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Analytical and Bioanalytical Chemistry (v.370, #8)
Development of a low-cost microfluidic capillary-electrophoresis system coupled with flow-injection and sequential-injection sample introduction (review) by Z.-L. Fang; Qun Fang (pp. 978-983).
Microfabrication techniques used for the production of MEMS (micro electro-mechanical systems) have been successfully used to produce highly efficient microfluidic capillary electrophoresis chip systems. A limitation of this approach are the difficulties associated with the creation of the micrometer-sized structures in glass or other substrates, which currently involve specialized and expensive lithographic and etching processes. A further limitation is that hitherto most microfluidic chips are not designed for continuous introduction of a series of different samples, which limits the overall throughput of such systems. This article reviews the development of a microfluidic system for rapid CE separations, produced at a low cost of less than a dollar each, using equipment and materials readily available in the ordinary laboratory. Applications of the system, after coupling to flow-injection and/or sequential-injection sample introduction, for the determination of FITC- labeled amino acids by laser-induced fluorescence, trace metals by chemiluminescence, carbohydrates by amperometry, and inorganic and organic anions by indirect UV absorbance are exemplified to illustrate the performance and versatility of the microfluidic system.
Analytical methodologies for aluminium speciation in environmental and biological samples – a review by S.-p. Bi; Xiao-di Yang; Fu-ping Zhang; Xian-long Wang; Gong-wei Zou (pp. 984-996).
It is recognized that aluminium (Al) is a potential environmental hazard. Acidic deposition has been linked to increased Al concentrations in natural waters. Elevated levels of Al might have serious consequences for biological communities. Of particular interest is the speciation of Al in aquatic environments, because Al toxicity depends on its forms and concentrations. In this paper, advances in analytical methodologies for Al speciation in environmental and biological samples during the past five years are reviewed. Concerns about the specific problems of Al speciation and highlights of some important methods are elucidated in sections devoted to hybrid techniques (HPLC or FPLC coupled with ET–AAS, ICP– AES, or ICP–MS), flow-injection analysis (FIA), nuclear magnetic resonance (27Al NMR), electrochemical analysis, and computer simulation. More than 130 references are cited.
A new method of Fourier-transform smoothing with ratio spectra derivative spectrophotometry by Zhenyu Lin; Jianhua Liu; Guonan Chen (pp. 997-1002).
A new method which combines Fourier-transform smoothing with ratio spectra derivative spectrophotometry has been developed for analysis of binary and ternary mixtures. The method proposed has been applied to the simultaneous determination of a binary mixture of 0.974–2.026 × 10–2 g L–1 nitrate and nitrite and to a ternary mixture of 1.840–3.888 × 10–3 g L–1 histidine, phenylalanine, and tryptophan. The relative standard derivations are between 0.07 and 5.3% for the former system, and between 0.5 and 7.3% for the latter. The results show that the method proposed is more accurate than multiple linear regression.
Identification of a new gene product of PKIβ by HPLC–ESIMS by H. Song; G. Yue; Z. Zhou; Y. Xie; P. Yang; Y. Mao; Y. Xu; H. Wang (pp. 1003-1008).
An identification method using high-performance liquid chromatography combined with electrospray mass spectrometry (HPLC–ESIMS) has been developed to verify an expressed gene product of kinase inhibitor (PKIβ). This protein was expressed in this university for the first time from a newly cloned gene in the cDNA library of human fetal brain. The measured MW (8468.9 Da) of PKIβ-78 was consistent with expectations. The gene product of PKIβ-78 was monitored by ESIMS to ensure there was no mis-expressed PKIβ-70 in the process of gene engineering. The peptide mapping of PKIβ-78 and its partial sequence were, furthermore, determined. By database searching based on the experimental MWs and partial sequences provided, it was verified that this gene product is a new protein. The pseudosubstrate site and leucine-rich site for the function region of PKIβ-78 are also confirmed.
Microdetermination of proteins with the arsenazo-DBN–Al(III) complex by Rayleigh light-scattering technique and application of the method by L. J. Dong; Run Ping Jia; Qian Feng Li; X. G. Chen; Zhi De Hu; M. A. Hooper (pp. 1009-1014).
The determination of proteins with arsenazo-DBN and Al3+ by Rayleigh light-scattering (RLS) is described. The weak RLS of arsenazo-DBN and BSA can be enhanced greatly by addition of Al3+ in the pH range 5.3–7.0; this resulted in two enhanced RLS signals at 420–440 nm and 460–480 nm. The reaction between arsenazo-DBN, Al3+, and proteins was studied and a new method was developed for quantitative determination of proteins. This method is very sensitive (0.34–41.71 μg mL–1 for bovine serum albumin, BSA, and 0.29–53.41 μg mL–1 for human serum albumin, HSA), rapid (< 2 min), simple (one step), and tolerant of most interfering substances. The effects of different surfactants were also examined. When these proteins were determined in four human serum samples the maximum relative error was not more than 2% and the recovery was between 97 and 103%.
A collision model for DNA separation by capillary electrophoresis in dilute polymer solution by Y. Jin; B.-C. Lin; Ying-sing Fung (pp. 1015-1022).
A theoretical description, based on chemical kinetics and electrochemistry, is given of DNA separation in dilute polymer solution by capillary electrophoresis. A self-consistent model was developed leading to predictions of the DNA electrophoretic velocity as a function of the experimental conditions – polymer concentration, temperature, and electric field strength. The effect of selected experimental variables is discussed. The phenomena discussed are illustrated for the example of 100 bp DNA ladder separation in dilute HPMC solution by capillary electrophoresis. This model is the first single model that can fully explain the dependence of DNA electrophoretic velocity on electrophoretic conditions.
Solid-substrate room-temperature phosphorescence study on zinc(II) and tin(IV) protoporphyrins and their interaction with DNA by A.-j. Tong; L. Liu; Lei Liu; Long-di Li; Carmen W. Huie (pp. 1023-1028).
The solid-substrate room-temperature phosphorescence (SS-RTP) of two commercially available metalloporphyrin compounds, zinc(II) protoporphyrin (ZnPP) and tin(IV) protoporphyrin (SnPP) has been studied. Strong and stable RTP signals of the two metalloporphyrins in neutral to weakly basic solutions can be simply induced on filter paper without addition of external heavy atom perturbers. Their emission bands appeared at 723 nm for ZnPP and 718 nm for SnPP at an excitation wavelength of 417 nm. Compared with SnPP, ZnPP is a better RTP probe for DNA because its RTP enhancement effect is much higher under the same experimental conditions. The interaction of ZnPP with DNA at pH 8.5 gives an apparent binding constant of 9.1 × 103 which is similar to that of the cationic porphyrin absorption probe CuTMPyP (copper (II)- tetrakis(4-N-methylpyridyl)porphine complex). Hydrogen bonding between the monocarboxylic acid substituent of ZnPP and the base pairs of DNA plays a crucial role in the binding.
Renewable amperometric immunosensor for complement 3 assay based on the sol–gel technique by Guo-dong Liu; Tong-Sheng Zhong; Sha-Sheng Huang; G.-L. Shen; R.-Q. Yu (pp. 1029-1034).
A renewable amperometric immunosensor based on the sol–gel technique has been constructed by dispersing graphite, complement 3 (C3) antiserum, and sol–gel at low temperature. The prepared immunosensor is rigid, porous, and has a renewable external surface. A competitive binding assay has been used to determine C3 in human serum with the aid of C3 labeled with horseradish peroxidase. The enzyme-labeled antigen can readily diffuse toward the encapsulated antibody, which retains its binding properties. The experimental conditions for the assay with the biocomposite, including the loading of C3 antiserum in the biocomposite, the amount of labeled C3 in incubation solution, incubation time, and temperature, have been optimized. Using C3 labeled with horseradish peroxidase, and o-AP as the substrate, amperometric detection at –150 mV (relative to the SCE) results in a linear detection range of 1.17–35.1 μg mL–1, with a detection limit of 0.56 μg mL–1. Serum samples have been assayed and the results demonstrate the feasibility of the proposed immunosensor for clinical analysis. The surface of the immunosensor can be renewed simply by polishing to obtain a fresh immunocomposite ready to use in a new competitive assay.
Development and certification of a coal fly ash certified reference material for selected polycyclic aromatic hydrocarbons by C. Xueli; X. Xiaobai; C. Wenxuan; X. Zhiqun (pp. 1035-1040).
The development and certification of a coal fly ash certified reference material (CRM) for polycyclic aromatic hydrocarbons (PAH) is described; this is the first natural matrix CRM for organic environmental analysis in China. The homogeneity and stability of this material have been tested by HPLC. The concentrations of several PAH were determined by use of two independent, different methods – solvent extraction–HPLC analysis with UV detection coupled with fluorescence detection (FLD) and solvent extraction, isolation with a silica column, and GC analysis with flame ionization detection (FID). Five certified values were determined: phenanthrene 7.1 ± 2.6 μg g–1, anthracene 2.0 ± 0.8 μg g–1, fluoranthene 7.4 ± 1.9 μg g–1, pyrene 7 ± 2 μg g–1, and benzo[a]pyrene 1.3 ± 0.3 μg g–1. Reference values for several other PAH are also suggested.
Speciation of rare earth elements in soil by sequential extraction then HPLC coupled with visible and ICP–MS detection by Qiuquan Wang; B. Huang; Zhenwei Guan; Limin Yang; Bin Li (pp. 1041-1047).
The distribution of rare earth elements (REE) in a pooled soil sample collected from Zhangzhou, Fujian Province, China, was screened by a five-step sequential extraction procedure coupled with ICP–MS determination after preconcentration of REE and removal of the matrix by extraction with 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (HPMBP). The results showed that the distribution of REE in the different fractions of the pooled soil sample studied followed the order soluble species (46.76%) > species bound to organic matter (22.08%) > species in the residue (16.77%) > species bound to Fe–Mn oxides (2.02%). An effective method for speciation of REE, which utilized weak cation-exchange HPLC separation hyphenated with post-column derivatization and visible or on line ICP–MS detection, was, moreover, developed and successfully applied to the speciation of REE in the soluble extract of the pooled soil sample. The stability of known complexes of lanthanum during the HPLC separation was investigated with fluoride, citrate, and ethylenediamine tetraacetic acid (EDTA) chosen as ligands modeling those in the soil. REE in the soluble extract of the pooled soil sample were subsequently classified into three types of species –≤ + 1 charged complexes (negatively charged, neutral, and +1 charged), + 2 charged complexes, and “free” REE species. This method is expected to be useful for identification of bioavailable (or toxic) species of REE in environmental samples.
Optical rubbery ormosils sensor for the detection of ammonia by X. Chen; Y. Dai; Z. Li; Z. Zhang; X. Wang (pp. 1048-1051).
Rubbery ormosil films with immobilized aminofluorescein (AF) were investigated to develop an optochemical sensor for the determination of ammonia in water. The gel precursors with tetramethoxysilane (TMOS) and dimethyldimethoxysilane (DiMeDMOS) were deposited on glass supports, and characterized in terms of response to pH, and to dissolved ammonia at constant pH. After preconditioning the sensing film was stable for 6 months. The detection limit for ammonia in water was 0.2 μg mL–1 (S/N 2), the response being linearly dependent on concentration in the range of 0.5 to 80 μg mL–1 ammonia. The response time was less than 5 min. The effects of sodium chloride concentration, temperature, and coexisting metal ions and compounds were investigated.
Kinetic studies on the mechanism of atomization in electrothermal atomic absorption spectrometry with and without chemical modifiers by Xiu-Ping Yan; Zhe-Ming Ni (pp. 1052-1060).
Over the past decade a few methods for determining kinetic data for atom formation from the absorption signal in electrothermal atomic absorption spectrometry (ETAAS) have been developed in the author’s laboratory. These approaches include improvement to the Smets method, and development of new methods for simultaneous determination of kinetic order and activation energy for atom formation at increasing or constant temperatures. The steady-state approximation and first-order kinetic assumption for atom formation have been avoided during their derivation. One of the most distinct features of these new methods is their suitability for quantitative determination of the kinetic order for atom formation from absorption signals under normal analytical conditions, even for atomization processes with fractional reaction orders and/or with multiple mechanisms. The application of the developed methods to the study of the mechanism of atomization in the graphite-furnace atomizer, with and without chemical modifiers, is reviewed with emphasis on research work in the authors’ laboratory.
A simple flow-injection on-line clean-up system for microwave plasma-torch atomic emission spectrometry by Liwei Zhao; Minjing Li; Xiaohang Xie; Daqian Song; Yuan Tian; Lijuan Zhang; Danhong Jin; Hanqi Zhang; Q. Jin (pp. 1061-1064).
A simple flow injection (FI) on-line clean-up system has been developed for microwave plasma-torch atomic emission spectrometry (MPT–AES). A non-selective strongly acidic cation-exchange resin was used to achieve the goal of “on-line clean-up”. Ag and Zr, which form halogen-complex anions in halide acid media, and Cr, Mo, and P, which exist as acid group anions or acids (neutral) in acidic solution, were determined and the interfering matrix cations were removed on-line. Satisfactory analytical results were obtained from some practical samples by use of this procedure.
Flow-injection chemical vapor-generating procedure for the determination of Au by atomic absorption spectrometry by Xiaoguang Du; Shukun Xu (pp. 1065-1070).
Volatile Au species in an acidified medium were generated at room temperature by reduction with NaBH4 in acidified aqueous medium using a flow-injection chemical vapor-generation atomic absorption spectrometric (FI–CVG–AAS) system in the presence of micro amounts of sodium diethyldithiocarbamate (DDTC). Precision of 2.0% RSD (n = 11, 2.0 mg L–1 level) was obtained at a sample throughput of 180 h–1. A detection limit of 24 ng mL–1 (3σ) was obtained with 300 μL sample solution. The method was used for the determination of gold in ore sample digests, and the results obtained agreed well with those obtained by flame AAS.
The kinetics and thermodynamics of surfactants in solvent sublation by Y.-j. Lu; Yue-song Wang; Y. Xiong; Xi-hai Zhu (pp. 1071-1076).
Solvent sublation has been performed on very dilute solutions of one cationic surfactant, hexadecylpyridinium chloride (HPC), and one anionic surfactant, dodecylbenzenesulfonic acid (LBS). Some thermodynamic values were obtained, e.g. molecular areas, A0, which are 50.0 and 47.7 Å2/molecule, respectively, for HPC and LBS, and free adsorption energies, ΔG°ads, which are –33.17 and –43.58 kJ mol–1, respectively, for HPC and LBS. The kinetics were determined for a range of temperatures and gas flow-rates. Although the processes of solvent sublation of the two surfactants obey first-order kinetics, the respective adsorption mechanisms of HPC and LBS in the solvent sublation process were different. The pH and the presence of KCl and ethanol had no effect on the solvent sublation of LBS. The apparent active energy was calculated as 8.11 kJ mol–1.
Nickel and strontium nitrates as modifiers for the determination of selenium in wine by Zeeman electrothermal atomic absorption spectrometry by J. Cvetković; Trajče Stafilov; Dragan Mihajlović (pp. 1077-1081).
A mixed matrix modifier of nickel and strontium nitrates was used as a chemical modifier for the determination of selenium in wines by Zeeman electrothermal atomic absorption spectrometry. Wine samples were heated on a boiling water bath with small amounts of nitric acid and hydrogen peroxide. For complete elimination of interference, especially from sulfates and phosphates, selenium is complexed with ammonium pyrolidinedithiocarbamate (APDTC), extracted into methyl isobutyl ketone (MIBK), and measured by ETAAS.The graphite furnace temperature program was optimized for both aqueous and organic solutions. Pyrolysis temperatures of 1300 °C and 800 °C were chosen for aqueous and organic solutions, respectively; 2700 °C and 2100 °C were used as optimum atomization temperatures for aqueous and organic solutions, respectively. The optimum modifier mass established is markedly lower than those presented in the literature. The platform atomization ensures pretreatment stabilization up to 1100 °C and 1600 °C, respectively, for organic and aqueous selenium solutions. The procedure was verified by the method of standard addition. The investigated wine samples originated from the different regions of the Republic of Macedonia. The selenium concentration varied from not detectable to 0.93 μg L–1.
Adsorptive behavior and electrochemical determination of the anti-fungal agent ketoconazole by T-z. Peng; Qiong Cheng; Catherine F. Yang (pp. 1082-1086).
The adsorptive properties and electrochemical behavior of ketoconazole, an oral anti-fungal agent, are demonstrated at a glassy carbon electrode. The adsorption of the compound obeys the Frumkin isotherm with an interaction factor (α) of 0.985 and adsorptive coefficient (β) of 1.98 × 106 L mol–1. The Gibbs energy of adsorption (ΔG) is –3.59 × 104 J mol–1 at 25 °C. A very sensitive electroanalytical method has been developed for determination of the drug with a detection limit of 4.0 × 10–11 mol L–1. Relationships between stripping current and concentration of ketoconazole were linear in the range 10–6–10–10 mol L–1 with different preconcentration periods. The method has been used to measure the ketoconazole content of tablets.
A study on the detection of weak photoacoustic signals by stochastic resonance by Huiyu Song; Xueguang Shao; Qingde Su (pp. 1087-1090).
On the basis of stochastic resonance (SR) theory a study of the detection of photoacoustic (PA) signals without a lock-in amplifier is presented. The applicability of the SR method is investigated both with simulated signals calculated by use of a Gaussian model and with experimental PA signals. Signal-to-noise ratio (SNR) is improved substantially by adjusting the properties of a non-linear system to realize SR. By use of this method the lock-in amplifier can be removed from a PA spectrometer.
Highly selective thiocyanate poly(vinyl chloride) membrane electrode based on a cadmium–Schiff’s base complex by M. R. Ganjali; Tahereh Poursaberi; Fatemeh Basiripour; Masoud Salavati-Niassari; Mohammad Yousefi; Mojtaba Shamsipur (pp. 1091-1095).
A PVC membrane electrode based on a cadmium–salen (N,N′-bis-salicylidene-1,2ethylenediamine) complex as an anion carrier is described. The electrode has an anti-Hofmeister selectivity sequence with a preference for thiocyanate at pH 1.5–11.0. It has a linear response to thiocyanate from 1.0 × 10–6 to 1.0 × 10–1 mol L–1 with a slope of 59.1 ± 0.2 mV per decade, and a detection limit of 7 × 10–7 mol L–1. This electrode has high selectivity for thiocyanate relative to many common organic and inorganic anions. The proposed sensor has a fast response time of approximately 15 s. It was applied to the determination of thiocyanate in a milk sample.
Solid-phase microextraction–gas chromatographic determination of volatile monoaromatic hydrocarbons in soil by B. Zygmunt; Jacek Namiesnik (pp. 1096-1099).
Benzene, toluene, ethylbenzene, three isomers of xylene, and cumene have been isolated and enriched from soil samples by a combination of water extraction at room and elevated temperature and headspace–solid-phase microextraction before their gas chromatographic–mass spectrometric (GC–MS) determination. The conditions used for all stages of sample preparation and chromatographic analysis were optimized. Analytes sampled on a polydimethylsiloxane-coated solid-phase microextraction fiber were thermally desorbed in the split/splitless injector of a gas chromatograph (GC) coupled with a mass spectrometer (MS). The desorption temperature was optimized. The GC separation was performed in a capillary column. Detection limits were found to be of the order of ca. 1 ng g–1. Relative recoveries of the analytes from soils were found to be highly dependent on soil organic-matter content and on compound identity; they ranged from ca 92 to 96% for sandy soil (extraction at room temperature) and from ca 27 to 55% for peaty soil (extraction at elevated temperature). A few real-world soil samples were analyzed; the individual monoaromatic hydrocarbon content ranged from below detection limits to 6.4 ng g–1 for benzene and 8.1 for the total of p- + m-xylene.
The determination of low lead levels in the bone of lead-depleted mice by graphite furnace atomic absorption spectrometry by I. Iavicoli; G. Carelli; N. Castellino; G. Schlemmer (pp. 1100-1104).
Low lead levels in the femurs of mice fed with a lead-depleted diet have been determined by use of electrothermal atomic absorption spectrometry with Zeeman-effect background correction. The method is based on the use of Mg(NO3)2/Pd as matrix modifier which enables significant reduction of the spectral interferences prevalent if chemical modifiers based on NH4H2PO4 with either Ca or Mg are used for samples rich in Ca3(PO4)2 matrix. The method was developed and validated by use of the NIST standard reference material 1486 bone. Bones were decomposed in a pressurized microwave-heated system using 70% nitric acid. Forty-three mice femurs, with a mass of 74.62 ± 12.54 mg, were dissolved in concentrated nitric acid. The lead results found in SRM 1486 (1.25 ± 0.15 μg g–1, n = 9) were in good agreement with the certificate (1.335 ± 0.014 μg g–1). Recoveries of 200 ng lead added to the SRM before or after digestion were 99.0 ± 1.4% and 98.5 ± 1.6%, respectively. The lead detection limit in bone samples is 0.06 μg g–1 dry mass. This method is, therefore, suitable for the determination of very low lead levels (0.06–0.20 μg Pb kg–1 bone) in the femurs of mice fed a diet with lead level of < 20μg kg–1.
Determination of Mn, Fe, and Cu in chemically-treated wood pulps by the XRF addition method by Jaakko Rämö; M. Sillanpää; Tapio Klasila; Sulo Piepponen (pp. 1105-1108).
A rapid X-ray fluorescence addition method has been developed for quantification of the technically most important metals in wood pulp matrix (Mn, Fe, and Cu). Pretreatment consisted of just two steps: first, acid was added to the sample to achieve homogeneous distribution of the metals; the pulp was then pressed lightly on to Mylar film. Total analysis time was less than 10 min. The concentration range investigated was up to 15 mg kg–1 for Mn and up to 5 mg kg–1 for Fe and Cu. Metal concentrations in Scandinavian pulps are not expected to exceed these amounts. The quantification limit was 2 mg kg–1 for all three metals. The reproducibilities and repeatabilities were concentration-dependent and varied between 3 and 19% and between 1 and 17%, respectively. The squares of the linear correlation coefficients between measured intensity and added metal concentration were 0.994, 0.950, and 0.932 for Mn, Fe, and Cu, respectively.
Group separation of trace rare-earth elements by countercurrent chromatography for their determination in high-purity calcium chloride by S. N. Ignatova; T. A. Maryutina; B. Ya. Spivakov; V. K. Karandashev (pp. 1109-1113).
A method has been developed for the separation of the entire group of rare-earth elements from high-purity calcium chloride by countercurrent chromatography, and subsequent determination of the elements by ICP– MS. A solution of diphenyl[dibutylcarbamoylmethyl]phosphine oxide in chloroform (0.5 mol L–1) has been chosen as reagent for the extraction and preconcentration of trace rare-earth elements from aqueous 5% CaCl2 solution, 3 mol L–1 in HNO3 and 0.1 mol L–1 in HClO4. The analytes are back-extracted into a small volume of water and the aqueous eluate is subjected to ICP–MS measurements. The performance characteristics of the procedure developed have been checked by use of the standard addition technique and a real CaCl2 sample (Merck product) has been analyzed. The results obtained demonstrate the applicability of countercurrent chromatography to the determination of ultratrace elements.
Direct determination of ultra-trace amounts of acetone by corona-discharge ion mobility spectrometry by T. Khayamian; M. Tabrizchi; N. Taj (pp. 1114-1116).
The capability of corona-discharge ion mobility spectrometry (CD-IMS) in the quantitative determination of acetone has been evaluated. Generally, in IMS the signal intensity of a product ion is not a linear function of the sample concentration. A linear calibration curve was, however, obtained by plotting ln(Ro +/Ro +–P+) against acetone concentration, where, Ro + is the original reactant ion density and P+ is the sum of all the product ion densities. The acetone detection limit was 60 ng m–3 and its dynamic range was three orders of magnitude.
Spectrofluorimetric determination of thiols by use of N-[P-(2-benzoxazolyl)- phenyl]maleimide by Shu-Cai Liang; Hong Wang; Zhi-Min Zhang; Y.-Y. Zhou; H.-S. Zhang (pp. 1117-1119).
The weak fluorescence of N-[P-2-benzoxazolyl)phenyl]maleimide (BOPM) can be greatly enhanced by thiol-containing compounds. A sensitive and simple spectrofluorimetric method based on the use of BOPM has been developed for the determination of thiols such as cysteine (Cys) and reduced glutathione (GSH). Calibration plots were linear in the concentration range from 0 to 1.6 × 10–7 mol L–1 for Cys and 0 to 1.7 × 10–7 mol L–1 for GSH. The detection limits (3σ) were 2.36 × 10–10 mol L–1 for Cys and 1.49 × 10–10 mol L–1 for GSH. Many other amino acids (present at 100-fold greater concentrations) did not interfere with the determination. The proposed method has been used for the determination of Cys in protein hydrolysate and cystine electrolyte or GSH in serum, with recoveries of 95.4–103.7%.