时间分辨荧光免疫分析法间接测定雌二醇

以氯磺酰基噻吩甲酰三氟丙酮（CTTA）为铕（Eu)的螯合剂,羊抗鼠（SAM）的IgG为二抗,用SAM－IgG-CTTA-Eu作标记二抗,建立了以竞争抑制为基础的时间分辨荧光免疫分析测定离雌二醇（E2）的新方法。同均相方法相比灵敏度有很大提高,测定雌二醇（E2）的线性范围为2．5－200pg/mL,检测限为2.5pg/mL。这一方法可望用于E2的临床检测。     

Liquid chromatography/mass spectrometry of pre-ionized Girard P derivatives for quantifying estrone and its metabolites in serum from postmenopausal women

An ultrasensitive stable isotope dilution liquid chromatography/selected reaction monitoring/mass spectrometry (LC/SRM/MS) assay has been developed for serum estrone, 16α‐hydroxyestrone, 4‐methoxyestrone, and 2‐ methoxyestrone. The enhanced sensitivity was obtained by the use of Girard P (GP) pre‐ionized derivatives coupled with microflow LC. The limit of detection for each estrogen using 0.5 mL of serum was 0.156 pg/mL and linear standard curves were obtained up to 20 pg/mL. Serum samples from 20 postmenopausal women (10 lifetime non‐smokers and 10 current smokers) were analyzed using this new assay. Mean serum concentrations of estrone and 2‐methoxyestrone were 14.06 pg/mL (±1.56 pg/mL) and 3.30 pg/mL (±1.00 pg/mL), respectively, for the 20 subjects enrolled in the study. The mean estrone concentration determined by our ultrasensitive and highly specific assay was significantly lower than that reported for the control groups in most previous breast cancer studies of postmenopausal women. In addition (and contrary to many reports) serum 16α‐hydroxyestrone was not detected in any of the subjects, and 4‐methoxyestrone was detected in only one of the subjects. Furthermore, there were no significant differences in the mean serum concentrations of estrone and 2‐methoxyestrone or the ratio of serum 2‐ methoxyestrone to estrone between the non‐smoking and smoking groups. Interestingly, the one subject with measurable serum 4‐methoxyestrone (2.3 pg/mL) had the lowest estrone and 2‐methoxyestrone concentrations. Using this assay it will now be possible to obtain definitive information on the levels of serum estrone, 4‐methoxyestrone, and 2‐methoxyestrone in studies of cancer risk using small serum volumes available from previous epidemiology studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous quantitation of the diastereoisomers of scholarisine and 19‐epischolarisine,vallesamine, and picrinine in rat plasma by supercritical fluid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study

Dengtaiye tablet has been used to treat chronic bronchitis cough. Scholarisine, 19‐epischolarisine, vallesamine, and picrinine are the representative constituents of Dengtaiye. A rapid and sensitive assay based on supercritical fluid chromatography with tandem mass spectrometry has been developed and validated for the determination of the diastereoisomers of scholarisine and 19‐epischolarisine, vallesamine, and picrinine in rat plasma using lamotrigine as internal standard. The analysis in a run time of only 6 min was performed on an ACQUITY UPC² Trefoil^TM BEH 2‐EP column (3.0 × 150 mm, 2.5 μm) at 50ºC. The mobile phase consisting of carbon dioxide and methanol (2 mM ammonium formate) was performed as follows: 15% methanol (2 mM ammonium formate) maintained at 0–2 min, 15–19% methanol (2 mM ammonium formate) at 2–4 min, 19–15% methanol (2 mM ammonium formate) at 4–6 min. The flow rate was 1.50 mL/min. The assay was linear over the concentration ranges 50–10000 pg/mL for scholarisine, 19‐epischolarisine, vallesamine, and picrinine with corresponding lower limits of quantitation of 50 pg/mL. Intra‐ and interday precisions were in the range 1.42–12.85% with accuracies in the range –11.71–2.48%. The method was successfully applied to a pharmacokinetic study involving a single oral administration of 108 mg/kg Dengtaiye tablet to rats.     

Development and validation of a NANOGold™ immunoassay for the detection of vascular endothelial growth factor (VEGF) in human serum using inductively coupled plasma mass spectrometry

This work aimed to develop and validate a NANOGold? based assay, quantified using inductively coupled plasma mass spectrometry (ICP‐MS), for the detection of human vascular endothelial growth factor (hVEGF) in serum. The initial assay range based on calibration standards was 62.5–2000 pg/mL with a detection limit of approximately 30 pg/mL. After validation using spiked validation controls, a quantification range between 175 and 1928 pg/mL was obtained. The inter‐assay precision was between 2.3 and 18.9% with accuracy between ?8.8 and ?3.1%. Additional performance parameters, including dilutional linearity, matrix specificity and time‐factored drift, were within ±20%, as defined by the validation acceptance criteria for the validation of macromolecule immunoassays used within our clinical environment. Serum samples from healthy donors were analysed to determine the endogenous levels of VEGF present; these ranged from 164 to 580 pg/mL with a mean of 273 pg/mL. The intra‐ and inter‐assay precision obtained from the healthy donor samples were 1.3–10.7% and 4.2–17.5%, respectively. This demonstration of a validated immunoassay opens further possibilities, utilising the simultaneous detection capabilities of ICP‐MS for the detection of multiple analytes in a single validated immunoassay, for routine use within a clinical environment. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a high-sensitivity liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with chemical derivatization for the determination of ethinyl estradiol in human plasma

An ultra-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of oral contraceptive ethinyl estradiol (EE) was developed and validated over the curve range of 2.5-500 pg/mL using 1 mL of human plasma sample. Ethinyl estradiol and the internal standard, ethinyl estradiol tetra-deuterated (EE-d4), were extracted from the plasma matrix with methyl t-butyl ether, derivatized with dansyl chloride and then back-extracted into hexane. The hexane phase was evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The chromatographic separation was achieved on a Luna C(18) column (50 x 2 mm, 5 micro m) with an isocratic mobile phase of 20:80 (v/v) water:acetonitrile with 1% formic acid. The offline derivatization procedure introduced the easily ionizable tertiary amine function group to EE. This greatly improved analyte sensitivity in electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 2.5 pg/mL. This high sensitivity method can be used for therapeutic drug monitoring or supporting bio-equivalence and drug-drug interaction studies in human subjects.     

Quantitative analysis of betulinic acid in mouse,rat and dog plasma using electrospray liquid chromatography/mass spectrometry

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.     

Simultaneous quantitation of testosterone and estradiol in human cell line (H295R) by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

The possible interaction of environmental contaminants with the endocrine system has been an environmental concern since the early 1990s. To examine these interactions test guidelines have been introduced by regulatory agencies to screen for possible endocrine active compounds. One of these guidelines is the EPA's OPPTS 890.1550 [Steroidogenesis (Human Cell Line‐H295R)]. This guideline requires the quantification of two major biomarkers (testosterone and estradiol) in various biological test systems. Traditional quantitation methodologies such as Radioimmunoassay (RIA) and Enzyme‐linked Immunosorbent Assay (ELISA) have been used to quantify low levels of steroids. However, those methodologies have drawbacks such as the radioactive safety, antibody availability, separate assay for each biomarker, and lack of selectivity. In the current study, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry method (LC/APPI‐MS/MS) has been developed and validated for the simultaneous quantitation of testosterone and estradiol in the H295R cell line. Briefly, the media from cultured cells was extracted with dichloromethane (CH₂Cl₂) containing internal standards of both testosterone‐d₃ and estradiol‐¹³C₃; then, the extracted organic layer was concentrated down to dryness. The final residue was derivatized with dansyl chloride solution, and directly analyzed by LC/APPI‐MS/MS. The calibration curves, with concentration ranging from 10 to 2500 pg/mL, were linear with coefficient >0.99. The lower limits of quantitation for both testosterone and estradiol were 10 pg/mL. This method was successfully validated to support requirements of the current EPA Steroidogenesis guideline. This type of method may also provide value for rapid and precise measurements of these two hormones in other in vitro or in vivo test systems. Copyright © 2011 John Wiley & Sons, Ltd.     

雌二醇的水凝胶毛细管电泳免疫分析

雌二醇是雌性激素中最重要和作用最强的一种激素 .临床上 ,雌二醇的长期监测可用于研究与激素相关的致癌物作用机理、绝经期妇女的雌激素补充及不孕病人的跟踪治疗等方面 .研究发现 ,人体中雌二醇含量与某些肿瘤如乳腺癌、子宫癌和肝癌等密切相关 [1] .由于雌二醇在体液中含量很低 ,难于测定 .免疫分析法的出现给雌二醇的测定带来了变革 ,提高了测定的灵敏度和特异性 .目前测定雌二醇的免疫分析方法有放射免疫分析法 ( RIA) [2 ]、酶免疫分析法 ( EIA) [3～ 7]、荧光免疫分析法 ( FIA) [8]和化学发光与生物发光免疫分析法 [9,10 ] .但 R…     

Different Biotinylation Strategies for Competitive Immunoassay of Estradiol

Study on biotinylation strategies for competitive immunoassay of estradiol (E2) was carried out. Two types of competitive enzyme immunoassay (EIA) with Biotin-Avidin amplification system were established and optimized.The E2-Biotin conjugate was used as a tracer in one assay, and biotinylated antibody was used as a tracer in the other. In both of EIAs, horseradish peroxidase-labelled Avidin (Avidin-HRP) was used with a spectrometric determination of enzyme activity. The precision, sensitivity and specificity were measured and compared. The results showed that although both were satisfactory in specificity, the EIA with hapten-Biotin showed to be superior to the EIA with biotinylated antibody in sensitivity and precision. The limit of detection of serum E2 was 8 and 50 pg/mL with E2-Biotin and biotinylated antibody as tracer, respectively.     

钒(Ⅴ)催化氧化甲基紫的反应动力学及其应用

在 H2 SO4 介质中 ,以抗坏血酸为活化剂 ,痕量的钒 ( )可强烈地催化溴酸钾氧化甲基紫的反应 ,研究了反应的最佳条件及动力学参数 ,探讨了反应机理 ,建立了测定超痕量钒的高灵敏方法 ,方法的线性范围 0 .0～ 2 50 pg/m L ,检出限为 6.5×1 0 - 13g/m L。方法用于井水、蔬菜及血清中痕量钒的测定 ,获得令人满意的结果     

高效液相色谱法测定血清中头孢噻肟浓度

用固相萃取法处理样品,以扑热息痛为内标物、甲醇－醋酸钠／醋酸缓冲溶液作流动相,采用反相高效液相色谱法测定血清中头孢噻肟的浓度。头孢噻肟和内标物的平均回收率分别为９６．７％和９７．７％,头孢噻肟血清浓度在１０ｍｇ／Ｌ至１５０ｍｇ／Ｌ范围内有良好线性关系,最低检测浓度为２ｍｇ／Ｌ,日内和日间相对标准偏差分别在３．０％和４．１％以内。结果表明方法准确、简便。     

Fluoroimmunoassay for tumor necrosis factor-α in human serum using Ru(bpy)3Cl2-doped fluorescent silica nanoparticles as labels

A novel fluoroimmunoassay (FIA) method was developed for the determination of tumor necrosis factor-α (TNF-α) in this study. The proposed method has the advantage of showing the specificity of immunoassays and sensitivity of fluorescent nanoparticles label technology. With the well-established inverse microemulsion polymerisation process, the tris(2′,2-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy)-doped fluorescent silica nanoparticles (RuDFSNs) were prepared. Then a RuDFSNs-labeled anti-TNF-α monoclonal antibody was prepared and used for FIA of TNF-α in human serum samples with a sandwich FIA by using the low fluorescent 96-well transparent microtiter plates. The assay response was linear from 1.0 to about 250.0 pg/mL with a detection limit of 0.1 pg/mL for TNF-α. The intra- and inter-assay precision are 4.9%, 4.4%, 4.6%; 6.1%, 5.9%, 5.3% for five parallel measurements of 2.0, 20.0, 200.0 pg/mL TNF-α respectively, and the recoveries are in the range of 96-104% for human serum sample measurements by standard-addition method. We also explored the application of fluorescence microscopy imaging in the study of the FIA for TNF-α with the fluorescent nanoparticle labels. The results demonstrate that the method offers potential advantages of sensitivity, simplicity and good reproducibility for the determination of TNF-α, and is applicable to the determination of TNF-α in serum samples and being capable of fluorescence microscopy imaging for the determination of TNF-α.     

Determination of clopidogrel in human plasma by liquid chromatography/tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid and sensitive LC/MS/MS assay was developed and validated for the determination of clopidogrel in human plasma. Clopidogrel was extracted by single liquid-liquid extraction with pentane, and chromatographic separations were achieved on a C(18) column. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), stability, accuracy and precision. The multiple reaction monitoring was based on m/z transition of 322.2 --> 211.9 for clopidogrel and 264.1 --> 125.1 for ticlopidine (internal standard). The total analytical run time was relatively short (3 min), and the LLOQ was 10 pg/mL using 0.5 mL of human plasma. The assay was linear over a concentration range from 10 to 10,000 pg/mL (r > 0.999). The intra- and inter-day accuracies were 101.3-108.8 and 98.4-103.5%, respectively, and the intra- and inter-day assay precisions were 1.9-5.5 and 4.4-8.1%, respectively. The developed assay method was applied to a pharmacokinetic study in human volunteers after oral administration of clopidogrel at a dose of 150 mg.     

Nanocarbon Materials Modified with the Zinc Complex of Protoporphyrin IX,Recognized Antibiotics in Water Samples

The complex between protoporphyrin IX and zinc was immobilized on nanocarbon paste and on nanodiamond paste to design two stochastic microsensors. The microsensors were used for the recognition and analysis of antibiotics: amoxicillin, ampicillin, and biotin in water samples. Stochastic sensors provided different signatures for the three antibiotics making possible their simultaneous recognition and assay in water samples. Low limits of determination 0.3 pg/mL for amoxicillin and ampicillin, and 0.21 pg/mL for biotin were obtained when nanocarbon paste was used, and 0.03 pg/mL for amoxicillin, 0.30 pg/mL for ampicillin, and 2.14 fg/mL for biotin were obtained when nanodiamond paste was used. Recoveries higher than 99.32 % with RSD lower than 1.00 % were obtained for the assay of the antibiotics in water samples.     

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography–electrospray tandem mass spectrometry

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra ^® MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r ²?=?0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.     

Quantification of femtomolar concentrations of the CYP3A substrate midazolam and its main metabolite 1'-hydroxymidazolam in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1′-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 μm UPLC? column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1′-hydroxymidazolam were quantified using deuterium- and ¹³C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05–250 pg/mL) and 1′-hydroxymidazolam (0.25–125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1′-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1–100 μg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1′-hydroxymidazolam for at least 10 h, even after the administration of only 1 μg of midazolam.     

Development and validation of a sensitive ELISA for quantitation of Grastim (rhG-CSF) in rat plasma: Application to a pharmacokinetic study

Grastim is bacterially produced recombinant counterpart of human granulocyte colony stimulating factor (G-CSF). It has biological activity similar to that of endogenous G-CSF. In the present work a sensitive, accurate, precise and enzyme-linked immunosorbent assay (ELISA) for the quantitation of G-CSF in rat plasma was developed and validated. The ELISA method employed a technique in which anti-human-G-CSF was adsorbed onto 96-well maxisorp plates and used to capture the G-CSF in rat plasma samples. The captured G-CSF was then detected using streptavidin-HRP amplification system. Absolute recovery was >90% from rat plasma. The validation includes assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLOQ), storage stability (30 days) in rat plasma and assay specificity. The standard curve for G-CSF was linear (R2 > 0.996) in the concentration range 4.88-625 pg/mL. The LLOQ was established at 4.88 pg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 15, 250 and 500 pg/mL, were in the range 3.00-8.66% relative standard deviation (RSD) and 1.03-4.69% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.28-110.79% of the nominal values. The assay shows dilutional linearity and specificity. Stability of G-CSF was established for 30 days at -80 degrees C and through three freeze-thaw cycles. The validated assay was successfully employed for the assessment of pharmacokinetic disposition of G-CSF in rats.     

Determination of phenanthrene by antibody-coated competitive real-time immuno-PCR assay

A reliable selective and sensitive antibody-coated competitive real-time immuno-PCR (RT-IPCR) assay for the determination of phenanthrene (PH) was developed. Phenanthrene butanoic acid (γ-oxo-PHA) was synthesized as the hapten of PH. An active ester method was used to couple the PHA to bovine serum albumin to form an artificial immune antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies, with which a novel RT-IPCR assay for determination of PH was developed. Under the optimized assay conditions, PH can be determined in the concentration range from 10 fg/mL to 100 pg/mL with a detection limit of 5 fg/mL. The cross-reactivities of the anti-PH antibody to seven structurally related compounds were below 12.5%. Some environmental water samples were analyzed with satisfactory results, which showed good accuracy and suitability to analyze PH in environmental water. Compared with high-performance liquid chromatography, the recovery was lower or higher with agitation but would still be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions.     

Trace LC/MS/MS quantitation of 17β‐estradiol as a biomarker for selective estrogen receptor modulator activity in the rat brain

A sensitive LC/MS/MS method has been developed by derivatization of 17β‐estradiol (E2) with dansyl chloride to quantitate 17β‐E2 in female rat serum. The use of E2‐d₅ minimized interferences from endogenous 17β‐E2 in order to achieve a limit of quantitation (LOQ) of 2.5 pg/ml using 150 µl of female rat serum. The recovery of the dansyl derivative was 95% or greater in quality control samples. The intra and interday assay precision was better than 8.2 and 6.2%, respectively, with accuracies ranging from 97 to 101% in the quality control samples. The assay was used for the quantitation of serum E2 as a biomarker for the estrogen receptor (ER) antagonist activity of small molecule SERMs (selective estrogen receptor modulators) in the female rat brain. The study revealed that a statistically significant upregulation of serum 17β‐E2 occurred for rats dosed with SERMs that are known to penetrate the brain and disrupt the hypothalamic‐pituitary‐ovarian (HPO) axis. Variations in 17β‐E2 in ascending dose studies also correlated with the corresponding trends in CYP17a1 levels, an mRNA biomarker for ovarian hyperstimulation. This biomarker assay has provided a useful screen for medicinal chemistry optimization to produce SERMs that do not interfere with negative feedback of estrogens on the brain and for biological hypothesis testing. Copyright © 2009 John Wiley & Sons, Ltd.     

Dual‐column capillary microextraction (CME) combined with electrothermal vaporization inductively coupled plasma mass spectrometry (ETV‐ICP‐MS) for the speciation of arsenic in human hair extracts

In this work, dual‐column capillary microextraction (CME) system consisting of N‐(2‐aminoethyl)‐3‐aminopropyltrimethoxysilane (AAPTS)‐silica coated capillary (C1) and 3‐mercaptopropyl trimethoxysilane (MPTS)‐silica coated capillary (C2) was developed for sequential separation/preconcentration of arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] in the extracts of human hair followed by electrothermal vaporization inductively coupled plasma mass spectrometry (ETV‐ICP‐MS) detection with iridium as permanent modifier. Various experimental parameters affecting the dual‐column microextraction of different As species had been investigated in detail. It was found that at pH 9, As(V) and MMA could be quantitatively retained by C1 and only As(III) could be quantitatively retained by C2. With the aid of valve switching, As(V)/MMA(V) retained on C1 and As(III) retained on C2 could be sequentially desorbed by 10 µl of 0.01 mol l^?1 HNO₃ [for As(V)], 0.1 mol l^?1 HNO₃ [for MMA(V)] and 0.2 mol l^?1 HNO₃‐3% thiourea (m/v) [for As(III)], respectively, the eluents were immediately introduced into the Ir‐coated graphite tubes for further ETV‐ICP‐MS detection. With two‐step ETV pyrolysis program, Cl^? in the sample matrix could be in situ removed, and the total As in the human hair extracts or digested solution could be interference‐free, determined by ETV‐ICP‐MS. DMA(V) in the human hair extracts was obtained by subtraction of total As in the human hair extracts from other three As species. Under the optimized conditions, the detection limits (3 σ) of the method were 3.9 pg ml^?1 for As(III), 2.7 pg ml^?1 for As(V), 2.6 pg ml^?1 for MMA(V) and 124 pg ml^?1 for total As with the relative standard deviations less than 7.0% (C = 0.1 ng ml^?1, n = 7), and the enrichment factor was 286, 262 and 260 for As(III), As(V) and MMA(V), respectively. The developed method was successfully applied for the speciation of arsenic in the extracts of human hair. Copyright © 2009 John Wiley & Sons, Ltd.