. In X-ray
diffraction, it was shown to have only two broad and diffuse peaks, with
maximum at 25° 2θ. No other
features were obvious and it was clearly not apatite. This pattern is typical
for substances that lack long range periodic regularity. It was found that
immediately after being mixed, the spontaneously formed precipitate was a
non-crystalline, or amorphous, calcium phosphate with calcium to phosphorus
molar ratio (Ca/P) of 1.50. After several hours, it could convert to poorly
crystalline apatite on ageing. Afterwards, this solid converts slowly to
crystalline apatite (Ca/P = 1.67) by an autocatalytic mechanism [3].
In 1965, Eanes et al. identified ACP as a bone component [2]. ACP in bone, along with the apatite, might account for the broad diffraction pattern and variable composition of bone minerals. An age-dependent change in the ACP content of bone was also described, with the proportion of ACP decreasing with age [3,4]. In 1975, ACP was found in the mineralized cytoplasmic structure isolated from the blue crab hepatopancreas, with a very similar short-range atomic structure to synthetic amorphous calcium phosphate [5].
In 1965, Eanes et al. identified ACP as a bone component [2]. ACP in bone, along with the apatite, might account for the broad diffraction pattern and variable composition of bone minerals. An age-dependent change in the ACP content of bone was also described, with the proportion of ACP decreasing with age [3,4]. In 1975, ACP was found in the mineralized cytoplasmic structure isolated from the blue crab hepatopancreas, with a very similar short-range atomic structure to synthetic amorphous calcium phosphate [5].
After the
discovery of amorphous calcium phosphate, the early studies were focused on the
structure of ACP. It was suggested that synthetic ACP particles, which appear
as 300- 1000 Å spheres in the electron microscope, consist of a random assembly
of ion clusters 9.5 Å in diameter, dimensions consistent with the chemical composition
of Ca9(PO4)6 [5]. And the 15-20% of
water found in synthetic amorphous calcium phosphate was shown to be mostly in
the interstices between, and not within, the individual Ca9(PO4)6
clusters [6]. Aggregated ACP
particles readily dissolve and crystallize to form apatite, a thermodynamically
stable phase. The typical radial distribution of noncrystalline ACP cluster
structures, calculated from the x-ray diffraction patterns, is only two broad
and diffuse peaks showing the rapid drop-off of atomic periodicity. Short-range
order exists in these amorphous structures but no long-range order such as that
in crystalline hydroxyapatite [6]. Infrared analysis
showed a similar lack of crystalline order about the PO4 anions in
the ACP structure [7].
It is now generally agreed that, both in vitro and in vivo, precipitation reactions at sufficiently high supersaturation and pH result in the initial formation of an amorphous calcium phosphate with a molar calcium/phosphate ratio of about 1.5, with a range of 1.34-1.50 in different pH and 1.50-1.67 when adding different amount of carbonates [8]. However, Wuthier et al reported that ACP, with Ca/PO4 molar ratio as low as 1.15, precipitated at more acidic preparative pHs, i.e.6.9 [9].
More importantly, it has been shown that ACP particles are nanometer particles. Primary particle sizes of ACP is about 40-100 nm. The morphology of ACP solids appears to be a curvilinear shape when viewed by TEM, rather than the faceted, angular shape of crystalline calcium phosphates. However, this curvilinear appearance has only been clearly established with dried ACP [10]. The initial flocculates collected immediately after precipitation of highly hydrated ACP have a low-contrast disk-shaped appearance. High-contrast spherical particles begin to appear as ACP suspensions age, and become the dominant shape with time [11].
The disordered structure makes ACP highly reactive with body fluid, resulting fast apatite reprecipitation. Accordingly, ACP has been evidenced to have better in vivo osteoconductivity than hydroxyapatite and better biodegradability than tricalcium phosphate [10]. The ACP precipitate, with little long-range order, is a highly unstable phase and hydrolyzes almost instantaneously to more stable phases. In the presence of other ions or under in vivo conditions, ACP may persist for appreciable periods due to kinetic stabilization [12]. Although the exact mechanism of stabilization of ACP is not understood, the presence of Mg2+, F-, carbonate, pyrophosphate, diphosphonates, or polyphosphorylated metabolites or nucleotides, in sufficient quantity will prevent the transformation of synthetic ACP to hydroxyapatite [13,14].
It is now generally agreed that, both in vitro and in vivo, precipitation reactions at sufficiently high supersaturation and pH result in the initial formation of an amorphous calcium phosphate with a molar calcium/phosphate ratio of about 1.5, with a range of 1.34-1.50 in different pH and 1.50-1.67 when adding different amount of carbonates [8]. However, Wuthier et al reported that ACP, with Ca/PO4 molar ratio as low as 1.15, precipitated at more acidic preparative pHs, i.e.6.9 [9].
More importantly, it has been shown that ACP particles are nanometer particles. Primary particle sizes of ACP is about 40-100 nm. The morphology of ACP solids appears to be a curvilinear shape when viewed by TEM, rather than the faceted, angular shape of crystalline calcium phosphates. However, this curvilinear appearance has only been clearly established with dried ACP [10]. The initial flocculates collected immediately after precipitation of highly hydrated ACP have a low-contrast disk-shaped appearance. High-contrast spherical particles begin to appear as ACP suspensions age, and become the dominant shape with time [11].
The disordered structure makes ACP highly reactive with body fluid, resulting fast apatite reprecipitation. Accordingly, ACP has been evidenced to have better in vivo osteoconductivity than hydroxyapatite and better biodegradability than tricalcium phosphate [10]. The ACP precipitate, with little long-range order, is a highly unstable phase and hydrolyzes almost instantaneously to more stable phases. In the presence of other ions or under in vivo conditions, ACP may persist for appreciable periods due to kinetic stabilization [12]. Although the exact mechanism of stabilization of ACP is not understood, the presence of Mg2+, F-, carbonate, pyrophosphate, diphosphonates, or polyphosphorylated metabolites or nucleotides, in sufficient quantity will prevent the transformation of synthetic ACP to hydroxyapatite [13,14].
It has been stated
that ACP likely plays a special role as a precursor to bioapatite and as a
transient phase in biomineralization [15]. In solutions, ACP
is readily converted to stable crystalline phases such as octacalcium phosphate
or apatitic products. One biomineralization strategy that has received
significant attention in recent years is mineralization via transient precursor
phases [16]. Transient
amorphous mineral phases have been detected in biomineral systems in different
phyla of the animal kingdom [17]. ACP has been
previously reported in the otoliths of blue sharks and also shown to form as a
precursor phase of carbonated hydroxyapatite in chiton teeth [18]. The presence of
an abundant ACP phase has also been demonstrated in the newly formed zebrafish
fin bony rays [19]. The disordered
phase is a precursor of crystalline carbonated hydroxyapatite. It was found
that the initially extracted amorphous mineral particles transformed into a
crystalline mineral phase with time, and the proportion of crystalline mineral
increased during bone maturation [19]. The transient ACP
phase may conceivably be deposited directly inside the gap regions of collagen
fibrils, but it may also be delivered as extrafibrillar particles [19]. This is
consistent with the study that collagen mineralization via a transient ACP
precursor phase in vitro to
produce aligned intrafibrillar carbonated apatite crystals [20].
Several studies in different systems in vivo also have reported the presence of transient precursor calcium phosphate phases in the deposition of carbonated hydroxyapatite. Beniash performed a comprehensive analysis of the mineral phases in the early secretory enamel of the mandibular mouse incisors using four physical characterization methods. It was suggested that the outer, younger, early secretory enamel contained a transient disordered ACP phase, which transformed with time into the final apatitic crystalline mineral [17].
A variety of proteins and ions have been proposed to be involved in the biomineralization of ACP to HAP [21,22]. Dentin matrix protein1 (DMP1) is one of such biomineralization proteins [23]. In the report of He, it has been shown that two peptide motifs identified in DMP1 [motif-A (ESQES) and motif-B (QESQSEQDS)] enhanced in vitro HAP formation when immobilized on a glass substrate. It was demonstrated in another study that the synthesized artificial protein composed of these peptide motifs of DMP1 facilitated reorganization of the internal structure of amorphous particles into ordered crystalline states, i.e., the direct transformation of ACP to HAP, thereby acting as a nucleus for precipitation of crystalline calcium phosphate [24].
Several studies in different systems in vivo also have reported the presence of transient precursor calcium phosphate phases in the deposition of carbonated hydroxyapatite. Beniash performed a comprehensive analysis of the mineral phases in the early secretory enamel of the mandibular mouse incisors using four physical characterization methods. It was suggested that the outer, younger, early secretory enamel contained a transient disordered ACP phase, which transformed with time into the final apatitic crystalline mineral [17].
A variety of proteins and ions have been proposed to be involved in the biomineralization of ACP to HAP [21,22]. Dentin matrix protein1 (DMP1) is one of such biomineralization proteins [23]. In the report of He, it has been shown that two peptide motifs identified in DMP1 [motif-A (ESQES) and motif-B (QESQSEQDS)] enhanced in vitro HAP formation when immobilized on a glass substrate. It was demonstrated in another study that the synthesized artificial protein composed of these peptide motifs of DMP1 facilitated reorganization of the internal structure of amorphous particles into ordered crystalline states, i.e., the direct transformation of ACP to HAP, thereby acting as a nucleus for precipitation of crystalline calcium phosphate [24].
Studies on the
preparation of hydroxyapatite [Ca10(PO4)6-(OH)2],
the synthetic prototype of bone mineral, showed that the precipitation of
initial solid phase from a calcium phosphate solution depends on the degree of
its supersaturation [8]. A noncrystalline
ACP precursor, approximating Ca9(PO4)6 in
composition forms under conditions of high supersaturation [1,15]. This precursor
ACP, unless stabilized in some way, transforms to thermodynamically more stable
calcium phosphate phases or will be taken place by an autocatalytic
solution-mediate crystallization process. On the other hand, the first solid to
form in low supersaturated solutions is hydroxyapatite with Ca/P ratio of 1.67
obtained without precursor phases. Therefore, ACP is considered as a
"mandatory precursor to apatite", and apatite can be formed in dilute
solutions without going through this precursor [15]. The pH value also
affects the initial solid phase in the precipitation of calcium and phosphate ions.
Octacalcium phosphate (OCP) is the crystalline phase that initially forms when
the reaction pH is less than 9.25, whereas apatite preferentially forms at
higher pHs [25]. It is known that
ACP is often the first-formed deposit in vitro, at neutral pH and moderate
supersaturation [26]. Transformation
mechanism of ACP to apatite at physiological pH has been described as
followings: firstly ACP dissolution, then a transient OCP solid phase reprecipitation
through nucleation growth, and finally hydrolysis of the transient OCP phase
into the thermodynamically more stable apatite by a topotactic reaction, which
usually takes tens of hours [26].
Based on the analysis of the measured precipitate induction time and the structure of the developing solid phase, Feenstra proposed that OCP might be an intermediate in the conversion of ACP to apatitic calcium phosphate [27]. Since OCP or apatite crystals are generally found in association with ACP spherules, it is possible that ACP acts as a template for the growth of these crystal phases. Their formation, however, appears to take place by consuming ions largely supplied from the surrounding solution, rather than from direct hydrolysis of the solid amorphous material. At pH 10, transformation of ACP to poorly crystalline HAP may proceed without changes in the local calcium environment, but with the development of longer range order in the structure.
However, in contrast to these results at pH = 10, under physiological conditions the picture is quite different. Tung used a titration method to study the conversion of high-concentration ACP slurry to an apatite. There was a typical conversion kinetics clearly indicating two processes: the first process consumes acid, with the conversion of ACP to an OCP-like intermediary and the second process consumes base with the conversion of the OCP-like intermediate to apatite or, possibly, direct conversion of ACP to apatite. It was proposed that a stoichiometric HAP could be formed when there is no OCP-like intermediate phase, and a nonstoichiometric apatite product could be formed when an OCP-like intermediate phase is involved [28].
Based on the analysis of the measured precipitate induction time and the structure of the developing solid phase, Feenstra proposed that OCP might be an intermediate in the conversion of ACP to apatitic calcium phosphate [27]. Since OCP or apatite crystals are generally found in association with ACP spherules, it is possible that ACP acts as a template for the growth of these crystal phases. Their formation, however, appears to take place by consuming ions largely supplied from the surrounding solution, rather than from direct hydrolysis of the solid amorphous material. At pH 10, transformation of ACP to poorly crystalline HAP may proceed without changes in the local calcium environment, but with the development of longer range order in the structure.
However, in contrast to these results at pH = 10, under physiological conditions the picture is quite different. Tung used a titration method to study the conversion of high-concentration ACP slurry to an apatite. There was a typical conversion kinetics clearly indicating two processes: the first process consumes acid, with the conversion of ACP to an OCP-like intermediary and the second process consumes base with the conversion of the OCP-like intermediate to apatite or, possibly, direct conversion of ACP to apatite. It was proposed that a stoichiometric HAP could be formed when there is no OCP-like intermediate phase, and a nonstoichiometric apatite product could be formed when an OCP-like intermediate phase is involved [28].
ACP has been
widely applied in biomedical field due to its excellent bioactivity, high cell
adhesion, adjustable biodegradation rate and good osteoconduction [29-32]. As discussed
above, the first quantitative studies on synthetic ACP were done in the mid
1960s [1]. From then on, more
and more attention has been attracted in the development and the application of
ACP-containing products, especially in orthopedic and dental fields. It is also
used as filler in ionomer cements to fill carious lesions or as a colloidal
suspension in toothpastes, chewing gums or mouthwashes to promote
demineralization of carious lesions and/or to prevent tooth demineralization.
6.1 CPP-ACP
Casein
phosphopeptides (CPP) contain the cluster sequence of -Ser (P)-Ser (P)-Ser
(P)-Glu-Glu from casein [33,34]. Through these
multiple phosphoseryl residues, CPP has a remarkable ability to stabilize
clusters of ACP into CPP-ACP complexes, preventing their growth to the critical
size required for nucleation, phase transformation and precipitation. In the
United States, up to now, this product is primarily used for abrasive
prophylaxis pastes and secondarily used for the treatment of tooth sensitivity
especially after in-office bleaching procedures, ultrasonic scaling, hand
scaling or root planing. However, its use for remineralizing dentin and enamel
and preventing dental caries is an off-label application. Outside the United
States, this product is marketed as GC Tooth Mousse [35,36].
A clinical trial of a mouthwash containing CPP-ACP showed that the contents of calcium and inorganic phosphate in supragingival plaque increased after use of the mouthwash for a three-day period [37]. Rose measured the affinity of Streptococcus mutans to CPP-ACP. It was demonstrated that CPP-ACP bound with about twice the affinity to the bacterial cells [38]. Hence, CPP-ACP binds well to plaque, providing a large calcium reservoir within plaque and slowing diffusion of free calcium. Additional evidence reported also by Rose indicates that CPP-ACP would compete with calcium for plaque Ca binding sites. As a result, this will reduce the amount of calcium bridging between the pellicle and adhering bacterial cells and between bacterial cells themselves [39]. This is likely to restrict mineral loss during a cariogenic episode and provide a potential source of calcium for the inhibition of demineralization and assist in subsequent remineralization.
A human in situ caries model has been used by Reynolds to study the ability of 1.0% CPP, 60-mM CaCl2 and 36-mM sodium phosphate, pH 7.0, solution to prevent enamel demineralization [40]. Two exposures of CPP-ACP solution per day to one side of the enamel slabs produced 51 ± 19% reduction in enamel mineral loss compared to the control side. Plaque exposed to CPP-ACP had 2.5 times more Ca and phosphorus than control plaque [36]. Reynolds also used an in vitro model system to study the effects of CPP-ACP solutions on remineralization of artificial lesions in human third molars. After a ten-day remineralization period, all solutions deposited mineral into the bodies of the lesions, with 1.0% CPP-ACP (pH 7.0) solution replacing 63.9 ± 20.1% of mineral lost at an averaged rate of 3.9 ± 0.8 × 10-8 mol hydroxyapatite/m2/s. The remineralizing capacity was greater in the solutions with higher levels of CPP-stabilized free calcium and phosphate ions [41].
CPP-ACP and fluoride were shown to have additive effects in reducing caries experience [42]. Thus CPP-ACFP would add into the current fluoride-containing dentifrices as a toothpaste additive to improve the efficacy. Recent studies indicate that CPP-ACP can be incorporated into confectionery and drinks without adverse organoleptic effects [43]. CPP-ACP is a natural derivative of milk, therefore could have an important role as a food additive for the prevention of dental caries [44]. However, in 2008 Azarpazhooh systemically reviewed 98 articles on the clinical efficacy of casein derivatives and concluded that there was insufficient evidence (in quantity, quality or both) in existing clinical trials to make a recommendation regarding the long-term effectiveness of casein derivatives, specifically CPP-ACP, in preventing caries in vivo and treating dentin hypersensitivity or dry mouth [34].
A clinical trial of a mouthwash containing CPP-ACP showed that the contents of calcium and inorganic phosphate in supragingival plaque increased after use of the mouthwash for a three-day period [37]. Rose measured the affinity of Streptococcus mutans to CPP-ACP. It was demonstrated that CPP-ACP bound with about twice the affinity to the bacterial cells [38]. Hence, CPP-ACP binds well to plaque, providing a large calcium reservoir within plaque and slowing diffusion of free calcium. Additional evidence reported also by Rose indicates that CPP-ACP would compete with calcium for plaque Ca binding sites. As a result, this will reduce the amount of calcium bridging between the pellicle and adhering bacterial cells and between bacterial cells themselves [39]. This is likely to restrict mineral loss during a cariogenic episode and provide a potential source of calcium for the inhibition of demineralization and assist in subsequent remineralization.
A human in situ caries model has been used by Reynolds to study the ability of 1.0% CPP, 60-mM CaCl2 and 36-mM sodium phosphate, pH 7.0, solution to prevent enamel demineralization [40]. Two exposures of CPP-ACP solution per day to one side of the enamel slabs produced 51 ± 19% reduction in enamel mineral loss compared to the control side. Plaque exposed to CPP-ACP had 2.5 times more Ca and phosphorus than control plaque [36]. Reynolds also used an in vitro model system to study the effects of CPP-ACP solutions on remineralization of artificial lesions in human third molars. After a ten-day remineralization period, all solutions deposited mineral into the bodies of the lesions, with 1.0% CPP-ACP (pH 7.0) solution replacing 63.9 ± 20.1% of mineral lost at an averaged rate of 3.9 ± 0.8 × 10-8 mol hydroxyapatite/m2/s. The remineralizing capacity was greater in the solutions with higher levels of CPP-stabilized free calcium and phosphate ions [41].
CPP-ACP and fluoride were shown to have additive effects in reducing caries experience [42]. Thus CPP-ACFP would add into the current fluoride-containing dentifrices as a toothpaste additive to improve the efficacy. Recent studies indicate that CPP-ACP can be incorporated into confectionery and drinks without adverse organoleptic effects [43]. CPP-ACP is a natural derivative of milk, therefore could have an important role as a food additive for the prevention of dental caries [44]. However, in 2008 Azarpazhooh systemically reviewed 98 articles on the clinical efficacy of casein derivatives and concluded that there was insufficient evidence (in quantity, quality or both) in existing clinical trials to make a recommendation regarding the long-term effectiveness of casein derivatives, specifically CPP-ACP, in preventing caries in vivo and treating dentin hypersensitivity or dry mouth [34].
6.2 ACP-filled polymeric
composites
ACP has been
evaluated as a filler phase in bioactive polymeric composites [45]. Skrtic has
developed unique biologically active restorative materials containing ACP as
filler encapsulated in a polymer binder, which may stimulate the repair of
tooth structure because of releasing significant amounts of calcium and
phosphate ions in a sustained manner [46-49]. In addition to
excellent biocompatibility, the ACP-containing composites release calcium and
phosphate ions into saliva milieus, especially in the oral environment caused
by bacterial plaque or acidic foods. Then these ions can be deposited into
tooth structures as apatitic mineral, which is similar to the hydroxyapatite
found naturally in teeth and bone [50,51].
However, it was reported that the orthodontic ACP-containing adhesive showed lower bond strength. Dunn conducted an in vitro study to compare ACP-containing vs. conventional resin-based orthodontic adhesives [52]. Foster also compared the shear bond strength of orthodontic brackets using ACP-containing adhesive with a conventional adhesive and a resin-modified glass ionomer. In both studies, ACP-containing adhesive was demonstrated with lower, but clinically satisfactory bond strength as an orthodontic adhesive [53]. When comparing four new ACP-containing bonding systems, including Aegis Ortho, with a conventional bracket bonding system (Transbond XT), it was found that the traditional bonding systems achieved greater bond strengths than the newer ACP-containing ones. According to the study, however, Aegis Ortho had bond strengths sufficient for orthodontic use at 24-hour post-cure time. But the bracket might drift because of low viscosity of the material during laboratory bonding. The authors also found that Aegis Ortho had lower flexural strength, which would explain for the material failure at the adhesive-bracket interface rather than the enamel adhesive interface [54].
Compared with more commonly used silanated glass or ceramic filler, more hydrophilic and biodegradable ACP-filled composites exhibited inferior mechanical properties, durability and water sorption characteristics [55]. The uncontrolled aggregation of ACP particulates along with poor interfacial interaction plays a key role in adversely affecting their mechanical properties [56]. Their clinical applicability may be compromised by relatively poor filler/matrix interfacial adhesion and also by excessive water sorption that occurs in both resin and filler phases of these composites [42,46].
However, it has been demonstrated that it is possible to improve the remineralizing potential of ACP composites by introducing Si or Zr elements during low-temperature synthesis of the filler. Si- and Zr- ACPs enhanced the duration of mineral ion release through their ability to slow down the intra-composite ACP to HAP conversion [57]. It was also possible that non-ionic and anionic surfactants and poly (ethylene oxide) (PEO) introduced during the preparation of ACP play a role on the particle size distribution and compositional properties of ACP fillers [58]. The hydrophilic PEO is widely used in water compatible polymer systems because of its proven ability to undergo multiple hydrogen bonding interactions and stabilize cations by multiple chelation. The incorporated PEO in ACP fillers would also be expected to affect ACP's tendency to form aggregates and the water content of the ACP-containing composites. These properties would eventually affect both ion release kinetics and mechanical stability of composites [59]. It was found that surfactants introduced during the precipitation of ACP stabilized the amorphous solid phase against the conversion to apatite. The particle size of ACP was moderately reduced because of the introduction of anionic surfactant. Addition of PEO resulted in more pronounced ACP agglomeration but no changes of ACP's water content. Both surfactants and PEO lead to no changes in dry biaxial flexure strength of composites compared to the control Zr-ACP composites. However, their strength was drastically reduced in contrast to the control after prolonged exposure to aqueous milieu.
However, it was reported that the orthodontic ACP-containing adhesive showed lower bond strength. Dunn conducted an in vitro study to compare ACP-containing vs. conventional resin-based orthodontic adhesives [52]. Foster also compared the shear bond strength of orthodontic brackets using ACP-containing adhesive with a conventional adhesive and a resin-modified glass ionomer. In both studies, ACP-containing adhesive was demonstrated with lower, but clinically satisfactory bond strength as an orthodontic adhesive [53]. When comparing four new ACP-containing bonding systems, including Aegis Ortho, with a conventional bracket bonding system (Transbond XT), it was found that the traditional bonding systems achieved greater bond strengths than the newer ACP-containing ones. According to the study, however, Aegis Ortho had bond strengths sufficient for orthodontic use at 24-hour post-cure time. But the bracket might drift because of low viscosity of the material during laboratory bonding. The authors also found that Aegis Ortho had lower flexural strength, which would explain for the material failure at the adhesive-bracket interface rather than the enamel adhesive interface [54].
Compared with more commonly used silanated glass or ceramic filler, more hydrophilic and biodegradable ACP-filled composites exhibited inferior mechanical properties, durability and water sorption characteristics [55]. The uncontrolled aggregation of ACP particulates along with poor interfacial interaction plays a key role in adversely affecting their mechanical properties [56]. Their clinical applicability may be compromised by relatively poor filler/matrix interfacial adhesion and also by excessive water sorption that occurs in both resin and filler phases of these composites [42,46].
However, it has been demonstrated that it is possible to improve the remineralizing potential of ACP composites by introducing Si or Zr elements during low-temperature synthesis of the filler. Si- and Zr- ACPs enhanced the duration of mineral ion release through their ability to slow down the intra-composite ACP to HAP conversion [57]. It was also possible that non-ionic and anionic surfactants and poly (ethylene oxide) (PEO) introduced during the preparation of ACP play a role on the particle size distribution and compositional properties of ACP fillers [58]. The hydrophilic PEO is widely used in water compatible polymer systems because of its proven ability to undergo multiple hydrogen bonding interactions and stabilize cations by multiple chelation. The incorporated PEO in ACP fillers would also be expected to affect ACP's tendency to form aggregates and the water content of the ACP-containing composites. These properties would eventually affect both ion release kinetics and mechanical stability of composites [59]. It was found that surfactants introduced during the precipitation of ACP stabilized the amorphous solid phase against the conversion to apatite. The particle size of ACP was moderately reduced because of the introduction of anionic surfactant. Addition of PEO resulted in more pronounced ACP agglomeration but no changes of ACP's water content. Both surfactants and PEO lead to no changes in dry biaxial flexure strength of composites compared to the control Zr-ACP composites. However, their strength was drastically reduced in contrast to the control after prolonged exposure to aqueous milieu.
6.3 ACP in bone repair
materials
Various compounds
from calcium phosphate family have been extensively investigated as hard tissue
repair materials due to their excellent biocompatibility [60]. It has been shown
that the rate of new bone formation coincides more closely with the resorption
rate of poorly crystalline apatites and ACP [61]. Additionally, ACP
showed better osteoconductivity in vivo than apatite and its biodegradability
was higher than that of tricalcium phosphate [25].
Clinically, it is widely accepted to use autograft and allograft materials to repair bone defects [29]. Recently, materials with ACP, hydroxyapatite and other calcium phosphate family members have been extensively investigated for alternative bone repair due to the limitations of traditional materials such as potential immunogenicity, insufficient supply and so on [62,63]. ACP and ACP/biopolymer composites have emerged as a new class of bone tissue engineering scaffold materials. Their excellent biocompatibility and osteoconductibility make them great materials for bone substitution and repair.
It has been shown that bone-like apatite materials have optimal surface characteristics for osteoblast cells to adhere, proliferate and differentiate, as a result, to favor bone formation and regeneration. An amorphous carbonated calcium phosphate ceramic was encapsulated within bioresorbable PLAGA microspheres and sintered to form a bioresorbable, highly porous, 3-dimensional scaffold. These noncrystalline and carbonated materials may be ideal for tissue ingrowth and potentially suitable for bone repair applications [64].
ACP was also incorporated into porous poly (L-lactic acid) (PLLA) to create a desired pore wall surface within bone tissue engineering scaffolds [65]. After being soaked in PBS, ACP aggregates in the composite experienced a fast and in situ transformation into bone-like apatite. The cell culture results also demonstrated that ACP/PLLA composite had an enhancement in cytocompatibility [65]. It has been demonstrated that ACP/PLLA material, which can experience morphological variations in the microstructure is also supposed to be a suitable candidate as scaffold for cartilage tissue engineering [63,65].
Clinically, it is widely accepted to use autograft and allograft materials to repair bone defects [29]. Recently, materials with ACP, hydroxyapatite and other calcium phosphate family members have been extensively investigated for alternative bone repair due to the limitations of traditional materials such as potential immunogenicity, insufficient supply and so on [62,63]. ACP and ACP/biopolymer composites have emerged as a new class of bone tissue engineering scaffold materials. Their excellent biocompatibility and osteoconductibility make them great materials for bone substitution and repair.
It has been shown that bone-like apatite materials have optimal surface characteristics for osteoblast cells to adhere, proliferate and differentiate, as a result, to favor bone formation and regeneration. An amorphous carbonated calcium phosphate ceramic was encapsulated within bioresorbable PLAGA microspheres and sintered to form a bioresorbable, highly porous, 3-dimensional scaffold. These noncrystalline and carbonated materials may be ideal for tissue ingrowth and potentially suitable for bone repair applications [64].
ACP was also incorporated into porous poly (L-lactic acid) (PLLA) to create a desired pore wall surface within bone tissue engineering scaffolds [65]. After being soaked in PBS, ACP aggregates in the composite experienced a fast and in situ transformation into bone-like apatite. The cell culture results also demonstrated that ACP/PLLA composite had an enhancement in cytocompatibility [65]. It has been demonstrated that ACP/PLLA material, which can experience morphological variations in the microstructure is also supposed to be a suitable candidate as scaffold for cartilage tissue engineering [63,65].
ACP is usually
formed as a metastable phase when calcium and phosphate ions in aqueous
solution react to precipitate. The x-ray diffraction pattern, structure,
morphology and infrared analysis results of ACP solids show typical
noncrystalline characters within short-range order, instead of long-range
periodic regularity. ACP act as an important intermediate product for in vitro
and in vivo apatite formation. A variety of proteins and ions can increase the
stability of ACP. ACP becomes increasingly significant in orthopedics and
dentistry because of their excellent biocompatibility and mechanical
properties. It is believed that ACP will be used even more extensively in the
future due to due to the fast development of tissue engineering techniques and
applied material science.
The authors
declare that they have no competing interests
JZ, YL, WS and HZ
have all been involved in drafting this review and have given final approval of
the version to be published.
WS would like to
thank Worldwide Universities Network Development Fund (No.201001168) and The
Natural Scientific Fund of Jiangsu (No. BK2010118)
References
1. Boskey AL: Amorphous calcium
phosphate: the contention of bone.
2. Eanes ED, Gillessen IH, Posner AS: Intermediate
states in the precipitation of hydroxyapatite.
3. Betts F, Blumenthal NC, Posner AS, Becker
GL, Lehninger AL: Atomic structure of intracellular amorphous calcium
phosphate deposits.
Proc Natl Acad Sci 1975, 72:2088-2090.
PubMed Abstract |
Publisher Full Text
| PubMed Central Full Text
4. Posner AS, Betts F, Blumenthal NC: Formation and
structure of synthetic and bone hydroxyapatite.
5. Blumenthal NC, Betts F, Posner AS: Stabilization of
amorphous calcium phosphate by Mg and ATP.
6. Termine JD, Eanes ED: Comparative
chemistry of amorphous and apatitic calcium phosphate preparations.
7. Eanes ED, Termine JD, Nylen MU: An electron
microscopic study of the formation of amorphous calcium phosphate and its
transformation to crystalline apatite.
8. Feenstra TP, De Bruyn PL: Formation of
Calcium Phosphates in Moderately Supersaturated Solutions.
9. Wuthier RE, Rice GS, Wallace JE, Weaver RL,
LeGeros RZ, Eanes ED: In vitro precipitation of calcium phosphate under
intracellular conditions: formation of brushite from an amorphous precursor in
the absence of ATP.
10. Dorozhkin SV: Amorphous calcium (ortho) phosphates.
11. Harries JE, Hukins DW, Holt C, Hasnain SS: Conversion of
Amorphous Calcium Phosphate into Hydroxyapatite.
12. Boskey AL, Posner AS: Magnesium Stabilization of Amorphous
Calcium Phosphate: A Kinetic Study.
THE END
ARTICLE-05
Simple isatin derivatives as free radical scavengers: Synthesis,
biological evaluation and structure-activity relationship
Oxidative stress
has been implicated as a major role in the onset and progression of a vast
variety of clinical abnormalities including neurodegenerative disorders. Free
radicals play important roles in many physiological and pathological conditions
[1]. In general, the
generation and scavenging of oxygen free radicals is balanced and any imbalance
or excessive amounts of active radicals may contribute to disease development.
It has been found that free radical reactions can produce deleterious
modifications in membranes, proteins, enzymes, and DNA [2], increasing the
risk of diseases such as cancer [3], Alzheimer's [4], Parkinson's [5], angiocardiopathy [6], arthritis [7], asthma [8], diabetes [9], and degenerative
eye disease [10]. Therefore, it is
important to find effective scavengers of free radicals for prevention and
treatment of such disorders.
Isatin is an endogenous indole present in mammalian tissues and fluids [11]. The substance was initially discovered as a component of endogenous monoamine oxidase (MAO) inhibitory activity, tribulin, and subsequently identified as a selective inhibitor of MAO B [12]. Further investigations have shown that isatin acts as an antagonist of both atrial natriuretic peptidestimulated and nitric oxide-stimulated guanylate cyclase activity [13-15]. Isatin has a distinct and discontinuous distribution in rat brain and other tissues; the highest concentrations in the brain are found in the hippocampus and cerebellum [10]. Many Isatin derivatives, such as isatin hydrazono, isatin Mannich bases, isatin based spiroazetidinones and 3-(methylene)indolin-2-ones, have also been reported to possess neuroprotection activity [16-19].
To develop more potent small molecules with enhanced free radical scavenger properties, a series of N-substituted isatin derivatives was synthesized by substitution reactions (as shown in Scheme 1), and the cytoprotective effect on the apoptosis of PC12 cells induced by H2O2 was screened.
Scheme 1
Synthesis of N-substituted isatin derivatives.
Isatin is an endogenous indole present in mammalian tissues and fluids [11]. The substance was initially discovered as a component of endogenous monoamine oxidase (MAO) inhibitory activity, tribulin, and subsequently identified as a selective inhibitor of MAO B [12]. Further investigations have shown that isatin acts as an antagonist of both atrial natriuretic peptidestimulated and nitric oxide-stimulated guanylate cyclase activity [13-15]. Isatin has a distinct and discontinuous distribution in rat brain and other tissues; the highest concentrations in the brain are found in the hippocampus and cerebellum [10]. Many Isatin derivatives, such as isatin hydrazono, isatin Mannich bases, isatin based spiroazetidinones and 3-(methylene)indolin-2-ones, have also been reported to possess neuroprotection activity [16-19].
To develop more potent small molecules with enhanced free radical scavenger properties, a series of N-substituted isatin derivatives was synthesized by substitution reactions (as shown in Scheme 1), and the cytoprotective effect on the apoptosis of PC12 cells induced by H2O2 was screened.
Scheme 1
Synthesis of N-substituted isatin derivatives.
Chemistry
The N-substituted isatin derivatives were
synthesized by reactions of substitution reaction. The reaction between isatin
and halohydrocarbon has been reported being carried out in the presence of
NaOEt using EtOH as solvent or in the presence of NaH using DMF as solvent [16]. The reactants and
the solvents involved in the reactions must be anhydrous. To develop a simple
method to synthesize N-substituted
isatin derivatives, we firstly screened the effect of the base and solvent on
the yield of the reaction of isatin and bromoethane (C2H5Br),
and the results was shown in Table 1.
Table 1. The substitution reaction between isatin and bromoethane
In this reaction, the protons transfers from N-H (a Brösted acid) to a Brösted or Lewis base via the hydrogen-bonded covalent and ionic complexes [20], producing the isatin anion which is the nucleophilic reactant to the halohydrocarbon. Higher solvent polarity can promote the proton-transfer equilibrium and leads to the higher yield [20]. From this table, it can be found that K2CO3-DMF system was an effective promotion for this reaction and other base-solvent systems were not effective with the yield no more than 60%. The possible reason might be that weak base can not help the proton transfer at the beginning effectively, but the too strong bases will lead to the substitution reaction between bromoethane and OH-. DMF exhibits the highest yield of 89% with K2CO3 for its highest solvent polarity, so the K2CO3-DMF was selected as the reactant reaction system in the following synthesis, and the results were shown in Table 2.
Table 2. Synthesis of N-substituted isatin derivatives
Table 1. The substitution reaction between isatin and bromoethane
In this reaction, the protons transfers from N-H (a Brösted acid) to a Brösted or Lewis base via the hydrogen-bonded covalent and ionic complexes [20], producing the isatin anion which is the nucleophilic reactant to the halohydrocarbon. Higher solvent polarity can promote the proton-transfer equilibrium and leads to the higher yield [20]. From this table, it can be found that K2CO3-DMF system was an effective promotion for this reaction and other base-solvent systems were not effective with the yield no more than 60%. The possible reason might be that weak base can not help the proton transfer at the beginning effectively, but the too strong bases will lead to the substitution reaction between bromoethane and OH-. DMF exhibits the highest yield of 89% with K2CO3 for its highest solvent polarity, so the K2CO3-DMF was selected as the reactant reaction system in the following synthesis, and the results were shown in Table 2.
Table 2. Synthesis of N-substituted isatin derivatives
Bioactivity
The chemical
modification of lead compound 1, focusing on the N-substituent, was carried out to further improve the free
scavenging ability. A series of new N-substituted isatin derivatives (compounds 2-12) was synthesized. The free radical
scavenging properties of these derivatives were evaluated to elucidate
structure-activity relationships. The protective effect on the apoptosis of
PC12 cells induced by H2O2 by free radical scavenging of
these compounds against H2O2 were evaluated by cell
survival assay in PC12 cells using a reported method [21]. The results were
given in Table 3.
Table 3. Inhibitory and protective effects of N-substituted isatin derivatives
From the table, we can find almost all of the compounds showed potent activity at the condensation of 2 μg/ml, which were more effective than VE ((±) α-Tocophreol with the percentage of 22.5%). There is a noteworthy phenomenon that the activities of all compounds at the condensation of 2 μg/ml are more potent than that at the condensation of 20 μg/ml, and the mechanism will be interesting for the further investigation. Compound 3 and 8 exhibited the most potent activity with the protective effect of 69.8% and 69.5% at the condensation of 2 μg/ml respectively, which are more potent than that at the condensation of 20 μg/ml.
Almost all of these compounds were weakly cytotoxic to PC12 cells at the concentrations of 2-20 μg/ml except compound 11 and 12. Almost all compounds are cytotoxic to PC12 cells at the concentrations of 200 μg/ml, the PC12 cells inhibitory effects are more than 40%. Based on the factors, we can conclude the addition of halogenous atom in the substituents (compound 11 and 12) enhance the cytotoxicity at the concentrations of 2-20 μg/ml.
The substitution reaction between isatin and halohydrocarbon (C1 to C6) gave compounds 2-7, which provided the appropriate material for the structure-activity relationship analyses. The cytoprotective activities of N-substituted isatin derivatives with the alkyl group containing one to six carbon atoms were shown in Figure 1. The activity approximately declines as the increase of the chain of the alkyl group. With a further analysis, it was found that there was a clear odd-even effect in these activities. The activities of N-substituted isatin derivatives with odd carbon atoms alkyl group (one, three and five carbon atoms, corresponding compound 2, 4 and 6, marked with solid pillars in Figure 1) decline as the chain of the alkyl group increases, and the same regulation can be found in the activities of the N-substituted isatin derivatives with even carbon atoms alkyl group (two, four and six carbon atoms, corresponding compound 3, 5 and 7, marked with virtual pillars in Figure 1). This regulation exhibits both under the condensation of 2 μg/ml and 20 μg/ml, and the activities of N-substituted isatin derivatives with even carbon atoms alkyl group are more potent than the that of N-substituted isatin derivatives with parallel odd carbon atoms alkyl group. Besides, by the structure-activity relationship analyses, it was found that the unsaturated bond of the substituent (compound 8-10) can improve the activity compared with the other substituents with similar carbon atoms.
Figure 1. The cytoprotective activities
of N-substituted isatin derivatives with the alkyl group containing 1-6
carbon atoms (The corresponding compounds are compounds 2-7.).
Table 3. Inhibitory and protective effects of N-substituted isatin derivatives
From the table, we can find almost all of the compounds showed potent activity at the condensation of 2 μg/ml, which were more effective than VE ((±) α-Tocophreol with the percentage of 22.5%). There is a noteworthy phenomenon that the activities of all compounds at the condensation of 2 μg/ml are more potent than that at the condensation of 20 μg/ml, and the mechanism will be interesting for the further investigation. Compound 3 and 8 exhibited the most potent activity with the protective effect of 69.8% and 69.5% at the condensation of 2 μg/ml respectively, which are more potent than that at the condensation of 20 μg/ml.
Almost all of these compounds were weakly cytotoxic to PC12 cells at the concentrations of 2-20 μg/ml except compound 11 and 12. Almost all compounds are cytotoxic to PC12 cells at the concentrations of 200 μg/ml, the PC12 cells inhibitory effects are more than 40%. Based on the factors, we can conclude the addition of halogenous atom in the substituents (compound 11 and 12) enhance the cytotoxicity at the concentrations of 2-20 μg/ml.
The substitution reaction between isatin and halohydrocarbon (C1 to C6) gave compounds 2-7, which provided the appropriate material for the structure-activity relationship analyses. The cytoprotective activities of N-substituted isatin derivatives with the alkyl group containing one to six carbon atoms were shown in Figure 1. The activity approximately declines as the increase of the chain of the alkyl group. With a further analysis, it was found that there was a clear odd-even effect in these activities. The activities of N-substituted isatin derivatives with odd carbon atoms alkyl group (one, three and five carbon atoms, corresponding compound 2, 4 and 6, marked with solid pillars in Figure 1) decline as the chain of the alkyl group increases, and the same regulation can be found in the activities of the N-substituted isatin derivatives with even carbon atoms alkyl group (two, four and six carbon atoms, corresponding compound 3, 5 and 7, marked with virtual pillars in Figure 1). This regulation exhibits both under the condensation of 2 μg/ml and 20 μg/ml, and the activities of N-substituted isatin derivatives with even carbon atoms alkyl group are more potent than the that of N-substituted isatin derivatives with parallel odd carbon atoms alkyl group. Besides, by the structure-activity relationship analyses, it was found that the unsaturated bond of the substituent (compound 8-10) can improve the activity compared with the other substituents with similar carbon atoms.
Figure 1. The cytoprotective activities
of N-substituted isatin derivatives with the alkyl group containing 1-6
carbon atoms (The corresponding compounds are compounds 2-7.).
Experimental
All starting
materials and solvents (A.R. grade) were commercially available and were used
without further purification. NMR spectra were recorded using a Bruker Drx-400
spectrometer operating at 400 MHz for 1H. Mass spectra were recorded
on a Micromass Platform spectrometer using a direct-inlet system operating in
the electron impact (EI) mode at 75 eV. Elemental analyses were obtained using
a Carlo Erba 1106 elemental analyzer.
General synthesis of N-alkyl
substituted isatin derivatives
Isatin (1 mmol)
and halohydrocarbon (1.2 mmol) were dissolved in DMF (20 ml), and 3 mmol
anhydrous K2CO3 was added. The mixture was stirred under
room temperature until the disappearance of isatin, as evidenced by thin-layer
chromatography. The solvent was removed in vacuo and the residue was separated
by column chromatography (silica gel, petroleum ether/ethyl acetate = 20:1),
giving N-alkyl
substituted isatin compound (compound 2-12).
1-Methylindoline-2,3-dione
(Compound 2)
1H-NMR (D6-DMSO, 400 MHz): 7.66
(1 H, td, J = 1.2, 7.6
Hz), 7.52 (1 H, d, J =
7.6 Hz), 7.12 (2 H, t, J =
7.6 Hz), 3.12 (3 H, s); MS (EI) m/z: 161 (M+); Anal. Found: C, 67.01; H, 4.40; N,
8.66 (%). Calc. for (C9H7NO2): C, 67.07; H,
4.38; N, 8.69 (%).
1-Ethylindoline-2,3-dione
(Compound 3)
1H-NMR (CDCl3, 400 MHz): 7.57 (2
H, m), 7.09 (1 H, t, J =
7.6 Hz), 6.89 (1 H, d, J =
7.6 Hz), 3.76 (2 H, q, J =
7.6 Hz), 1.29 (3 H, t, J =
7.6 Hz); MS (EI) m/z:
175 (M+); Anal. Found: C, 68.59; H, 5.22; N, 8.01 (%). Calc. for (C10H9NO2):
C, 68.56; H, 5.18; N, 8.00 (%).
1-Propylindoline-2,3-dione
(Compound 4)
1H-NMR (D6-DMSO, 400 MHz): 7.58
(1 H, d, J = 6.8 Hz),
7.55 (1 H, t, J = 7.6 Hz),
7.09 (1 H, t, J = 7.6 Hz),
6.88 (1 H, d, J = 8 Hz),
3.67 (2 H, t, J = 7.2 Hz),
1.72 (2 H, m, J = 7.2-7.6
Hz), 0.98 (3 H, t, J =
7.6 Hz); MS (EI) m/z:
189 (M+); Anal. Found: C, 69.88; H, 5.89; N, 7.35 (%). Calc. for (C11H11NO2):
C, 69.83; H, 5.86; N, 7.40 (%).
1-Butylindoline-2,3-dione
(Compound 5)
1H-NMR (CDCl3, 400 MHz) δ: 7.60 (2 H, m), 7.12 (1 H, t, J = 7.6 Hz), 6.91 (1 H, d, J = 8.4 Hz), 3.73 (2 H, t, J = 7.6 Hz), 1.69 (2 H, m), 1.42 (2 H, m),
0.98 (3 H, t, J = 7.2 Hz);
MS (EI) m/z: 203 (M+);
Anal. Found: C, 70.90; H, 6.59; N, 6.90 (%). Calc. for (C12H13NO2):
C, 70.92; H, 6.54; N, 6.89 (%).
1-Pentylindoline-2,3-dione
(Compound 6)
1H-NMR (CDCl3, 400 MHz) δ: 7.60 (2 H, m), 7.12 (1 H, t, J = 7.6 Hz), 6.91 (1 H, d, J = 8.0 Hz), 3.72 (2 H, t, J = 7.6 Hz), 1.71 (2 H, m), 1.37 (4 H, m),
0.91 (3 H, t, J = 6.8 Hz);
MS (EI) m/z: 217 (M+);
Anal. Found: C, 71.88; H, 7.00; N, 6.44 (%). Calc. for (C13H15NO2):
C, 71.87; H, 6.96; N, 6.45 (%).
1-Hexylindoline-2,3-dione
(Compound 7)
1H-NMR (CDCl3, 400 MHz) δ: 7.60 (2 H, m), 7.11 (1 H, t, J = 7.6 Hz), 6.90 (1 H, d, J = 7.6 Hz), 3.72 (2 H, t, J = 7.6 Hz), 1.70 (2 H, m), 1.31-1.38 (6 H,
m), 0.89 (3 H, t, J =
6.4 Hz); MS (EI) m/z:
231 (M+); Anal. Found: C, 72.72; H, 7.40; N, 6.01 (%). Calc. for (C14H17NO2):
C, 72.70; H, 7.41; N, 6.06 (%).
1-Allylindoline-2,3-dione
(Compound 8)
1H-NMR (D6-DMSO, 400 MHz): 7. 63
(1 H, t, J = 7.6 Hz),
7.55 (1 H, d, J = 7.2 Hz),
7.12 (1 H, t, J = 7.6 Hz),
7.04 (1 H, d, J = 7.6 Hz),
5.84 (1 H, m, J = 5.2-5.6
Hz), 5.32 (1 H, d, J =
17.2 Hz), 5.18 (1 H, d, J =
10.4 Hz), 4.30 (2 H, d, J =
4.8 Hz); MS (EI) m/z:
187 (M+); Anal. Found: C, 70.60; H, 4.84; N, 7.49 (%). Calc. for (C11H9NO2):
C, 70.58; H, 4.85; N, 7.48 (%).
1-Benzylindoline-2,3-dione
(Compound 9)
1H-NMR (D6-DMSO, 400 MHz) δ: 7.56 (2 H, m), 7.42 (2 H, d, J = 7.6 Hz), 7.30 (2 H, t, J = 7.6 Hz), 7.27 (1 H, m), 7.10 (1 H, t, J = 7.6 Hz), 6.96 (1 H, m), 4.90 (2 H, s); MS
(EI) m/z: 233 (M+);
Anal. Found: C, 75.99; H, 4.65; N, 5.92 (%). Calc. for (C15H11NO2):
C, 75.94; H, 4.67; N, 5.90 (%).
Ethyl
2-(2,3-dioxoindolin-1-yl)acetate (Compound 10)
1H-NMR (CDCl3, 400 MHz) δ: 7.62 (1 H, d, J = 7.6 Hz), 7.57 (1 H, t, J = 7.6 Hz), 7.14 (1 H, t, J = 7.6 Hz), 6.77 (1 H, d, J = 7.6 Hz), 4.47 (2 H, s), 4.22 (2 H, q, J = 7.2 Hz), 1.26 (3 H, t, J = 7.2 Hz); MS (EI) m/z: 233 (M+); Anal. Found: C,
61.84; H, 4.72; N, 6.00 (%). Calc. for (C12H11NO4):
C, 61.80; H, 4.75; N, 6.01 (%).
1-(2-Chloroethyl)indoline-2,3-dione
(Compound 11)
1H-NMR (D6-DMSO, 400 MHz) δ: 7.67 (1 H, td, J = 8, 1.2 Hz), 7.56 (1 H, dd, J = 7.6, 1.2 Hz), 7.29 (1 H, d, J = 8.0 Hz), 7.14 (1 H, dd, J = 7.6, 0.8 Hz), 4.10 (2 H, t, J = 6.4 Hz), 3.70 (2 H, t, J = 6.4 Hz); MS (EI) m/z: 211 (M+); Anal. Found: C,
58.86; H, 3.99; N, 13.70 (%). Calc. for (C10H8ClNO2):
C, 58.82; H, 3.95; N, 13.72 (%).
1-(2-Bromoethyl)indoline-2,3-dione
(Compound 12)
1H-NMR (D6-DMSO, 400 MHz) δ: 7.67 (1 H, td, J = 8, 1.2 Hz), 7.57 (1 H, dd, J = 7.6, 1.2 Hz), 7.29 (1 H, d, J = 8.0 Hz), 7.14 (1 H, dd, J = 7.6, 0.8 Hz), 4.11 (2 H, t, J = 6.4 Hz), 3.71 (2 H, t, J = 6.4 Hz); MS (EI) m/z: 254 (M+); Anal. Found: C,
47.31; H, 3.19; N, 5.50 (%). Calc. for (C10H8BrNO2):
C, 47.27; H, 3.17; N, 5.51 (%).
The authors
declare that they have no competing interests.
GC has formulated
the research idea and prepared the manuscript draft version, YW prepared the
manuscript for submission and coordinated further formalities, SM and QS
carried out the chemical and biological studies, XH conceived of the study,
participated in its design and coordination. All authors have read and approved
the final manuscript.
This work was
financially supported by the grant from Scientific Research Program Funded by
Shaanxi Provincial Education Department (Program No.11JK0560).
References
1. Vendemiale G, Grattagliano I, Altomare E: An update on the
role of free radicals and antioxidant defense in human disease.
2. Valko M, Rhodes CJ, Moncol J, Izakovic M,
Mazur M: Free radicals, metals and antioxidants in oxidative stress-induced
cancer.
3. Cooke MS, Evans MD, Dizdaroglu M, Lunec J: Oxidative DNA
damage: mechanisms, mutation and disease.
4. Richardson JS: Free radicals in
the genesis of Alzheimer's disease.
Annals of the New York Academy of Sciences 2006, 695:73-76.
5. Olanow CW: An introduction
to the free radical hypothesis in Parkinson's disease.
6. Bankson DD, Kestin M, Rifai N: Role of free
radicals in cancer and atherosclerosis.
7. Jaswal S, Mehta HC, Sood AK, Kaur J: Antioxidant
status in rheumatoid arthritis and role of antioxidant therapy.
8. Mak JC, Chan YMM: Reactive oxidant
species in asthma.
9. Shen SS, Nauduri D, Anders MW: Targeting
antioxidants to mitochondria: A new therapeutic direction.
10. Glover V, Halket JM, Watkins PJ, Clow A, Goodwin BL, Sandler M: Isatin: Identity
with the purified endogenous monoamine oxidase inhibitor tribulin.
THE END
