Journal of Chromatography B (v.917-918, #C)

Troubleshooting carry-over of LC–MS/MS method for rifampicin, clarithromycin and metabolites in human plasma by D.H. Vu; R.A. Koster; A.M.A. Wessels; B. Greijdanus; J.W.C. Alffenaar; D.R.A. Uges (1-4).
► “Carry-over” of rifampicin and 25 desacetyl rifampicin was originated in the column. ► Replacing the polar end-capped phase column excluded the “carry-over”. ► The new method is more convenience for clinical practice than previous method.Clarithromycin and rifampicin are used for the treatment of Mycobacteria. Pharmacokinetic drug interaction is possibly due to the influence of the two drugs on the liver enzymes. Using a Hypurity Aquastar C18 column (50 mm × 2.1 mm × 5 μm) for liquid chromatography including a polar end-capped phase for the determination of clarithromycin, rifampicin and their metabolites together in plasma using LC–MS/MS resulted in a substantial carry-over. As a consequence, the throughput of the method is not assured. Using a step-by-step troubleshooting procedure, such carry-over was found originating from column memory effect. With the use of another type of C18 column, the carry-over is eliminated. Due to the absence of carry-over, the analytical concentration ranges are extended and are therefore more appropriate for the analysis of patient samples. The method was re-validated for linearity, reproducibility and dilution integrity.
Keywords: Rifampicin; Clarithromycin; Mycobacteria; Carry-over; LC–MS/MS;

► For first time, USAE-ME coupled with UV–vis spectrophotometry for determination of an inorganic spicies (thiocyanate ion). ► The method was developed for analysis of drinking mineral water, urine and saliva samples. ► High enrichment factor and ease of operation are the main advantages of proposed method. ► Hyphenation of USAE-ME with ordinary UV–vis spectrophotometry improved the sensitivity significantly.Ultrasound-assisted emulsification microextraction (USAE-ME) procedure coupled with UV–vis spectrophotometric measurement has been developed for determination of thiocyanate ion (SCN) in water and biological fluids samples. The method is based on protonation of SCN ions in acidic medium and extraction of thiocyanic acid into fine droplets of chloroform as an extraction solvent contains rhodamine B (RhB). The RhB was protonated in presence of thiocynanic acid to form highly colored ion-pair complex of [thiocynate][RhBH+] in chloroform, which used for subsequent spectrophotometric determination of SCN ions. Experimental parameters for both spectrophotometric reaction and USAE-ME procedure have been optimized. Under optimized conditions the calibration curve for SCN showed good linearity in the range of 38.0–870.0 ng mL−1 (R 2  = 0.9967). The limit of detection (S/N = 3) and preconcentration factor were 5.0 ng mL−1 and 40, respectively. Relative standard deviation for determination of 200 ng mL−1 of SCN was 2.8% (n  = 5). The proposed method has been successfully applied for determination of SCN ion in tap water, mineral bottled water and human saliva and urine samples with an average recovery of 99.2%.
Keywords: Ultrasound-assisted emulsification microextraction; Thiocynate ion; Rhodamine B; UV–vis spectrophotometry; Urine; Saliva;

► Pressurized liquid extraction has been developed to the HPLC quantitative determination of seven tetracyclines in egg, fish and shrimp. ► The extraction procedure is simple and rapid. ► The optimized procedure has been successfully applied to real samples. ► The method is robust and useful for monitoring and quantification of tetracycline residues.A simple and especially rapid method, pressurized liquid extraction, has been developed and applied to the quantitative determination of oxytetracycline, tetracycline, chlortetracycline, minocycline, methacycline, demeclocycline and doxycycline in egg, fish and shrimp. The procedure consisted of a trichloracetic acid/methanol extraction conducted at elevated temperature (60 °C) and pressure (65 bar), without further clean-up, the extraction solution was concentrated and finally for high-performance liquid chromatography analysis. The limits of detection were 5.0–10.0 μg/kg and the limits of quantification were 10.0–15.0 μg/kg for tetracyclines in egg, fish and shrimp using UV detection. The analytical limits CCα and CCβ were also calculated. The recoveries of tetracyclines spiked at levels of 15–300 μg/kg, averaged 75.6–103.5% with the relative standard deviation values less than 11%. The optimized procedure has been successfully applied to real samples in our laboratories. It demonstrated that the new method was robust and useful for monitoring and quantification of 7 tetracycline residues in food of animal origin.
Keywords: Tetracyclines; Residues; Pressurized liquid extraction; Food of animal origin; HPLC-UV;

Quantification of intact carboplatin in human plasma ultrafitrates using hydrophilic interaction liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study by Hajime Ito; Hiroaki Yamaguchi; Asuka Fujikawa; Narumi Shiida; Nobuaki Tanaka; Jiro Ogura; Masaki Kobayashi; Takehiro Yamada; Nariyasu Mano; Ken Iseki (18-23).
► We developed a HILIC/MS/MS method for the determination of carboplatin in human plasma ultrafiltrates. ► Human plasma ultrafiltrates samples were precipitated by acetonitrile containing internal standard and further diluted with acetonitrile. ► This method was applied to a clinical pharmacokinetic study of carboplatin in a cancer patient.Carboplatin is a platinum agent that is used for treatment of non-small-cell lung cancer and ovarian cancer. A sensitive and selective analytical method for the quantification of carboplatin in human plasma ultrafiltrates using liquid chromatography–tandem mass spectrometry was developed. Human plasma ultrafiltrates were precipitated by acetonitrile containing carboplatin-d4 as an internal standard and were further diluted with acetonitrile. Chromatographic separation was performed on a Accucore HILIC (50 mm × 2.1 mm i.d., 2.6 μm) column using mobile phase (acetonitrile–water–acetic acid = 90:10:0.1, v/v/v) at the flow rate of 0.2 mL/min. Detection was performed on electrospray ionization triple quadrupole tandem mass spectrometer using low-energy collision induced dissociation (CID-MS/MS) analysis operating in the selected reaction monitoring (SRM) scan mode. The lower limit of quantification for carboplatin was 0.025 μg/mL. This method covered a linearity range of 0.025–50 μg/mL. The intra-day precision and inter-day precision (R.S.D.) ranged from 1.5 to 4.3%, and the accuracy (R.E.) was within ±2.9%. The present method was applied to a clinical pharmacokinetic study of carboplatin in a cancer patient.
Keywords: Carboplatin; Hydrophilic interaction liquid chromatography–tandem mass spectrometry; Plasma ultrafiltrates;

► Derivatization with improved chromatographic retention and separation. ► The derivatization was performed without prior sample clean-up. ► The method was validated over the concentration range of 8.78–103.78 μg/mL. ► This method was compared to a commercially available colorimetric assay kit. ► The method can also be adapted for determination of urea in plasma samples.A sensitive, selective, and quantitative method for the determination of urea has been developed and validated in human epithelial lining fluid (ELF; the supernatant from bronchoalveolar lavage). The method employs a simple derivatization of urea with camphanic chloride to improve the chromatographic retention and separation. The derivatization was performed after drying an aliquot of ELF (20 μL) without prior sample clean-up. Ultra High Performance Liquid Chromatography (UHPLC) on a HSS-T3 stationary phase column with 1.8 μm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration range of 8.78–103.78 μg/mL, however the dynamic range can be further lowered if needed. The results from assay validation show that the method is rugged, precise, accurate, and well-suited to support analysis of urea in ELF samples. In addition, the relatively small sample volume (20 μL) and a run time of 1.5 min facilitate automation and allow for high-throughput analysis. This derivatization method was compared to a commercially available colorimetric assay kit, and it was used in a preclinical non-GLP mouse study where urea measurements were used as marker of bronchoalveolar lavage fluid dilution.
Keywords: Urea; Derivatization; Camphanic chloride; Mass spectrometry; UHPLC; Epithelial lining fluid; ELF; Bronchoalveolar lavage fluid; BALF;

► The L2 (252–273) from E. coli ribosomal protein L2 was used as a purification tag. ► Magnetic nanoparticles had a higher adsorption loading than the other adsorbents. ► Tag-free target protein purification by L2 (252–273)-SUMO fusion technology. ► The tag-free recombinant EGFP with a purity of greater than 93% was obtained.A basic polypeptide L2 (252–273) derived from Escherichia coli ribosomal protein L2 was used as a purification tag. In order to develop faster, less expensive methods for expression and purification of proteins, the L2 (252–273)-small ubiquitin like modifier (SUMO) fusion expression system was constructed. We comparatively analyzed the adsorption properties of the deleted protein of L2 (L2 (252–273)) on diatomite and superparamagnetic carboxymethyl chitosan nanoparticles. The time required to reach adsorption equilibrium of L2 (252–273) fusion protein on diatomite was shorter than that of L2 (252–273) fusion protein on magnetic particles. The maximum adsorption capacity of L2 (252–273) fusion protein on magnetic particles was about 5 times larger than that of L2 (252–273) fusion protein on diatomite. SUMO was introduced as a specific protease cleavage site between the target protein and the purification tags. The enhanced green fluorescent protein (EGFP) as a model protein was fused with the L2 (252–273)-SUMO fusion protein and purified by a simple method which involves the electrostatic adsorption of L2 (252–273) fusion proteins on superparamagnetic carboxymethyl chitosan nanoparticles and the L2 (252–273)-SUMO fusion partner was removed based on the robust cleavage by the poly lysine tagged SUMO protease. The high purity of tag-free EGFP (>93%) was obtained. Our results preliminary proved that the system was an effective fusion expression system for the production of recombinant proteins in E. coli.
Keywords: Ribosomal protein L2; Small ubiquitin-related modifier; Diatomite; magnetic particles; Cation exchangers; Recombinant protein purification;

► LC/MS-method for analysis of amlodipine and atorvaststin in plasma has been developed. ► Method of protein direct precipitation with acetonitrile was used for extraction. ► Method was validated through investigation of seven parameters. ► CADUET (Pfizer), new combination of the two drugs was analyzed for the first time. ► The method was successfully applied in a clinical bioequivalence study.A simple liquid chromatography/ion trap mass spectrometry method for the quantification of amlodipine and atorvastatin with its metabolites, ortho and para hydroxy atorvastatin, simultaneously in human plasma was developed. Analytes with internal standard were extracted by protein direct precipitation with acetonitrile. Adequate chromatographic separation was achieved using Phenomenex Synergi 4u polar-RP 80A (150 mm × 4.6 mm, 4 μm) column in the isocratic elution mode and the eluent was water/methanol (14:86%, v/v) adjusted by trichloroacetic acid to pH 3.2 which was delivered isocratically at constant flow rate of 0.50 mL/min. Standard solutions for the analytes were prepared using amlodipine besylate, atorvastatin calcium, ortho-hydroxy atorvastatin dihydrate monosodium salt, para-hydroxy atorvastatin disodium salt, and pravastatin sodium as an internal standard. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to USFDA guideline. Standard calibration levels were prepared by pooled human plasma to attain final dynamic range of 0.2–20.0 ng/mL for amlodipine, 1.5–150 ng/mL for atorvastatin, 1.0–100.0 ng/mL for ortho-hydroxy atorvastatin and 0.2–20.0 ng/mL for para-hydroxy atorvastatin. Clinical bioequivalence study was successfully investigated by the application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products 10 mg amlodipine/80 mg atorvastatin in a single dose. In this study, 29 healthy volunteers were participated in randomized, two periods, double blend, open label cross over design. Pharmacokinetic parameters of C max, AUC0–t and AUC0– were calculated to compare a test product with CADUET® reference product.
Keywords: Amlodipine; Atorvastatin; Ortho- and para-metabolites; LC–MS/MS; Protein precipitation; CADUET; Clinical study; Human plasma;

Display Omitted► Overview of analytical techniques for the quantification of clopidogrel and clopidogrel carboxylic acid in biological samples. ► New developments addressing the determination of the active clopidogrel metabolite. ► Thorough comparison of validation data obtained by the reviewed literature reports.Clopidogrel has been applied in antiplatelet therapy since 1998 and is the thienopyridine with the largest clinical experience. By 2011, clopidogrel (Plavix®) was the second top-selling drug in the world. Following complete patent expiry in 2012/2013 its use is expected to grow even further from generics entering the market. Prefaced by a brief description of clopidogrel metabolism, this review analyzes analytical methods addressing the quantification of clopidogrel and its metabolites in biological samples. Techniques that have been applied to analyze human plasma or serum are predominantly LC–MS and LC–MS/MS. The lowest level of clopidogrel quantification that has been achieved is 5 pg/mL, the shortest runtime is 1.5 min and almost 100% recovery has been reported using solid-phase extraction for sample preparation.
Keywords: Clopidogrel; Clopidogrel carboxylic acid; Clopidogrel active metabolite; Quantification; Review;

Identification and quantification of drug–albumin adducts in serum samples from a drug exposure study in mice by L. Switzar; L.M. Kwast; H. Lingeman; M. Giera; R.H.H. Pieters; W.M.A. Niessen (53-61).
► A generic strategy was developed for the analysis of drug–albumin adducts in serum. ► Identification and localization of low-abundant NAPQI-albumin adducts in mouse serum. ► Quantification of low levels of drug–protein adduct formation in vivo.The formation of drug–protein adducts following the bioactivation of drugs to reactive metabolites has been linked to adverse drug reactions (ADRs) and is a major complication in drug discovery and development. Identification and quantification of drug–protein adducts in vivo may lead to a better understanding of drug toxicity, but is challenging due to their low abundance in the complex biological samples. Human serum albumin (HSA) is a well-known target of reactive drug metabolites due to the free cysteine on position 34 and is often the first target to be investigated in covalent drug binding studies. Presented here is an optimized strategy for targeted analysis of low-level drug–albumin adducts in serum. This strategy is based on selective extraction of albumin from serum through affinity chromatography, efficient sample treatment and clean-up using gel filtration chromatography followed by tryptic digestion and LC–MS analysis. Quantification of the level of albumin modification was performed through a comparison of non-modified and drug-modified protein based on the relative peak area of the tryptic peptide containing the free cysteine residue. The analysis strategy was applied to serum samples resulting from a drug exposure experiment in mice, which was designed to study the effects of different acetaminophen (APAP) treatments on drug toxicity. APAP is bioactivated to N-acetyl-p-benzoquinoneimine (NAPQI) in both humans and mice and is known to bind to cysteine 34 (cys34) of HSA. Analysis of the mouse serum samples revealed the presence of extremely low-level NAPQI-albumin adducts of approximately 0.2% of the total mouse serum albumin (MSA), regardless of the length of drug exposure. Due to the targeted nature of the strategy, the NAPQI-adduct formation on cys34 could be confirmed while adducts to the second free cysteine on position 579 of MSA were not detected.
Keywords: Drug–protein adducts; Adverse drug reaction (ADR); Serum albumin; NAPQI; Protein quantification; LC–MS(/MS);

Determination of tissue distribution of potent antitumor agent ureidomustin (BO-1055) by HPLC and its pharmacokinetic application in rats by Shin-I. Chien; Jiin-Cherng Yen; Rajesh Kakadiya; Ching-Huang Chen; Te-Chang Lee; Tsann-Long Su; Tung-Hu Tsai (62-70).
► Ureidomustin hydrochloride (BO-1055) was designed as a water-soluble nitrogen-mustard. ► An HPLC with photodiode array was used to perform the preclinical pharmacokinetics of ureidomustin. ► Ureidomustin appeared to be a two-compartment model in the rats. ► The results demonstrate a rapid distribution and a slow elimination after ureidomustin administration.Ureidomustin hydrochloride (BO-1055) was designed as a water-soluble nitrogen-mustard, which exhibited potent anticancer activity and was selected as a candidate for preclinical studies. However, up to date, there is rarely an easy and economic method to quantize ureidomustin in the biological samples. The aim of this study is to develop a simple yet valid quantization method to tackle this challenge. Here we present a combined high-performance liquid chromatography with photodiode array (HPLC-PDA) method in quantizing the ureidomustin in the plasma and various organs of Sprague-Dawley rats. The method was validated in terms of precision, accuracy, and extraction recovery. Furthermore, the established method was applied to study pharmacokinetics of ureidomustin in the rat's plasma and verified via a liquid chromatography tandem mass spectrometry (LC–MS/MS) method. Calibration curves of the plasma and organ samples were falling at the range between 0.5–50 μg/mL and 0.1–50 μg/mL (r 2  ≥ 0.999 and CV ≤ ±15%), respectively. The limits of detection (LOD) were 0.1 μg/mL for plasma samples and 0.05 μg/mL for organ samples, while the detection limits of quantification (LOQ) were 0.5 μg/mL for plasma samples and 0.1 μg/mL for organ samples. The average recovery of ureidomustin was about 83%. These results demonstrated a linear pharmacokinetic pattern at dosages of 10 and 30 mg/kg. The pharmacokinetic data revealed that ureidomustin was best fitted to a two-compartment model with a rapid distribution phase and a slow elimination phase. Besides, after a short intravenous administration time at the dose of 10 mg/kg, ureidomustin was found to be quickly distributed to all organs in rats, accumulated mainly in the kidney, and only a limited amount was detected in the brain.
Keywords: Alkylating agent; Bioassay; Organ distribution; Pharmacokinetics;

► A QuEChERS-DLLME-UPLC-MS/MS has been developed for rapid determination of multiclass pesticide residues in ginseng. ► The procedure to increase the sensitivity was concentration by DLLME method instead of evaporation by nitrogen. ► This method was simple, rapid and cheap.A procedure involving acetonitrile-based extraction combined with dispersive liquid–liquid microextraction (DLLME) and detection by ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was used for determination of 39 pesticides in ginseng. The extraction of pesticide residues in ginseng was performed with acetonitrile, applying QuEChERS methodology, and the extract was further disposed by DLLME method before analyzed by UHPLC–MS/MS. The average recoveries ranged from 70 to 120% for 82% of the analytes with RSD lower than 15%. The calibration curves obtained with blank matrices were linear with a correlation coefficient of over 0.99. The limits of detection were between 0.01 and 1.0 μg/kg. Matrix effects were studied by comparing solvent calibration curves and matrix-matched calibration curves. The results indicate the feasibility of this method for the determination of 39 pesticides in ginseng.
Keywords: Pesticides; Ginseng; QuEChERS; DLLME; UHPLC–MS/MS;

An HPLC–MS/MS method for simultaneous determination of decitabine and its valyl prodrug valdecitabine in rat plasma by Youxi Zhang; Jin Sun; Yikun Gao; Ying Kong; Youjun Xu; Weiru Jia; Chuanrong Liao; Peng Zhang; He Lian; Xiaopeng Han; Dongpo Li; Yajie Geng; Zhonggui He (78-83).
► A C18 column (150 mm × 4.6 mm, 3.5 μm) was applied to obtain satisfactory separation. ► A cation exchange solid phase extraction provided high recovery and clean sample. ► The single analysis time was short and close to 15 min. ► The LLOQ was 10 ng/mL for decitabine using only 50 μL of rat plasma. ► The method was sufficiently sensitive to measure low concentration of decitabine.A simple and sensitive HPLC–MS/MS method was developed and validated for the simultaneous determination of decitabine and valdecitabine in rat plasma. The analytes were separated on a C18 column (150 mm × 4.6 mm, 3.5 μm) and a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was applied for detection. A clean solid-phase extraction procedure with cation exchange cartridge was employed to extract the analytes from rat plasma with high recovery of decitabine (>82%). The calibration curves were linear over a concentration range of 10–10,000 ng/mL for decitabine and 5–500 ng/mL for valdecitabine. The lower limit of quantitation (LLOQ) of decitabine and valdecitabine was 10 and 5 ng/mL, respectively. The intra-day and inter-day precisions were less than 15% and the relative error (RE) was all within ±15%. The validated method was successfully applied to a pharmacokinetics study in rats after either decitabine or valdecitabine orally administrated to the Sprague-Dawley rats.
Keywords: Decitabine; Valdecitabine; HPLC–MS/MS; Rat plasma; Simple solid-phase extraction; Pharmacokinetics;

► Simultaneous determination of Eleutheroside B and Eleutheroside E in rat plasma. ► Method was validated and can be successfully applied for pharmacokinetic study. ► Lower absolute bioavailability of Eleutheroside B and Eleutheroside E was observed. ► Difference in PK behavior between the single substances and an aqueous extract.Eleutheroside B and Eleutheroside E, two kinds of the major bioactive saponins of Eleutherococcus senticosus, play a pivotal role in biologic activity. In this study, a specific and sensitive high performance liquid chromatography–electrospray ionization-tandem mass spectrometry method (HPLC–MS/MS) was developed and validated for simultaneous determination of Eleutheroside B and Eleutheroside E in rat plasma. The analytes were extracted from rat plasma via a simple protein precipitation procedure with methanol and polygonin was used as internal standard. Chromatographic separation was achieved on a C18 column using a gradient elution program with acetonitrile and water containing 0.1% ammonium hydroxide solution as the mobile phase, with a flow rate of 0.2 mL/min. The detection was performed on a triple–quadrupole tandem mass spectrometer by multiple reactions monitoring (MRM) mode in a negative ion mode via electrospray ionization (ESI). The transition monitored were m/z 371 [M−H]  → 209 for Eleutheroside B, m/z 741[M−H]  → 579 for Eleutheroside E and m/z 389[M−H]  → 277 for internal standard. Linear calibration curves were obtained in the concentration range of 1–2000 ng/mL for both (Eleutheroside B and Eleutheroside E), with a lower limit of quantification of 1 ng/mL. Extraction recovery was over 80% in plasma. The intra- and inter-day precision (RSD) values were below 12% and accuracy (RE) was −2.80 to 5.70% at three QC levels for both. The assay was successfully applied to study pharmacokinetics behavior in rats after oral and intravenous administration of the single substances (Eleutheroside B and Eleutheroside E). And further research was performed by comparing the difference in pharmacokinetic behavior between the single substances and an aqueous extract of E. senticosus after oral administration. Significant difference in pharmacokinetic characteristics between the single substances and an aqueous extract was found in rat, which would be beneficial for the pre-clinical research and clinical use of E. senticosus.
Keywords: Eleutheroside B; Eleutheroside E; Eleutherococcus senticosus; LC–MS/MS; Pharmacokinetics;

► An HPLC–HG–AFS method for determination of 4 arsenic species in dog plasma was developed. ► The comparative pharmacokinetic study of arsenic species was carried out after oral administration of Realgar and NHJDP. ► DMA and As(V) were found to be the main arsenic species in dog plasma after oral administration of both Realgar and NHJDP. ► The concentration–time profiles and pharmacokinetic parameters of DMA in beagle dogs were presented.A high performance liquid chromatography–hydride generation–atomic fluorescence spectrometry (HPLC–HG–AFS) method was developed for the simultaneous determination of four arsenic species (As(III), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and arsenate As(V)) in dog plasma. Good separation of the four arsenic species was achieved within 15 min on an anion-exchange column with isocratic elution using 15 mmol/L KH2PO4 (pH 5.9) as eluent at a flow rate of 1.0 mL/min. The assay was linear over the range of 1.25–200, 1.56–200, 1.34–172, and 2.50–200 ng/mL with the detection limits of 0.80, 1.00, 0.86 and 2.00 ng/mL for As(III), DMA, MMA and As(V), respectively. The method was validated for selectivity, precision, accuracy and recovery and then applied to a comparative pharmacokinetic study of the arsenic species in beagle dogs after a single oral administration of Realgar (24.32 mg/kg, equivalent to 11.31 mg As/kg) alone or Niu Huang Jie Du Pian (a patent traditional Chinese medicine (TCM), 380 mg/kg, equivalent to 28.45 mg As/kg), respectively. DMA was found to be the predominant species in the dog plasma after dosing, with As(V) appeared as the quickly eliminating one. No traces of MMA and As(III) were detected at any sampling time points. The main pharmacokinetic parameters found for DMA p.o. administration of Realgar and Niu Huang Jie Du Pian were as follows: C max (14.7 ± 4.2) and (57.0 ± 32.0) ng/mL, T max (2.4 ± 0.5) and (2.5 ± 0.5) h, AUC0–36 (151.1 ± 12.9) and (635.9 ± 418.2) ng h/mL, AUC0–∞ (206.0 ± 44.5) and (687.2 ± 425.1) ng h/mL, t 1/2 (16.2 ± 7.9) and (9.4 ± 2.2) h, respectively. The influence of compounding in Niu Huang Jie Du Pian on the pharmacokinetics of arsenics was shown with increased transformation of DMA and its faster elimination rate.
Keywords: Realgar; Niu Huang Jie Du Pian; Liquid chromatography–hydride generation–atomic fluorescence spectrometry; Arsenic speciation; Dog plasma; Pharmacokinetics;

► A novel UPLC–MS/MS method was established and fully validated. ► It was used for simultaneous quantitation of 7 plasma sulfur mustard biomarkers. ► The method has been proved to be simple, rapid and sensitive. ► It is suitable for early monitoring sulfur mustard poisoning. ► The plasma profiles of these biomarkers in rats were reported for the first time.Sulfur mustard (SM) is a hazardous chemical warfare agent that has been used in several military conflicts. SM is also considered as a major threat to civilians because of its existing stockpiles and easy production. Analysis of exposure biomarkers in biological samples collected from suspected victims is a useful tool for early diagnosis of SM poisoning. In this study, a sensitive and rapid quantitative method with ultra high performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for simultaneous determination of seven SM plasma biomarkers, including its oxidative, hydrolysis and β-lyase metabolites. A simple one-step protein precipitation with acetonitrile–methanol (4:1) was used for sample preparation. A full validation was conducted with respect to specificity, linearity, recovery, matrix effect, precision, accuracy and stability. The lower limits of quantification for the seven metabolites ranged from 0.01 μg L−1 to 5 μg L−1. The intraday relative standard deviation was less than 7.0%, and the interday deviation was less than 9.1%. The recoveries varied in the range from 82.8% to 118%. This method has been successfully applied to a toxicokinetic study for obtaining the plasma profiles of seven metabolites in SM-exposed rats, following a single subcutaneous dose of 3.3 mg kg−1. All the targeted compounds were detected in rat plasma. bis-β-Chloroethyl sulfoxide (SMO), thiodiglycol (TDG), thiodiglycol sulfoxide (TDGO), 1,1′-sulfonylbis-[2-S-(N-acetylcysteinyl)ethane (SBSNAE), 1,1′-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) were found to be the major metabolites in rat plasma. The time windows for the detection of these metabolites were varied in the range of 5 min to 48 h after exposure. The method provides a useful tool for short-term diagnosis of SM poisoning.
Keywords: UPLC–MS/MS; Sulfur mustard; Biomarker; Metabolite; Plasma; Quantification; Toxicokinetics;