Journal of Chromatography B (v.945-946, #C)

To investigate the pharmacokinetics of silibinin and silibinin hemisuccinate in human plasma, two high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) methods were developed and validated. The methods require a small volume of sample (100 μL), and the recovery of the analytes was complete with a good reproducibility (CV% 1.7–9.5), after a simple protein precipitation. Naringenin was used as internal standard. The chromatographic methods provided a good separation of diastereoisomers A and B of both silibinin and silibinin hemisuccinate onto a Chromolith Performance RP18e 100 mm × 3 mm column, with a resolution of peaks from plasma matrix in less than 6 min. The methods precision values expressed as CV% were always ≤6.2% and the accuracy was always well within the acceptable 15% range. Quantification was performed on a triple-quadrupole tandem mass spectrometer by Selected Reaction Monitoring (SRM) mode, in a negative ion mode, via electrospray ionization (ESI). The lower limit of quantitation was set at 5.0 ng/mL (silibinin) and 25.0 ng/mL (silibinin hemisuccinate), and the linearity was validated up to 1000.0 and 12,500.0 ng/mL, for silibinin and silibinin hemisuccinate, respectively, with correlation coefficients (R 2) of 0.991 or better. The methods were suitable for pharmacokinetic studies and were successfully applied to human plasma samples from subjects treated intravenously with Legalon® SIL at the dose of 20 mg/kg, expressed as silibinin.
Keywords: Silibinin; Silibinin hemisuccinate; HPLC–MS/MS; Validation; Pharmacokinetics;

Tyrosinase immobilized enzyme reactor: Development and evaluation by Karina Bora de Oliveira; Keylla Lençone Mischiatti; José Domingos Fontana; Brás Heleno de Oliveira (10-16).
Immobilized enzyme reactors of tyrosinase (tyr-IMERs) for use on-line in HPLC system were prepared by different procedures and then compared. The enzyme, obtained from Agaricus bisporus, was immobilized on epoxy-silica which was prepared using different conditions. Enzyme immobilization was conducted by both in situ and in batch techniques. The different procedures were compared in terms of protein and activity retention, IMERs activity, kinetics and stability. The influence of immobilization procedure on enzyme activity and the behavior of the IMERs against a standard inhibitor were also investigated. In situ immobilization on epoxy-silica, synthesized using microwave assistance, provided the best conditions to prepare tyrosinase IMERs. The tyr-IMERs were successfully tested with known and potential inhibitors of tyrosinase, and the results showed that they can be used for the screening of inhibitors of that enzyme.
Keywords: Tyrosinase; Agaricus bisporus; Immobilized enzyme reactor; Enzyme immobilization;

Matrine (MT), oxymatrine (OM) and sophoridine (SP) are three bioactive alkaloids in Sophora alopecuroides. In the present study, the chromatographic characteristics of six widely used macroporous resins, namely NKA, NKA-9, HPD-100, HPD-722, HPD-750, and AB-8, respectively, towards the separation and enrichment of the three alkaloids from the aqueous extract of S. alopecuroides are critically evaluated. The results indicated that AB-8 resin offered the best absorption and desorption capacity and its adsorption data fitted best to the Freundlich isotherm. Dynamic adsorption and desorption experiments on packed columns of AB-8 resin have been investigated for optimization of chromatographic parameters. The adsorption of the alkaloids on the resin was best achieved by 5 bed volume (BV) of sample solution of pH 10 with a flow rate of 2 BV/h. The desorption of the compounds from the resin was effectively completed by using 5 BV of 80% ethanol in water at a flow rate of 2 BV/h. After one run of adsorption and desorption, the contents of MT, OM, and SP were increased from 9.30, 8.39 and 9.84% to 22.22, 21.44 and 28.02%, the recovery were 69.4, 78.3 and 72.6%, respectively. This method would provide useful information to the industrial production of the alkaloids from S. alopecuroides
Keywords: Sophora alopecuroides; Macroporous resin; Matrine; Oxymatrine; Sophoridine; Separation and enrichment;

A novel analytical approach based on molecularly imprinted solid phase extraction (MISPE) coupled with dispersive liquid–liquid microextraction (DLLME), and injector port silylation (IPS) has been developed for the selective preconcentration, derivatization and analysis of 3-phenoxybenzoic acid (3-PBA) using gas chromatography–tandem mass spectrometry (GC–MS/MS) in complex biological samples such as rat blood and liver. Factors affecting the synthesis of MIP were evaluated and the best monomer and cross-linker were selected based on binding affinity studies. Various parameters of MISPE, DLLME and IPS were optimized for the selective preconcentration and derivatization of 3-PBA. The developed method offers a good linearity over the calibration range of 0.02–2.5 ng mg−1 and 7.5–2000 ng mL−1 for liver and blood respectively. Under optimized conditions, the recovery of 3-PBA in liver and blood samples were found to be in the range of 83–91%. The detection limit was found to be 0.0045 ng mg−1 and 1.82 ng mL−1 in liver and blood respectively. SRM transition of 271 → 227 and 271 → 197 has been selected as quantifier and qualifier transition for 3-PBA derivative. Intra and inter-day precision for five replicates in a day and for five, successive days was found to be less than 8%. The method developed was successfully applied to real samples, i.e. rat blood and tissue for quantitative evaluation of 3-PBA. The analytical approach developed is rapid, economic, simple, eco-friendly and possess immense utility for the analysis of analytes with polar functional groups in complex biological samples by GC–MS/MS.
Keywords: Molecularly imprinted polymer; Dispersive liquid–liquid microextraction; Injector port silylation; Gas chromatography–tandem mass spectrometry;

Liquid chromatography and ion trap mass spectrometry for simultaneous and multiclass analysis of antimicrobial residues in feed water by Chusak Ardsoongnearn; Ongart Boonbanlu; Sunan Kittijaruwattana; Leena Suntornsuk (31-38).
This work firstly reported the development of liquid chromatography coupled to an ion trap mass spectrometer (LC–MS ion trap) for the simultaneous determination of nitrofurans (e.g. nitrofurazone (NFZ), nitrofurantoin (NFT), furazolidone (FZD) and furaltadone (FTD)), nitroimidazoles (e.g. metronidazole (MNZ), ronidazole (RNZ) and dimetridazole (DMZ)) and chloramphenicol (CAP) in feed water. Isotope-labeled internal standards for the corresponding target analytes were employed to prevent matrix effects that might lead to signal suppression/enhancement. High performance liquid chromatography (HPLC) analysis was performed on a Prodigy ODS-3 column, 2.0 mm × 150 mm, 5 μm with a guard cartridge at a flow rate of 0.2 mL/min, column oven temperature of 40 °C, and an injection volume of 10 μL. Solid phase extraction (SPE) procedures, factors affecting HPLC separation (e.g. buffer pH and concentrations) and mass spectrometry (MS) parameters were optimized. After an off-line SPE by the OASIS HLB cartridges (with an enrichment factor of 400), the eight antimicrobial agents were separated in 18 min using a gradient elution of acetonitrile in acidified water (pH 5.0). MS detection was by an ion trap MS coupled with electrospray ionization (ESI) in tandem mass spectrometry mode (MS/MS) using the nebulizer gas at 35 psi, drying gas at 9 L/min and drying temperature of 325 °C. Method linearity was good (r 2  = 0.979–0.999) with acceptable precision (% RSDs = 3.4–26.6%) and accuracy (%recovery = 88.4–110.1%). Very low limits of detection (LOD) and quantitation (LOQ) were achieved in ranges of 0.002–0.06 μg/L and 0.005–0.25 μg/L, respectively. The established method is successfully employed by the Department of Livestock Development of Thailand for the monitoring of the drug residues in feed waterbecause of its convenience, reliability and high sensitivity.
Keywords: Nitrofurans; Nitroimidazoles; Chloramphenicol; HPLC-MS; Feed water;

Efficient conversion of myricetin from Ampelopsis grossedentata extracts and its purification by MIP-SPE by Shian Zhong; Yanyue Kong; Ling Zhou; Chengyun Zhou; Xiaona Zhang; Yan Wang (39-45).
In this study, we developed an efficient conversion process of dihydromyricetin to myricetin from Ampelopsis grossedentata extracts. The content of myricetin increased from 2.38% to 85.57%, demonstrating the successful dehydrogenation of dihydromyricetin. Molecularly imprinted polymers (MIPs) were prepared by surface imprinting method using silica microspheres as the support matrices and myricetin as template. The MIPs were applied for the selective adsorption of myricetin. The chemical structure of the MIPs was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Static, dynamic and selective adsorption experiments showed that the MIPs exhibited good adsorption ability, rather fast template rebinding kinetics, and appreciate selectivity over structurally related compounds. Accordingly, the MIPs were applied as the selective sorbent in SPE to purify myricetin obtained through dehydrogenation, followed by HPLC-UV analysis. The recoveries of myricetin and dihydromyricetin were 92.7% and 55.6%, respectively. This study demonstrates the feasibility of using the developed MIP-SPE method to purify and enrich myricetin in the natural products.
Keywords: Ampelopsis grossedentata; Dehydrogenation; Molecularly imprinted microspheres; Myricetin; SPE;

A novel and sensitive solid phase extraction method based on the adsorption of cetyltrimethylammonium bromide on the surface of Fe3O4 nanoparticles was developed for extraction and preconcentration of ultra-trace amounts of mefenamic acid in biological fluids. The remarkable properties of Fe3O4 nanoparticles including high surface area and strong magnetization were utilized in this SPE procedure so that a high enrichment factor (98) and satisfactory extraction recoveries (92–99%) were obtained using only 50 mg of magnetic adsorbent. Furthermore, a fast separation time (about 15 min) was achieved for a large sample volume (200 mL) avoiding time-consuming column-passing process of conventional SPE. A comprehensive study on the parameters effecting the extraction recovery such of the amount of surfactant, pH value, the amount of Fe3O4 nanoparticles, sample volume, desorption conditions and ionic strength were also presented. Under the optimum conditions, the method was linear in the 0.2–200 ng mL−1 range and good linearity (r 2  > 0.9991) was obtained for all calibration curves. The limit of detection was 0.097 and 0.087 ng mL−1 in plasma and urine samples, respectively. The relative standard deviation (RSD %) for 10 and 50 ng mL−1 of the analyte (n  = 5) were 1.6% and 2.1% in plasma and 1.2% and 1.9% in urine samples, respectively. Finally, the method was successfully applied to the extraction and preconcentration of mefenamic acid in human plasma and urine samples.
Keywords: Magnetic nanoparticles; Mixed hemimicelles; Mefenamic acid; Solid phase extraction; HPLC;

Headspace solid-phase microextraction (HS-SPME) combined with gas chromatography/mass spectrometry (GC/MS) method was applied for the investigation of low molecular weight volatile organic metabolites (VOMs) in pleural effusion samples. Three important HS-SPME experimental parameters that influence extraction efficiency (fiber coating, extraction time and temperature of sampling) were optimized by a univariate optimization design. The highest extraction efficiency was obtained when sampling was performed at 50 °C for 10 min under agitation using a carboxen/polydimethylsiloxane (CAR/PDMS) fiber. A total of 36 volatile metabolites belonging to nine distinct chemical classes were identified in 40 pleural effusion samples (20 malignant effusions from lung cancer patients and 20 benign effusions from inflammatory patients). Ketones, alcohols, and benzene derivatives were the main chemical classes for the metabolomic profile of malignant effusions. The average peak areas of ketones and alcohols were much higher in malignant group compared to benign group. Together with phenols, they exhibit significant differences (P  < 0.05) between the two groups. Particularly, the average peak areas of cyclohexanone and 2-ethyl-1-hexanol in malignant effusions were significantly higher than those in benign ones. Furthermore, of the 36 identified metabolites, 5 compounds including cyclohexanone and 2-ethyl-1-hexanol were found to be statistically different (Student's t-test, P  < 0.05) between the two groups by statistical analysis based on the peak areas of all identified metabolites. Among them, cyclohexanone and 2-ethyl-1-hexanol might be considered as candidate biomarkers of lung cancer to differentiate malignant from benign effusions. The results show that HS-SPME-GC/MS is a simple, rapid, sensitive and solvent-free method for the determination of VOMs in pleural effusion samples. Pleural effusion is a valuable sample source for observation of changes in VOMs for differentiation between lung cancer patients and inflammatory individuals.
Keywords: Gas chromatography/mass spectrometry; Solid-phase microextraction; Volatile organic metabolites (VOMs); Pleural effusions; Lung cancer;

Relationship study of partition coefficients between ionic liquid and headspace for organic solvents by HS-GC by Meiping Ni; Ting Sun; Lin Zhang; Yan Liu; Meng Xu; Ye Jiang (60-67).
A general study was carried out to investigate the relationship between analytes (organic solvents) and matrix medium (ionic liquids, ILs) by headspace gas chromatography (HS-GC) in order to provide a guidance to choose a suitable matrix medium during the process of experiment. Thirteen ILs contained different cations or anions and two kinds of organic solvents (alkylogens and aprotic solvents which involved ability of pro-proton) performed different interactions with ILs were chosen in this study. The concentrations of analytes in headspace were determined by HS-GC and then logK (the logarithm of concentration radio between matrix medium and headspace) was calculated respectively. Factors which affect logK, such as logP O/W (the logarithm of the octanol/water partition coefficient for a solvent) for different cations (including parent nucleus and alkyl chains) and anions of ILs, were investigated. The results indicated that the longer alkyl chains, the lower polarity of parent nucleus and the higher polarity of anions performed the higher headspace efficiency for alkylogens. Meanwhile, the shorter alkyl chains and the lower polarity of parent nucleus make the higher headspace efficiency for aprotic solvents which involved ability of pro-proton. For both kinds of organic solvents, anions of ILs performed little influences to headspace efficiency. The relationship between ILs and organic solvents was primarily investigated and a helpful guidance was provided for the application of ILs as matrix medium to analyze solvents by HS-GC. The model was successfully used to determine the organic residual solvents in ketoconanzale to choose a suitable ionic liquid during the process of HS-GC.
Keywords: HS-GC; ILs; Partition coefficients; Organic solvents;

Enrichment and purification of total flavonoids from Flos Populi extracts were studied using five macroporous resins. The static tests indicated that NKA-9 resin was appropriate and its adsorption data were well fitted to the Langmuir and Freundlich isotherms. To optimize the separation process, dynamic adsorption and desorption tests were carried out. The optimal adsorption parameters were initial concentrations in sample solution of 7.64 mg/mL, pH of 5.0, sample loading amount of 2.3 BV, flow rate of 2 BV/h, temperature of 25 °C. The optimal desorption parameters were deionized water and 20% ethanol each 5 BV, then 60% ethanol of 10 BV, flow rate of 2 BV/h. After one run treatment with NKA-9 resin, the content of total flavonoids in the product increased from 11.38% to 53.41%, and the recovery yield was 82.24%. The results showed that NKA-9 resin revealed a good ability to enrichment total flavonoids from Flos Populi, and the method can be referenced for the enrichment of total flavonoids from other materials. The antioxidant activities of the purified flavonoids were further evaluated in vitro. It showed that the DPPH radical scavenging increased from 59.46% to 82.63% at different concentrations (0.06–0.14 mg/mL). At different concentrations (0.6–1.4 mg/mL), the hydroxyl radical scavenging increased from 35.39% to 74.12%. Moreover, the reducing ability and total oxidant capacity appeared to be dose-dependent of flavonoids. It indicated that the purified flavonoids can be used as a source of potential antioxidant.
Keywords: Total Flavonoids; Flos Populi; Macroporous resins; Enrichment; Antioxidant activity;

A new method was developed to determine polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in fishery and aquaculture products. Samples were extracted by an accelerated solvent extraction system and cleaned up by sequential solid phase extraction (SPE) including dispersive SPE (D-SPE) and tandem SPE. PBDEs and PCBs were analyzed by a large-volume injection gas chromatography triple quadrupole mass spectrometry (LVI-GC–QqQ-MS/MS). Good linearity (R 2  ≥ 0.9958) was achieved. Method detection limits (MDLs) were 0.16–3.3 pg g−1 (wet weight, ww) for PBDEs and 0.13–0.97 pg g−1ww for PCBs. Mean recoveries were 60–140% with relative standard deviations (RSDs) of less than 20% in weever fish, scallop and shrimp samples spiked at a lower level of 13–31 pg g−1  ww and a higher level of 50–125 pg g−1  ww. Certified reference materials were analyzed with acceptable results. The method reduced solvent consumption, analytical time and labor, and is suitable for the routine analysis of PBDEs and PCBs in fishery and aquaculture products.
Keywords: POPs; LVI-GC–QqQ-MS/MS; Tandem SPE; Food safety;

Development of a method for the analysis of drugs of abuse in vitreous humor by capillary electrophoresis with diode array detection (CE–DAD) by Jose Luiz Costa; Andre Ribeiro Morrone; Rodrigo Ribeiro Resende; Alice Aparecida da Matta Chasin; Marina Franco Maggi Tavares (84-91).
This work presents the development of an analytical method based on capillary electrophoresis with diode array detection for the analysis of drugs of abuse and biotransformation products in vitreous humor. Composition of the background electrolyte, implementation of an online pre-concentration strategy and sample preparation procedures were objects of study. The complete electrophoretic separation of 12 analytes (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), ketamine, cocaine, cocaethylene, lidocaine, morphine, 6-monoacetylmorphine and heroin) and the internal standard N-methyl-1-(3,4-methylenedioxyphenyl)-2-butamine (MBDB) was obtained within 13 min of run. The method was validated presenting good linearity (r 2  > 0.99), recovery ≥90%, precision better than 12% RSD and acceptable accuracy in the range of 86–118% at three concentration levels (50, 100 and 500 ng/mL). LODs and LOQs in the order of 1–5 ng/mL and 5–10 ng/mL, respectively, were obtained. After validation, the method was applied to eighty-seven vitreous humor samples and the results were compared to those obtained by a liquid chromatography tandem mass spectrometry (LC-MS/MS) screening method, routinely used by the forensic toxicology laboratory of the Sao Paulo State Police, Brazil. Cocaine was detected in 7.1%, cocaethylene in 3.6%, lidocaine in 2.4% and ketamine in 1.2% of the total number of analyzed samples.
Keywords: Capillary zone electrophoresis; Vitreous humor; Forensic toxicology; Cocaine; Liquid chromatography–mass spectrometry;

Characterization of aroma compounds of Chinese famous liquors by gas chromatography–mass spectrometry and flash GC electronic-nose by Zuobing Xiao; Dan Yu; Yunwei Niu; Feng Chen; Shiqing Song; Jiancai Zhu; Guangyong Zhu (92-100).
Aroma composition of five Chinese premium famous liquors with different origins and liquor flavor types was characterized by gas chromatography–mass spectrometry (GC–MS) and flash gas chromatographic electronic nose system. Eighty-six aroma compounds were identified, including 5 acids, 34 esters, 10 alcohols, 9 aldehydes, 4 ketones, 4 phenols, and 10 nitrous and sulfuric compounds. To investigate possible correlation between aroma compounds identified by GC–MS and sensory attributes, multivariate ANOVA-PLSR (APLSR) was performed. It turned out that there were 30 volatile composition, ethyl acetate, ethyl propanoate, ethyl 2-methyl butanoate, ethyl 3-methyl butanoate, ethyl lactate, ethyl benzenacetate, 3-methylbutyl acetate, hexyl acetate, 3-methyl-1-butanol, 1-heptanol, phenylethyl alcohol, acetaldehyde, 1,1-diethoxy-3-methyl butane, furfural, benzaldehyde, 5-methyl-2-furanal, 2-octanone, 2-n-butyl furan, dimethyl trisulfied and 2,6-dimethyl pyrazine, ethyl nonanoate, isopentyl hexanoate, octanoic acid, ethyl 5-methyl hexanoate, 2-phenylethyl acetate,ethyl oleate, propyl hexanoate, butanoic acid and phenol, ethyl benzenepropanoate, which showed good coordination with Chinese liquor characteristics. The multivariate structure of this electronic nose responses was then processed by principal component analysis (PCA) and hierarchical cluster analysis (HCA). According to the obtained results, GC–MS and electronic nose can be used for the differentiation of the liquor origins and flavor types.
Keywords: Chinese liquor; Aroma compounds; Gas chromatography–mass spectrometry; Electronic nose; PCA; HCA;

A contrast between the analytical performance characteristics using gas chromatography–mass spectrometry (GC–MS) liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography–ultraviolet (LC–UV) detection for the determination of the antiretroviral drug (ARV) nevirapine (NVP) in fortified human plasma after QuEChERS extraction has been made. Analytical performance characteristics, i.e. linearities, instrument detection limits (IDLs), limits of quantitation (LOQs), method detection limits (MDLs), % mean recoveries and the corresponding relative standard deviations (%RSDs) were estimated using techniques above. Using GC–MS, the correlation coefficients (r 2) were ≥0.990, which were deemed acceptable linearities. The MDLs ranged between 11.1–29.8 μg/L and 13.7–36.0 μg/L using helium and hydrogen carrier gases respectively. The LOQs ranged between 16.5–66.7 μg/L and 28.4–98.7 μg/L using helium and hydrogen carrier gases respectively with a % mean recovery of 83% and %RSD of 4.6%. Using LC–MS and LC–UV, the correlation coefficients (r 2) were ≥0.990. The MDLs were ranged between 3.14 and 47.1 μg/L. The LOQs ranged between 2.85 and 90.0 μg/L respectively. The MDLs using GC–MS, LC–MS and LC–UV were below the therapeutic range for NVP in human plasma is considered to be between 2300 μg/L (C min) and 8000 μg/L (C max). This study also demonstrated that helium can be substituted with hydrogen which is relatively cheaper and easily obtainable even by use of a generator.
Keywords: Nevirapine; GC–MS; LC–MS; LC–UV; Therapeutic drug monitoring;

An ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method has been developed for the simultaneous determination of four β-lactam antibiotics (amoxicillin, ampicillin, cefotaxime, and cefoperazone) and two β-lactamase inhibitors (tazobactam, sulbactam) in bovine milk. The analytes were extracted with water from bovine milk and purified with Oasis HLB solid phase extraction (SPE) cartridges. The analytes were determined in less than 3 min by UPLC-MS/MS in positive and negative electrospray ionization (ESI) modes, separately. The method was linear over the range of 1–100 μg/L for tazobactam, sulbactam, ampicillin, and cefoperazone, and 2–100 μg/L for amoxicillin and cefotaxime. The recoveries for all six analytes in bovine milk ranged from 82.5 to 98.3%. The limits of detection and the limits of quantitation were 0.1–0.2 μg/L and 0.3–0.5 μg/L, respectively. The intra- and inter-day precisions were less than 6% for each compound.
Keywords: β-Lactam antibiotics; β-Lactamase inhibitors; Ultra-high performance liquid chromatography; Tandem mass spectrometry; Bovine milk;

An automated two-dimensional method using Protein A chromatography followed by size exclusion HPLC was developed for analysis of aggregates of monoclonal antibodies in mammalian cell culture samples. The method development was intended to address the analysis of IgG2 monoclonal antibody products that are particularly prone to aggregation at pH < 3.5. An aqueous solution of sodium phosphate at pH 4.3 containing arginine was demonstrated in this work as an excellent eluent for Protein A chromatography. In addition, the arginine solution is a compatible mobile phase for the analysis of these samples by size exclusion HPLC, separating aggregates from the monomer of the monoclonal antibody. The effect of arginine concentration in the eluent on parameters such as protein recovery from Protein A chromatography and resolution of aggregates from the monomer are reported. The developed method was shown to provide accuracy of reported aggregates greater than 98%, and intermediate precision of 4.4% RSD. The method limit of quantitation for aggregates was determined to be 0.1%. Application of the method is demonstrated for analysis of aggregates in cell culture samples to aid in the development of cell culture conditions for the production of antibodies.
Keywords: Monoclonal antibody; Aggregates; Arginine; Protein A chromatography; Size-exclusion HPLC;

A simple, novel, rapid and sensitive supercritical fluid chromatography–tandem mass spectrometry (SFC-MS/MS) method was developed and validated for the determination of lacidipine in beagle dog plasma with nimodipine as internal standard. The method involved a simple liquid–liquid extraction method with tert-butyl methyl ether. The analytes were analyzed on an Acquity UPC2 with a HSS C18 SB column (3 mm × 100 mm, 1.8 μm) set at 50 °C. The mobile phase was carbon dioxide (≥99.99%) and methanol (92:8, v/v) at a flow rate of 2 ml/min, the compensation solvent was methanol with 2% formic acid at a flow rate of 0.2 ml/min and a total analysis time of 1.5 min for each sample. The multiple reaction-monitoring mode was used for quantification of ion transitions at m/z 473.32 → 354.10 and 419.00 → 343.10 for lacidipine and internal standard, respectively. The linearity range of proposed method was 0.10–100 ng/ml) (r 2  ≥ 0.9990). The intra- and inter-day precision values were less than 15% and accuracy was from −0.83% to 3.27% at all quality control levels. The proposed method was successfully applied to a pharmacokinetic study of lacidipine in beagle dogs.
Keywords: Lacidipine; Plasma; SFC–MS/MS; Pharmacokinetic;

A novel molecularly imprinted organic–inorganic hybrid polymer (MI-MAA/APTS) based on a dummy molecular imprinting technique and an organic–inorganic hybrid material technique was synthesised and used as a sorbent in solid-phase extraction for the selective isolation and determination of ofloxacin (OFL), lomefloxacin (LOM), and ciprofloxacin (CIP) in tilapia samples. The MI-MAA/APTS sorbent was prepared from 3-aminopropyltriethoxysilanes (APTS) as an inorganic source and methacrylic acid (MAA) as an organic source and exhibited high mechanical strength and special affinities to the analytes. A comparison of MI-MAA/APTS with other conventional sorbents (C18 and HLB) showed that MI-MAA/APTS displayed good selectivity and affinity for OFL, LOM, and CIP, and the recoveries of the analytes at three spiked levels were in the range of 85.1–101.0%, with the relative standard deviations ≤5.1%. The presented MI-MAA/APTS-SPE–HPLC method could be potentially applied to the determination of fluoroquinolones (FQs) in complex fish samples.
Keywords: Organic–inorganic hybrid materials; Molecular imprinting; Solid-phase extraction; Fluoroquinolones; Tilapia;

Glycosylation is a major biochemical attribute of therapeutic proteins and detailed analyses including the structures and sites of such modifications are often required for product quality control and assurance. Using liquid chromatography and tandem mass spectrometry techniques, we analyzed the O-linked glycosylation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derived from glycoengineered Pichia pastoris with regard to its nature, structure, occupancy, and location. Peptide mappings using protease and chemical cleavages were performed to determine the specific O-linked glycosylation site used by Pichia-derived rhG-CSF. Our results demonstrated that Thr134, the equivalent O-linked glycosylation site found on endogenous human G-CSF, is the only site modified with a single mannose, allowing glycoengineered P. pastoris to be used as a viable production platform for therapeutic rhG-CSF.
Keywords: O-linked mannosylation; rhG-CSF; LC–MS; HPAEC-PAD;

A quantitative LC–MS/MS method for determination of thiazolidinedione mitoNEET ligand NL-1 in mouse serum suitable for pharmacokinetic studies by Kiran K. Pedada; Xiang Zhou; Harini Jogiraju; Richard T. Carroll; Werner J. Geldenhuys; Li Lin; David J. Anderson (141-146).
Thiazolidinedione (TZD) compounds have shown promise as antidiabetic, antibiotics, antifungal and neuroprotective agents. The mitochondrial effect of a novel mitoNEET ligand, NL-1 {5-[(3,5-di-tert-butyl-4-hydroxyphenyl)methyl]-1,3-thiazolidine-2,4-dione}, and other TZD compounds, is a newly proposed mechanism for the neuroprotective action of these TZD compounds. In this work, a sensitive LC–MS/MS assay has been developed and validated for quantification of NL-1 in mouse serum. Sample preparation involved an acetonitrile protein precipitation procedure with addition of an internal standard NL-2 {5-[(4-hydroxy-3,5-dimethyl-phenyl)methyl]thiazolidine-2,4-dione}. LC–MS/MS analysis utilized a Columbus C-18 HPLC column (2 mm × 50 mm, 5 μm). Chromatography employed a multiple step gradient program that featured a steep linear gradient (25–95% in 0.5 min) of 15 μM ammonium acetate (additive for eliminating carry-over) in 2% methanol mixing with increasing proportions of 100% methanol. The HPLC was interfaced to a QTrap 5500 mass spectrometer (AB Sciex) equipped with an electrospray ionization source used in a negative ionization mode. Multiple reaction monitoring (MRM) of m/z 334 → 263 for NL-1 and m/z 250 → 179 for NL-2 was done. The method had a linear range of at least 1–100 ng/mL in serum. The intra-assay and inter-assay percent coefficient of variation (%CV) were less than 4% and accuracies (%RE) ranged from −2.7% to 2.0%. The analytical procedure gave 96–115% absolute extraction recovery of NL-1. The relative matrix effect was measured and found to be insignificant. The analyte in serum was confirmed to be stable during storage and treatment. The method is suitable for pharmacokinetic (PK) studies of the parent drug NL-1 based on the preliminary serum results from dosed NL-1 mouse studies.
Keywords: NL-1; Thiazolidinedione (TZD); mitoNEET; LC–MS/MS; Carry-over; Mouse serum;

Simultaneous detection of green tea catechins and gallic acid in human serum after ingestion of green tea tablets using ion-pair high-performance liquid chromatography with electrochemical detection by Keiko Narumi; Jun-Ichiro Sonoda; Keita Shiotani; Michihiro Shigeru; Masayuki Shibata; Akio Kawachi; Erisa Tomishige; Keizo Sato; Toshiro Motoya (147-153).
We developed an analytical method for the simultaneous determination of tea catechins and gallic acid (GA) in human serum using ion-pair high-performance liquid chromatography (HPLC) with electrochemical detection. GA was measured to estimate the amount of gallate moiety produced by degradation of gallated catechins ((−)-epicatechin-3-gallate, ECG; (−)-epigallocatechin-3-gallate, EGCG). Ethyl gallate was adopted as an internal standard to correct for the extraction efficiency. To maximize extraction efficiency, a hydrophobic polytetrafluoroethylene (PTFE) filter was selected for pre-treatment prior to separation. HPLC separation was performed using a C18 reversed-phase column with a gradient mobile phase of phosphate buffer (pH 2.5) containing tetrahexylammonium hydrogensulfate as an ion-pair reagent. Using this method, (−)-epicatechin (EC), (−)-epigallocatechin (EGC), ECG, EGCG, ethyl gallate, and GA were detected as single peaks. The resolution values for target analytes were 4.0–13.0 and the mean values of the absolute recoveries of catechins and GA were 77.3–93.9%. The detection limits for catechins and GA in serum were 0.4–3.1 ng/mL. The serum catechin levels of eight healthy volunteers after ingestion of a single dose of green tea tablets were measured using this method. The concentration of total catechins (free + conjugated forms) in serum peaked 60 min after ingestion. From these results, this method is thought to enable the simultaneous quantification of GA, the hydrolysis product of gallated catechins, and target catechins, and to be sufficiently sensitive for pharmacokinetic studies of catechins following oral administration of green tea.
Keywords: Green tea catechins; Gallic acid; HPLC; Ion-pair reagent; Electrochemical detection; Pharmacokinetic study;

Pharmacokinetics and excretion study of sophoricoside and its metabolite in rats by liquid chromatography tandem mass spectrometry by Xuran Zhi; Ning Sheng; Lin Yuan; Zhiyong Zhang; Peipei Jia; Xiaoxu Zhang; Lantong Zhang (154-162).
In this study, a new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of sophoricoside and its metabolite genistein in rat plasma, bile, urine and feces after oral administration of sophoricoside, using sulfamethalazole as internal standard (IS). The separation was performed on a reverse phase C18 column with gradient elution consisting of 0.2‰ aqueous formic acid and methanol (containing 0.2‰ formic acid). The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.2/268.2 for sophoricoside, 268.7/133.0 for genistein and 252.0/156.0 for IS. This developed method provides good linearity (r  > 0.9983), intra- and inter-day precisions (RSD < 8.31%) with accuracies (RE, −6.91 to 6.66%), stability (RE, −7.45 to 6.59%), extract recovery (76.24 to 93.30%) and matrix effect (81.06–106.2%) of the analytes in plasma, bile, urine and feces. The mean elimination half-life (t 1/2) of sophoricoside and genistein were 59.78 ± 7.19 and 103.14 ± 16.97 min, respectively. The results showed that sophoricoside was rapidly absorbed and then eliminated from rat plasma. The total recoveries of sophoricoside in bile, urine and feces were about 0.0111%, 1.76% and 11.13%. The amounts excreted of genistein were 0.42 ± 0.02 μg in bile, 10.15 ± 0.22 μg in urine and 2.92 ± 0.13 μg in feces. This is the first report to evaluate the pharmacokinetics and excretion of sophoricoside and its metabolite in rats after oral administration of sophoricoside monomer. The results provided a meaningful basis for the clinical application of sophoricoside.
Keywords: Pharmacokinetics; Excretion; Sophoricoside; Metabolite; Genistein; LC–MS/MS;

Xanthine oxidase (XOD) immobilized core–shell magnetic silica (Fe3O4@SiO2-XOD) nanoparticles coupled with high performance liquid chromatography–mass spectrometry (HPLC–MS) was developed to fish out and analyze XOD binders from two Fabaceae species, Puerariae lobata flower and Glycyrrhiza uralensis root. The prepared Fe3O4@SiO2-XOD nanoparticles exhibited good specificity for XOD binders, better dispersion in aqueous solution and reusability than those of Fe3O4-XOD nanoparticles. The amount of XOD immobilized onto Fe3O4@SiO2 nanoparticles was 339.9 μg/mg and the activity of Fe3O4@SiO2-XOD nanoparticles remained 95% after ten times usage. The optimum conditions of selective fishing were optimized, and finally incubating pH was set at 7, incubating temperature at 25 °C and adsorption time at 30 min. Twelve XOD binders were successfully identified from ethyl acetate extract of P. lobata flower and G. uralensis root. The developed method provides a rapid, purposeful and effective way to identify active compounds from natural complex mixtures.
Keywords: Xanthine oxidase; Magnetic nanoparticles; Selective fishing; Puerariae lobata flower; Glycyrrhiza uralensis root; HPLC–MS;

Validation of an LC–MS/MS method for the quantification of mycophenolic acid in human kidney transplant biopsies by Zaipul I. Md Dom; Benjamin D. Noll; Janet K. Coller; Andrew A. Somogyi; Graeme R. Russ; Dennis A. Hesselink; Teun van Gelder; Benedetta C. Sallustio (171-177).
Mycophenolic acid (MPA) has a low therapeutic index and large inter-individual pharmacokinetic variability necessitating therapeutic drug monitoring to individualise dosing after transplantation. There is an ongoing discrepancy as to whether plasma MPA concentrations sufficiently predict kidney rejection or toxicity and whether immunosuppressant concentrations within the graft tissue may better predict transplant outcomes. The aim of the study was to develop an LC–MS/MS method for the quantification of MPA concentrations in human kidney biopsies taken as part of routine clinical procedures. A total of 4 surplus human kidney biopsies obtained from 4 different kidney transplant recipients were available to use for this study. MPA was also quantified in 2 kidney samples from rats administered MPA to assess tissue extraction reproducibility. Human kidney biopsies and rat kidneys were homogenised mechanically and underwent liquid–liquid extraction before analysis by LC–MS/MS. MPA-free human kidney tissue was used in calibrators and quality control samples. Analyte detection was achieved from multiple reaction monitoring of the ammonium adducts of both MPA (m/z 321.1 → 207.3) and N-phthaloyl-l-phenylalanine (PPA, internal standard, m/z 296.2 → 250.2) using positive electrospray ionisation. The method was linear (calibration curves R 2  > 0.99, n  = 10), precise, and accurate with coefficients of variation and bias less than 15%. Extraction efficiencies for MPA and PPA were approximately 97% and 86%, respectively, and matrix effects were minimal. In 4 kidney transplant recipients, tissue MPA concentrations ranged from 1.3 to 7.7 ng/mg of tissue, however, the correlation between blood (C 0) and tissue MPA concentrations could not be established. The method was successfully applied to the quantification of MPA in human kidney biopsies without the need to alter current clinical protocols.
Keywords: Mycophenolic acid; LC–MS/MS; Tissue MPA concentrations; Transplantation;

Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist that has been illegally used as an animal feed additive for growth promotion in China. In this study, an immunoaffinity chromatography (IAC) column for selective extraction of PA from swine feed, meat and liver samples was developed. The IAC column was constructed by covalently coupling specific polyclonal antibody (Ab) against PA to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, selectivity, extraction recovery and stability. The maximum capacity of the ICA for PA extraction was found to be 239.4 ng. For selectivity testing, 100 ng of other three β-adrenergic agonists (clenbuterol, ractopamine and salbutamol) was separately loaded onto the column, and it was observed that the tested compounds could not be captured on the column, e.g. the column could only selectively recognize PA. The recovery of the IAC for PA extraction was found within 96.47–101.98% when 10, 50 and 100 ng PA were separately loaded onto IAC column. The IAC column was also applied to real sample extraction. Swine feed, meat and liver samples were collected and spiked with PA in range of 1.0–20 ng g−1. The spiked and unspiked samples were extracted by IAC column and measured by high performance liquid chromatography (HPLC). It was found that there was no detectable PA in the blank samples, and the extraction recoveries of the IAC for PA from the spiked samples were within 89.48–104.89%. The stability of the column was also tested. It was showed that after 35 times repeated usage, 60% of the maximum capacity was still remained. The proposed IAC was proven to be a feasible extraction method for PA from different matrices with the properties of high maximum capacity, selectivity, extraction efficiency and stability.
Keywords: Immunoaffinity chromatography (ICA); Phenylethylamine A; Agonist; Column; HPLC;

Simultaneous determination of imperatorin and its metabolite xanthotoxol in rat plasma by using HPLC–ESI-MS coupled with hollow fiber liquid phase microextraction by Juan Zhang; Min Zhang; Shan Fu; Tao Li; Shuang Wang; Minmin Zhao; Weijing Ding; Chunying Wang; Qiao Wang (185-192).
The objective of the present study was to develop a new method for the simultaneous quantitation of imperatorin and its metabolite xanthotoxol in rat plasma. The samples were prepared with hollow fiber liquid phase microextraction (HF-LPME). The optimized extraction procedure was acquired by assessing extraction solvent, length of the fiber, agitation rate, extraction temperature and time. A comparison of sample pretreatment ways between HF-LPME and deproteinization with methanol was performed, which demonstrated less ion suppression and better sensitivity of HF-LPME. Analytes were separated on a C18 column with a gradient elution consisted of methanol and water containing 1 mmol/L ammonium acetate. The detection was accomplished by electrospray ionization (ESI) source operating in the positive ionization mode. Selected-multiple-reaction monitoring (SMRM) scanning was employed, which guaranteed a higher sensitivity compared with MRM mode. Calibration curves were linear over investigated ranges with correlation coefficients greater than 0.9979. Precision varied from 0.26% to 14%, and the accuracy varied within ±5.5%. The developed method was successfully applied to the pharmacokinetic research of imperatorin and its metabolite xanthotoxol after oral administration of imperatorin to rats.
Keywords: Imperatorin; Xanthotoxol; Hollow fiber liquid phase microextraction; Pharmacokinetics; HPLC–ESI-MS;

SYL-1119 is a sphingosine-1-phosphate receptor 1 modulator for the treatment of autoimmune disease with better selectivity, while SYL-1119-P is its active phosphate. A sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of SYL-1119 and SYL-1119-P in rat plasma. SYL-1110, an analogue of SYL-1119, was used as the internal standard. Plasma samples were prepared by protein precipitation using acetonitrile. The analytes and internal standard were separated on a Zorbax SB-C18 column (3.5 μm, 100mm × 2.1 mm) with a gradient mobile phase consisting of methanol and water containing 0.1% formic acid at a flow rate of 0.2 ml/min with an operating temperature of 20 °C. The detection was performed on a triple quadrupole tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode of the transitions at m/z 364 → 259 for SYL-1119, m/z 444 → 259 for SYL-1119-P, and m/z 378 → 273 for the IS. Calibration curves were linear in the range of 0.2–50 ng/ml for SYL-1119 and 10–1000 ng/ml for SYL-1119-P. The lower limit of quantification (LLOQ) was 0.2 ng/ml for SYL-1119 and 10 ng/ml for SYL-1119-P. The intra- and inter-day precisions were 5.4–12.8% for two analytes with accuracies within ±10%. The recoveries for two compounds were 91.3–104.5%. The analytes were proved to be stable during all sample storage, preparation, and analytic procedures. The method was successfully applied to the pharmacokinetic study of SYL-1119 and SYL-1119-P in rats after oral administration of SYL-1119.
Keywords: SYL-1119; SYL-1119-P; LC–MS/MS; Pharmacokinetic;

The steroids, hydrocortisone, androstenedione, 17-α-hydroxyprogesterone, testosterone, 17-α-methyltestosterone, and progesterone were separated with microemulsion electrokinetic chromatography (MEEKC) and detected with UV absorption. The microemulsion phases were prepared from both artificial and vegetable oils, from them the first was made of alkane and alcohol and the latter from colza, olive, linseed, and walnut oils. The electrolyte solutions were made to emulsions using sodium dodecyl sulfate and alkaline tetraborate. The solution mixtures made from ethyl acetate, sodium dodecyl sulfate, 1-butanol, acetonitrile, and sodium tetraborate were used as the reference solutions to evaluate the performance of the vegetable oil emulsions. Our study showed that the lipophilic organic phase in the microemulsion did provide resolution improvements but not selectivity changes. The results also correlate with real interactions of the steroids with the lipophilic organic microemulsion phase. The quality of the oils between the manufacturers did not have importance, which was noticed from the equal behavior of the steroids in the vegetable oil emulsions. Detection limits of the steroids in vegetable oil emulsions were at the level of 0.20–0.43 μg/L. Thus, they were 2–10 times higher than the concentrations in the partial filling micellar electrokinetic chromatography (PF-MEKC), which we have obtained earlier. The repeatability (RSD%) of the electrophoretic mobilities of the steroids was between 0.50 and 3.70. The RSD% values between the inter-day separations were below 1%, but when walnut and olive oils were used the values exceeded even 10%.
Keywords: Steroid; Vegetable oils; Separation; Microemulsion; Capillary electrophoresis;

Rapid and simultaneous quantitation of prostanoids by UPLC–MS/MS in rat brain by Jafar Sadik B. Shaik; Tricia M. Miller; Steven H. Graham; Mioara D. Manole; Samuel M. Poloyac (207-216).
The metabolites of arachidonic acid (AA) produced from the cyclooxygenase (COX) pathway, collectively termed as prostanoids, and from the CYP 450 pathway, eicosanoids, have been implicated in various neuro-degenerative and neuroinflammatory diseases. This study developed a quantitative UPLC–MS/MS method to simultaneously measure 11 prostanoids including prostaglandins and cyclopentenone metabolites in the rat brain cortical tissue. Linear calibration curves ranging from 0.104 to 33.3 ng/ml were validated. The inter-day and intra-day variance for all metabolites was less than 15%. The extraction recovery efficiency and matrix (deionized water) effects measured at 12.5 ng/ml (750 pg on column) ranged from 88 to 100% and 3 to 14%, respectively, with CV% values below 20%. Additionally, applying the processing and extraction conditions of this method to our previous CYP450 eicosanoids method resulted in overall improvement in extraction recovery and reduction in matrix effects at low (0.417 ng/ml) and high (8.33 ng/ml) concentrations. In rat brain cortical tissue samples, concentrations of prostanoids ranged from 10.2 to 937 pmol/g wet tissue and concentration of eicosanoids ranged from 2.23 to 793 pmol/g wet tissue. These data demonstrate that the successive measurement of prostanoids and eicosanoids from a single extracted sample of rat brain tissue can be achieved with a UPLC–MS/MS system and that this method is necessary for evaluation of these metabolites to delineate their role in various neuroinflammatory and cerebrovascular disorders.
Keywords: Arachidonic acid; Cyclooxygenase; Eicosanoids; Ischemic stroke; Prostanoids; UPLC–MS/MS;

A fully automated and robust method featuring on-line solid-phase extraction (SPE) and large volume injection (LVI) gas chromatographic (GC) high resolution mass spectrometry (HRMS) is used to determine polychlorinated biphenyls (PCBs) and organochlorine pesticides, such as penta- and hexachlorobenzene (PeCBz, HxCBz), hexachlorocyclohexane isomers (HCH) and 4,4′-dichlorodiphenyldichloroethene (a metabolite of dichlorodiphenyltrichloroethane (DDT)), with only 200 μl of human blood, serum or plasma. After spiking the sample with 13C-labeled internal standards and precipitating the proteins, the sample is passed through a 10 mm × 2.0 mm ID SPE cartridge filled with C18 material that adsorbs the analytes. After washing and drying, the cartridge is extracted with hexane/dodecane (99/1, v/v); the extract is directly injected into a LVI where GC/HRMS analysis follows. The fully automated system utilizes a robotic autosampler and a modular SPE system including two high-pressure syringe pumps, an automatic SPE cartridge exchanger unit and 6 switchable valves. All sample preparation steps are performed within 20 min during the GC run of a previous sample, limiting the throughput with only the GC runtime. The contents are quantified using the isotope dilution method. Due to laboratory air contamination problems, we achieved LOQs of 0.017 (PeCBz), 0.009 (HxCBz), 0.007 (HCH), 0.016 (DDE), while for the six indicator PCBs, we achieved values of 0.030 (PCB-28), 0.044 (PCB-52), 0.024 (PCB-101), 0.009 (PCB-138), 0.015 (PCB-153) and 0.008 (PCB-180) μg/l serum. Under clean laboratory air conditions, these values may be improved. This method is recommended when high throughput is desirable and/or only small amounts of material are available, such as during studies involving children.
Keywords: On-line solid phase extraction; Large volume injection; Gas chromatography; Mass spectrometry; Human blood; Polychlorinated biphenyls; Organochlorine pesticides;

A sensitive HPLC–MS/MS method for the determination of dolutegravir in human plasma by Chantelle Bennetto-Hood; Glenn Tabolt; Paul Savina; Edward P. Acosta (225-232).
A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of dolutegravir (DTG) in plasma samples. This work describes an assay system requiring only a 20 μL aliquot of human plasma that is subjected to a simple acetonitrile protein precipitation containing a stably labeled isotope of DTG used as an internal standard. Chromatography was performed on an XBridge C18, 2.1 mm × 50 mm, reversed phase analytical column, using a 60:40 acetonitrile/water mobile phase containing 0.1% formic acid. Detection of the analyte and internal standard was achieved by ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 420.1/136.0 and 428.1/283.1 for DTG and DTG-IS, respectively. The dynamic range of this assay extends from 5 to 10,000 ng/mL, with a mean coefficient of determination (r, mean ± SD) of 0.9996 ± 0.0003. The mean precision values for calibration standards ranged from 0.7 to 4.1%, while accuracy values were 98.3 to 102.0%. Validation results demonstrated high accuracy (≤6.5% deviation) and high precision (≤9.1% CV) for the quality control samples. This assay system provides an accurate, precise, and sensitive method for DTG quantitation and was successfully applied to clinical research samples as part of a phase I/II pediatric clinical trial.
Keywords: HIV; Integrase inhibitor; Mass spectrometry; Human; Dolutegravir;

A method for quantitation of β-lyase metabolites of sulfur mustard (SM) adducts with glutathione has been developed and validated using gas chromatography–tandem mass spectrometry (GC–MS/MS). The linear range of quantitation was 0.1–1000 ng/mL in urine with a method detection limit of 0.02 ng/mL. The method was applied in a rabbit exposure model. Domestic rabbits were cutaneously exposed to neat liquid SM in three dosage levels, and the β-lyase metabolites in urine were determined as 1,1′-sulfonylbis[2-(methylthio)ethane] (SBMTE). The study showed that even though more than 99% of the total amount of β-lyase metabolites was excreted in the first week after exposure, the β-lyase metabolites of SM adducts with glutathione could be detected in urine from rabbits for up to 3 or 4 weeks after the SM cutaneous exposure. For high dosage group (15 mg/kg, 0.15 LD50), the mean concentration of SBMTE detected was 0.32 ng/mL on day 28. For middle (5 mg/kg, 0.05 LD50) and low (2 mg/kg, 0.02 LD50) dosage groups, the mean concentrations of SBMTE were 0.07 ng/mL and 0.02 ng/mL on day 21, respectively. The data from this study indicate that the method is sensitive and provides a relatively long time frame for the retrospective detection of SM exposure.
Keywords: Gas chromatography–tandem mass spectrometry; Sulfur mustard; β-Lyase metabolites; Urine; Rabbit cutaneous exposure model;

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the estimation of gestodene in human plasma. Gestodene was extracted from human plasma by using solid-phase extraction technique. Gestodene D6 was used as the internal standard. An Acquity HSS-T3 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 326.2 → 124.1 for gestodene and m/z 332.3 → 129.1 for gestodene D6. The method involves a solid phase extraction from plasma, rapid derivatization with hydroxylamine to form oxime, simple gradient chromatographic conditions and mass spectrometric detection that enables detection at sub-picogram levels. The proposed method has been validated for a linear range of 50–11957 pg/ml with a correlation coefficient ≥ 0.9994. The intra-run and inter-run precision and accuracy were within 10%. The overall recoveries for gestodene and gestodene D6 were 62.02% and 67.57% respectively. The total run time was 4.0 min. The developed method was applied for the determination of the pharmacokinetic parameters of gestodene following a single oral administration of a 2 × 0.06 mg gestodene tablets in 10 healthy female volunteers.
Keywords: Gestodene; Hydroxylamine; Oxime derivative; LC–MS/MS; Human plasma;