Journal of Chromatography B (v.1002, #C)

For the production of bio active compounds, e.g., active enzymes or antibodies, a conserved purification process with a minimum loss of active compounds is necessary. In centrifugal partition chromatography (CPC), the separation effect is based on the different distribution of the components to be separated between two immiscible liquid phases. Thereby, one liquid phase is kept stationary in chambers by a centrifugal field and the mobile phase is pumped through via connecting ducts. Aqueous two phase systems (ATPS) are known to provide benign conditions for biochemical products and seem to be promising when used in CPC for purification tasks. However, it is not known if active biochemical compounds can “survive” the conditions in a CPC where strong shear forces can occur due to the two-phasic flow under centrifugal forces. Therefore, this aspect has been faced within this study by the separation of active laccases from a fermentation broth of Pleurotus sapidus. After selecting a suitable ATPS and operating conditions, the activity yield was calculated and the preservation of the active enzymes could be observed. Therefore, CPC could be shown as potentially suitable for the purification of bio-active compounds.
Keywords: Centrifugal partition chromatography (CPC); Aqueous two-phase systems (ATPS); Downstream processing; Laccase from Pleurotus sapidus; Activity preservation;

Permeation profiles of resveratrol cream delivered through porcine vaginal mucosa: Evaluation of different HPLC stationary phases by Hudson Caetano Polonini; Pedro Paulo Soldati; Priscila Aparecida de Almeida; Carla Grazieli Azevedo da Silva; Carol Hollingworth Collins; Marcone Augusto Leal de Oliveira; Anderson de Oliveira Ferreira; Nádia Rezende Barbosa Raposo; Marcos Antônio Fernandes Brandão (8-12).
Trans-resveratrol affects biological systems in a multitude of ways, but its oral bioavailability is remarkably poor due to in vivo metabolization. This drawback has fomented the development of new strategies for systemic delivery, such as transmucosal delivery via the vaginal route, which is our main focus here. In this sense, our pioneering study purposed to evaluate the trans-resveratrol permeation efficacy through this route. For that, we used a previously validated method and tested it with three different stationary phases: a commercial C18 column and two laboratory-made chromatographic columns containing poly(methyloctadecylsiloxane) (PMODS) thermally immobilized onto zirconized silica (Zr-PMODS) or titanized silica (Ti-PMODS). The permeation experiments showed that resveratrol, in the formulation used, was not successfully delivered to the bloodstream – it was actually retained within the vaginal mucosa, which suggests a local use rather a systemic one.
Keywords: Transmucosal absorption; Resveratrol; Laboratory-made HPLC columns;

The aim of this study is to develop a sensitive UPLC-MS/MS method to quantify columbin in biological sample. Chromatographic separation was accomplished using Waters UPLC BEH C18 column with acetonitrile and 0.1% of formic acid in water as the mobile phases. The mass analysis was performed on an API 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) with positive scan mood. The one-step protein precipitation by methanol was used to extract the analyte from blood samples. The results showed that the linear response range for columbin was 1.22–2,500 nM. The intra and inter day variances were less than 15% and the accuracy was in acceptable range (85–115%). The analysis was done within 3.0 min, and only 50 μL of blood was needed. The validated method was used to determine the pharmacokinetic profile of columbin in Wistar rats, and its transport characteristics in the Caco-2 cell culture model. The results showed that columbin was poorly bioavailable (2.8% p.o. and 14% i.p.) in rats, but its transport was rapid across the Caco-2 cell monolayers, suggesting that extensive first-pass metabolism in the liver was the likely reason for its poor bioavailability. The results revealed that the validated method can be used for columbin analysis in both bioequivalent buffer and blood.
Keywords: Columbin; Pharmacokinetic; Caco-2 transport; UPLC-MS/MS;

Multi-residue analysis of veterinary drugs, pesticides and mycotoxins in dairy products by liquid chromatography–tandem mass spectrometry using low-temperature cleanup and solid phase extraction by Jie Xie; Tao Peng; Ailing Zhu; Jianli He; Qiaoying Chang; Xueyan Hu; Hui Chen; Chunlin Fan; Wenxiao Jiang; Min Chen; Jiancheng Li; Shuangyang Ding; Haiyang Jiang (19-29).
A multi-class multi-residue analysis method for determination of veterinary drugs, pesticides and mycotoxins in dairy products by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been established. These 17 classes, a total of 40 kinds of target compounds were chosen because their administration to food-producing animals is banned or regulated in China and may be potentially abused or misused. Samples were extracted with acetonitrile–ethyl acetate–acetic acid (49.5 + 49.5 + 1, v/v/v). Most of lipids in the extract were removed by low-temperature cleanup, prior to solid phase extraction on HLB cartridges. The quantification and confirmation of the 40 analytes were performed by LC–MS/MS with electro-spray ionization (ESI) interface in multiple reaction monitoring (MRM) mode. The limits of detection (LODs) and limits of quantification (LOQs) were 0.006–0.3 μg/kg and 0.02–1.0 μg/kg, respectively. The spiked recoveries in milk, yogurt, milk powder and cheese samples were from 67.3% to 106.9%. The repeatability and the within-laboratory reproducibility were less than 12.7% and 13.9%. Applying this method, our results revealed the presences of chloramphenicol, cimeterol, and flunixin at the concentration of 0.027–0.452 μg/kg in some samples.
Keywords: Veterinary drugs; Pesticides; Mycotoxins; LC–MS/MS; Low-temperature cleanup; Dairy products;

Enantioselective GC–FID and enantioselective GC–MS have been utilized under temperature gradient mode with differently substituted heptakis- and octakis-cyclodextrins to achieve the resolution of chiral terpenoids in the essential oil of indigenously grown cultivars of Mentha spicata. Modified cyclodextrins were derivatized in GC column for the separation of chiral terpenoids. A 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin doped into 14% cyanopropylphenyl/86%dimethylpolysiloxane (TBDE-β-CD) showed good enantioselectivity for all the studied chiral compounds excluding carvone. Carvone enantiomers were well resolved in 2,3-diacetoxy-6-tert-butyldimethylsilyl-β-cyclodextrin column (TBDA-β-CD) with enantioseparation (Es) of 1.006. A TBDE-β-CD provides maximum enantiomeric separation for β-pinene (Es 1.038), sabinene (Es 1.051), limonene (Es 1.045), isomenthone (Es 1.029) and α-terpineol (Es 1.014). Furthermore, enantiomer elution order reversal was observed for sabinene, menthone, terpinen-4-ol and menthol while changing from β- to γ-cyclodextrin phase. Carvone exhibits enantiomer elution order reversal by changing substituents i.e., methyl to acetyl at 2- & 3- position of the cyclodextrin derivative. Chiral constituents such as (+)-isomenthone, (−)-menthone, (1R,2S,5R)-(−)-menthol and (4S)-(+)-piperitone exist as a single enantiomer with >99% excess. Existence of (R)-(+)-limonene and (S)-(+)-carvone enantiomers has been proven first time in M. spicata essential oils and can be used as the marker for Indian origin.
Keywords: Cyclodextrin derivatives; Enantioselective GC–FID; Mentha spicata; (R)-(−)-carvone; (S)-(−)-limonene;

At-line coupling of LC–MS to bioaffinity and selectivity assessment for metabolic profiling of ligands towards chemokine receptors CXCR1 and CXCR2 by Marija Mladic; Danny J. Scholten; Wilfried M.A. Niessen; Govert W. Somsen; Martine J. Smit; Jeroen Kool (42-53).
This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation.The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40 pmol in CXCR1 binding and 8 pmol in CXCR2 binding. For CXCL8, the LOD was 5 pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314 ± 59 nM (CXCR1 binding) and 38 ± 11 nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9 ± 1.4 nM (CXCR1 binding) and 2.7 ± 1.3 nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC–MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described.
Keywords: LC–MS; Bioaffinity and selectivity assessment; MK-7123; CXCR1; CXCR2; Metabolic profiling;

Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC–MS/MS approach: Assay development, validation, and a case study by Ang Liu; Alexander Kozhich; David Passmore; Huidong Gu; Richard Wong; Frank Zambito; Vangipuram S. Rangan; Heather Myler; Anne-Françoise Aubry; Mark E. Arnold; Jian Wang (54-62).
Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC–MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.
Keywords: Antibody-conjugated payload; Hybrid approach; Immuno-capture; LC–MS/MS; Validation; Plasma;

Measurements of plasma concentrations of free normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MTY) constitute the most diagnostically accurate screening test for pheochromocytomas and paragangliomas. The aim of this article is to present the results from a validation of an analytical method utilizing high performance liquid chromatography with coulometric detection (HPLC-CD) for quantifying plasma free NMN, MN and MTY. Additionally, peak integration by height and area and the use of one calibration curve for all batches or individual calibration curve for each batch of samples was explored as to determine the optimal approach with regard to accuracy and precision.The method was validated using charcoal stripped plasma spiked with solutions of NMN, MN, MTY and internal standard (4-hydroxy-3-methoxybenzylamine) with the exception of selectivity which was evaluated by analysis of real plasma samples. Calibration curve performance, accuracy, precision and recovery were determined following both peak-area and peak-height measurements and the obtained results were compared. The most accurate and precise method of calibration was evaluated by analyzing quality control samples at three concentration levels in 30 analytical runs.The detector response was linear over the entire tested concentration range from 10 to 2000 pg/mL with R 2  ≥ 0.9988. The LLOQ was 10 pg/mL for each analyte of interest. To improve accuracy for measurements at low concentrations, a weighted (1/amount) linear regression model was employed, which resulted in inaccuracies of −2.48 to 9.78% and 0.22 to 7.81% following peak-area and peak-height integration, respectively. The imprecisions ranged from 1.07 to 15.45% and from 0.70 to 11.65% for peak-area and peak-height measurements, respectively. The optimal approach to calibration was the one utilizing an individual calibration curve for each batch of samples and peak-height measurements. It was characterized by inaccuracies ranging from −3.39 to +3.27% and imprecisions from 2.17 to 13.57%.The established HPLC-CD method enables accurate and precise measurements of plasma free NMN, MN and MTY with reasonable selectivity. Preparing calibration curve based on peak-height measurements for each batch of samples yields optimal accuracy and precision.
Keywords: High performance liquid chromatography; Coulometric detection; Plasma; Normetanephrine; Metanephrine; Methoxytyramine;

A sensitive method to simultaneously quantitate quetiapine and norquetiapine in rat plasma and brain tissue was developed using a one-step liquid–liquid extraction for sample preparation and LC-MS/MS for detection. The method provided a linear range of 1.0–500.0 ng/mL for each analyte in plasma and 3.0–1500.0 ng/g in brain tissue. The method was validated with precision within 15% relative standard deviation (RSD), accuracy within 15% relative error (RE), matrix effects within 10% and a consistent recovery. This method has been successfully applied in a preclinical study of quetiapine and norquetiapine to simultaneously determine their concentrations in rat plasma and brain tissue.
Keywords: Quetiapine; Norquetiapine; Plasma; Brain; LC-MS/MS;

Simultaneous quantitation of delamanid (OPC-67683) and its eight metabolites in human plasma using UHPLC–MS/MS by Min Meng; Benjamin Smith; Brad Johnston; Spencer Carter; Jerry Brisson; Sharin E. Roth (78-91).
Delamanid (OPC-67683) is a novel nitro-dihydroimidazo-oxazole derivative that is being developed by Otsuka Pharmaceutical Co., Ltd., Japan (referred to as Otsuka hereafter) for the treatment of tuberculosis (TB). An ultra-high performance liquid chromatographic–tandem mass spectrometry (UHPLC–MS/MS) method has been developed and validated for the determination of OPC-67683 and its eight metabolites, DM-6704, DM-6705, DM-6706, DM-6717, DM-6718, DM-6720, DM-6721 and DM-6722 in human plasma to support regulated clinical development. During method development several technical challenges such as poor chromatography, separation of structural isomers, conversion of the analytes, instability in matrix and long cycle time were encountered and overcome. A protein precipitation extraction (PPE) was used to extract plasma samples (50 μL) and the resulting extracts were analyzed using reversed phase UHPLC–MS/MS with a electrospray (ESI) and selected reaction monitoring (SRM). The method was fully validated over the calibration curve range of 1.00–500 ng/mL for all nine analytes with linear regression and 1/x 2 weighting according to regulatory guidance for bioanalysis. Based on three inter-day precision and accuracy runs, the between-run % relative standard deviation (RSD) for all nine analytes varied from 0.0 to 11.9% and the accuracy ranged from 92.7% to 102.5% of nominal at all quality controls (QC) concentrations, including the lower limit of quantitation (LLOQ) QC at 1.00 ng/mL. The extraction recovery of OPC-67683 and its eight metabolites were above 95%. Various short term and long term solution and matrix stability were established including the stability of OPC-67683 and its eight metabolites in human plasma for 708 days at −70 °C. Although this method has been used to support regulated clinic studies during the last decade and over ten thousand samples have been analyzed, this is the first time that the method development process and validation data have been published.
Keywords: Tuberculosis; Delamanid (code-named OPC-67683); Human plasma; Ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC–MS/MS); Selected reaction monitoring (SRM);

Vanillylmandelic acid (VMA), as one of the most important catecholamine metabolites, is commonly used to aid in diagnosis of pheochromocytoma. This study develops a rapid and simple high-throughput LC–MS/MS method for the measurement of urinary VMA. Without sample pretreatment, the urine specimens were mixed with internal standard (IS) solution for direct analysis by LC–MS/MS in two minutes. VMA and VMA-d3 were detected in the multiple-reaction monitoring mode using the specific transitions m/z 197.0 → 137.0 and 200.0 → 140.0, respectively. This method was validated for consistent linearity from 1.0 to 250.0 μM with CVs ≤ 3.12%, excellent recovery, good stability and low carryover. The lowest limit of quantification (LLOQ) was 0.125 μmol/L for VMA with CV of 14.1%, and the lower limit of detection (LOD) was 0.025 μmol/L. Intra-assay and inter-assay imprecision values (CVs) for VMA were all below 2.11%. Dilution linearity was investigated with a satisfied mean accordance of 105%. Method comparison of LC–MS/MS and microcolumn chromatography in our lab was performed and the reference interval was established in agreement with that of the Mayo Clinic. All these results demonstrate that this validated LC–MS/MS approach shows improved accuracy and reproducibility compared with microcolumn chromatography. The low sample volume, simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.
Keywords: Vanillylmandelic acid; Pheochromocytoma; LC–MS/MS;

A simple method based on matrix solid phase dispersion (MSPD) using molecularly imprinted polymers (MIPs) as sorbents for selective extraction of malachite green (MG) from aquatic products was developed. The MIPs were prepared by using carbon nanotube as support, MG as template, methacrylic acid as functional monomer, ethyleneglycol dimethacrylate as crosslinker and methylene chloride as solvent. The MIPs were characterized by Fourier transform infrared spectrometry and transmission electron microscopy. The isothermal adsorption, kinetics absorption and selective adsorption experiments were carried out. We optimized the extraction conditions as follows: the ratio of MIPs to sample was 2:3, the dispersion time was 15 min, washing solvent was 4 mL 50% aqueous methanol and elution solvent was 3 mL methanol-acetic acid (98: 2, v/v). Once the MSPD process was completed, the MG extracted from aquatic products was determined by high performance liquid chromatography. The detection limit of MG was 0.7 μg kg−1. The relative standard deviations of intra-day and inter-day were obtained in the range of 0.9%–4.7% and 3.4%–9.8%, respectively. In order to evaluate the applicability and reliability of the proposed method, it was applied to determine MG in different aquatic products samples including fish, shrimp, squid and crabs. The satisfied recoveries were in the range of 89.2%–104.6%. The results showed that this method is faster, simpler and makes extraction and purification in the same system.
Keywords: Carbon nanotube; Molecularly imprinted polymer; Matrix solid phase dispersion; Malachite green; High performance liquid chromatography;

A simple, rapid and sensitive reversed-phase high performance liquid chromatography (HPLC) method has been developed for the determination of methotrexate in human serum. After deproteinization of the serum with 40% silver nitrate solution, methotrexate and internal standard (IS) were separated on a reversed-phase column with a mobile phase consisting of 10 mM sodium phosphate buffer (pH6.40)-methanol (78:22%, v/v) and ultraviolet detection at 310 nm. The linearity is evaluated by a calibration curve in the concentration range of 0.05–10.0 μg/mL and presented a correlation coefficient of 0.9995. The absolute recoveries were 97.52 ± 3.9% and 96.87 ± 3.7% for methotrexate and ferulic acid (internal standard), respectively. The intra- and inter-day precision were less 6.19 and 5.89%, respectively (n  = 6). The limit of quantitation was 0.02 μg/mL and the limit of detection was 0.006 μg/mL. The complete analysis was achieved less than 10 min with no interference from endogenous components or 22 examined drugs. This method was validated by using serum samples from high-dose methotrexate treated patients with osteosarcoma, breast cancer, acute leukemia and lymphoma. The method was demonstrated to be a simple, rapid and reliable approach in quantification of methotrexate in serum samples from patients with high-dose methotrexate therapy.
Keywords: Method validation; HPLC; Methotrexate; Silver nitrate; Serum;

Separation and purification of epigallocatechin-3-gallate (EGCG) from green tea using combined macroporous resin and polyamide column chromatography by Xin Jin; Mingyan Liu; Zaixing Chen; Ruikun Mao; Qinghuan Xiao; Hua Gao; Minjie Wei (113-122).
Epigallocatechin-3-gallate (EGCG) is a major bioactive ingredient of green tea that produces beneficial neuroprotective effects. In this paper, to optimize the EGCG enrichment, thirteen macroporous resins with different chemical and physical properties were systemically evaluated. Among the thirteen tested resins, the H-bond resin HPD826 exhibited best adsorption/desorption capabilities and desorption ratio, as well as weakest affinity for caffeine. The absorption of EGCG on the HPD826 resin followed the pseudo-second-order kinetics and Langmuir isotherm model. The separation parameters of EGCG were optimized by dynamic adsorption/desorption experiments with the HPD826 resin column. Under the optimal condition, the content of EGCG in the 30% ethanol eluent increased by 5.8-fold from 7.7% to 44.6%, with the recovery yield of 72.1%. After further purification on a polyamide column, EGCG with 74.8% purity was obtained in the 40–50% ethanol fraction with a recovery rate of 88.4%. In addition, EGCG with 95.1% purity could be easily obtained after one-step crystallization in distilled water. Our study suggests that the combined macroporous resin and polyamide column chromatography is a simple method for large-scale separation and purification of EGCG from natural plants for food and pharmaceutical applications.
Keywords: Epigallocatechin-3-gallate; Green tea; Macroporous resin; Polyamide; Combined column chromatography;

1,3-Butadiene (BD) is a ubiquitous environmental pollutant found in tobacco smoke. In vivo, BD is mainly metabolized to form monohydroxybutenylmercapturic acid (MHBMA) and N-acetyl-S-(3, 4-dihydroxybutyl) cysteine (DHBMA). The accurate quantification of MHBMA and DHBMA in urine may provide important insights into the actual internal exposure of the general population to BD. 8-Hydroxy-2-deoxyguanosine (8-OHdG) is the biomarker of oxidative damage. In this study, a column-switching LC–MS/MS method was developed and validated for the simultaneous quantification of MHBMA, DHBMA derived from BD exposure and 8-OHdG in human urine. Urine samples were loaded on a LiChrospher® RP-8 ADS (25 μm) 25 × 4 mm RAM column for the extraction and clean-up of analytes. The separation was achieved using a SUPELCO® LC-18-DB column (75 mm × 3.0 mm, 3 μm). The analytes were ionized in negative electrospray ionization mode and analyzed in multiple reaction monitoring mode. Under optimum conditions, recoveries ranged from 78.9% to 101.7%, with relative standard deviations less than 11%. The limits of quantification ranged from 0.15 to 0.27 ng/mL, highlighting the high sensitivity of this simple method. The validated method was successfully applied to analysis urine samples from 56 non-smokers and 233 smokers who smoked cigarettes with 3 different tar yields. There was a correlation between urinary MHBMA, DHBMA and 8-OHdG. This method did not require any preparation process and efficiently removed interference from the matrix by using column-switching. The developed method is applicable to epidemiological studies.
Keywords: Biomarker; 1,3-Butadiene; Oxidative damage; Column-switching; LC–MS/MS;

Advances in the metabolic profiling of acidic compounds in children’s urines achieved by comprehensive two-dimensional gas chromatography by N. Pérez Vasquez; M. Crosnier de bellaistre-Bonose; N. Lévêque; E. Thioulouse; D. Doummar; T. Billette de Villemeur; D. Rodriguez; R. Couderc; S. Robin; C. Courderot-Masuyer; F. Moussa (130-138).
The main objective of this work was to evaluate a comprehensive two-dimensional gas chromatographic (GCxGC) coupled to quadrupole mass spectrometry (qMS) method in the field of biomarker candidates’ discovery. To this purpose we developed a GCxGC-qMS method suitable for the separation of organic acids and other classes of compounds with silylable polar hydrogen such as sugars, amino-acids, and vitamins. As compared to those obtained by a widely used 1D-GC method, the urinary chromatographic profiles performed by the proposed 2D-GC method exhibit higher resolution and sensitivity, leading to the detection of up to 92 additional compounds in some urine samples including some well-known biomarkers.In order to validate the proposed method we focused on three metabolites of interest with various functional groups and polarities including CH3-malonic acid (MMA: biomarker of methylmalonic acidemia), 3-hydroxy-3-methyl-glutaric acid (3-OHMGA: biomarker of 3-hydroxy-3-methylglutaric acidemia), and phenylpiruvic acid (PhPA: marker of phenylketonuria). While these three metabolites can be considered as representative of organic acids classically determined by 1D-GC, they cannot be representative of new detected metabolites. Thus, we also focused on quinolic acid (QUIN), taken as an example of biomarker not detected at basal levels with the classical 1D GC-qMS method. In order to obtain sufficient recoveries for all tested compounds, we developed a sample preparation protocol including a step of urea removal followed by two extraction steps using two solvents of different polarity and selectivity. Recoveries with the proposed method reached more than 80% for all targeted compounds and the linearity was satisfactory up to 50 μmol/L. The CVs of the within-run and within-laboratory precisions were less than 8% for all tested compounds. The limits of quantification (LOQs) were 0.6 μmol/L for MMA, 0.4 μmol/L for 3-OHMGA, 0.7 μmol/L for PhPA, and 1 μmol/L for QUIN. The LOQs of these metabolites obtained by a classical GC-MS method under the same chromatographic conditions were 5 μmol/L for MMA, 4 μmol/L for 3-OHMGA, 6 μmol/L for PhPA while QUIN was below the limit of detection. As compared to 1D-GC, these results highlight the enhanced detectability of urine metabolites by the 2D-GC technique. Our results also show that for each new detected compound it is necessary to develop and validate an appropriate sample preparation procedure.
Keywords: Two-dimensional gas chromatography; Organic acids; Urine; Metabolism; Biomarkers; Quinolinic acid;

High-performance liquid chromatography combined with intrinsic fluorescence detection to analyse melittin in individual honeybee (Apis mellifera) venom sac by Jiangtao Dong; Bihua Ying; Shaokang Huang; Shuangqin Ma; Peng Long; Xijuan Tu; Wenchao Yang; Zhenhong Wu; Wenbin Chen; Xiaoqing Miao (139-143).
Melittin is the major toxin peptide in bee venom, which has diverse biological effects. In the present study, melittin was separated by reverse-phase high-performance liquid chromatography, and was then detected using intrinsic fluorescence signal of tryptophan residue. The accuracy, linearity, limit of quantitation (LOQ), intra-day and inter-day precision of the method were carefully validated in this study. Results indicate that the intrinsic fluorescence signal of melittin has linear range from 0.04 μg/mL to 20 μg/mL with LOQ of 0.04 μg/mL. The recovery range of spiked samples is between 81.93% and 105.25%. The precision results are expressed as relative standard deviation (RSD), which is in the range of 2.1–7.4% for intra-day precision and 6.2–10.8% for inter-day precision. Because of the large linear dynamic range and the high sensitivity, intrinsic fluorescence detection (IFD) can be used for analyzing melittin contents in individual venom sac of honeybee (Apis mellifera). The detected contents of melittin in individual bee venom sac are 0.18 ± 0.25 μg for one-day old honeybees (n  = 30), and 114.98 ± 43.51 μg for 25-day old (n  = 30) honeybees, respectively. Results indicate that there is large bee-to-bee difference in melittin contents. The developed method can be useful for discovering the melittin related honeybee biology information, which might be covered in the complex samples.
Keywords: Melittin; HPLC; Intrinsic fluorescence; Bee venom; Individual honeybee;

Despite the biological importance of membrane proteins, their analysis has lagged behind that of soluble proteins and still presents a great challenge mainly because of their highly hydrophobic nature and low abundance. Sodium deoxycholate (SDC)-assisted digestion strategy has been introduced in our previous papers, which cleverly circumvents many of the challenges in shotgun membrane proteomics. However, it is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of 1% SDC. In this study, an enhanced SDC-assisted digestion method (ESDC method) was developed that incorporates the almost strongest ability of SDC with a high concentration (5%) to lyse membrane and extract/solubilize hydrophobic membrane proteins, and then dilution to 1% for more efficient digestion. The comparative study using rat liver membrane-enriched sample showed that, compared with previous SDC-assisted method and the “universal” filter-aided sample preparation (FASP) method, the ESDC method not only increased the identified number of total proteins, membrane proteins, hydrophobic proteins, integral membrane proteins (IMPs) and IMPs with more than 5 transmembrane domains (TMDs) by an average of 10.8%, 13.2%, 17.8%, 17.9% and 52.9%, respectively, but also enhanced the identified number of total peptides and hydrophobic peptides by averagely 12.5% and 14.2%. These results demonstrated that the ESDC method provides a substantial improvement in the recovery and identification of membrane proteins, especially those with high hydrophobicity and multiple TMDs, and thereby displaying more potential for shotgun membrane proteomics.
Keywords: SDC-assisted digestion; Shotgun analysis; Membrane proteome; Integral membrane proteins; Hydrophobic proteins; LC–MS/MS;

Validation of high-performance liquid chromatography–tandem mass spectrometry assays quantifying omacetaxine mepesuccinate and its 4′‑des-methyl and cephalotaxine metabolites in human plasma and urine by C.M. Nijenhuis; L. Lucas; H. Rosing; J.H.M. Schellens; J.H. Beijnen; S.H. Gorman; S.M. Burke; D.A. Campbell; M.W. Chapple; T.H. Yousey; D.E. Mulvana (152-159).
Omacetaxine mepesuccinate (hereafter called omacetaxine) is a modified cephalotaxine and is registered (Synribo®) for the treatment of adult patients with chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs). To evaluate the pharmacokinetics of omacetaxine, sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays for the quantification of omacetaxine and its inactive 4′-des-methyl (4′-DMHHT) and cephalotaxine metabolites in human plasma and urine were developed and validated.Since omacetaxine is mainly metabolised by esterases, the plasma samples were immediately stabilised after collection with an esterase inhibitor and stored at a nominal temperature of −80 °C. Urine samples were stored at −80 °C immediately after collection. Protein precipitation was applied as the sample pretreatment method for the plasma samples, and urine samples were processed using solid-phase extraction (SPE). For both assays, the dried and reconstituted extracts were injected on a XBridge BEH Phenyl column for analysis of all analytes. Gradient elution was applied with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionised using a turbospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer.The validated plasma assay quantifies all analytes in the concentration range of 0.1–100 ng/mL and the urine assay in the range of 0.1–50 ng/mL. At all concentrations, the accuracies were within ±15% of the nominal concentrations and precisions were ≤15%. The developed methods have successfully been applied in a human mass balance study of omacetaxine.
Keywords: Omacetaxine mepesuccinate; 4′-Des-methylhomoharringtonine; Cephalotaxine; HPLC–MS/MS; GLP;

An enantioselective high performance liquid chromatography method has been developed and validated by evaluating the suitability of newly introduced immobilized polysaccharide chiral stationary phases, the effect of different organic modifiers and temperature including the entropy and enthalpy on resolution of the (R,S)-(−) & (S,R)-(+) emtricitabine enantiomers on rat dried blood spots. Both the enantiomers were extracted from dried blood spots using ethanol: methanol (80:20 v/v) mixture and separated on an immobilized amylose tris-(3,5-dimethyl phenyl carbamate) chiral stationary phase using n-hexane:ethanol (65:35 v/v) as a mobile phase at a flow rate of 0.8 mL/min. The detection was carried out at 280 nm using photo diode array detector connected to a polarimeter in series to determine their order of eluton. The method was validated with respect to limits of detection and quantification, linearity, accuracy and precision. The calibration curves were linear over the concentration range of 0.5–500 μg/mL for both enantiomers and the correlation coefficient (r 2) was >0.998. The overall recovery of (R,S)- & (S,R)-enantiomers of emtricitabin from DBS were 90.4 and 90.6%, respectively. The limits of detection and quantification of enantiomers were 0.26, 0.30 and 0.85, 0.92 μg/mL for (R,S)- and (S,R)-emtricitabin enantiomers, respectively. The assay was specific and precise (RSD <10%). The stability of emtricitabin was also performed and the results were found to be well within the limits. The effect of hematocrit on extraction of emtricitabin enantiomers from dried blood spots was evaluated and no interference from endogenous substances was observed.
Keywords: Emtricitabin enantiomers; Immobilized; Amylose tris-(3,5-dimethylphenyl carbamate); Dried blood spots; Hematocrit;

Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum by Ryan C. Schofield; Lakshmi V. Ramanathan; Kazunori Murata; Marie Grace; Martin Fleisher; Melissa S. Pessin; Dean C. Carlow (169-175).
A rapid and simple turbulent flow liquid chromatography (TFC–LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N 10-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100 μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC–LC–MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000 nmol/L and for 7-OH MTX from 20 to 2000 nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10 nmol/L to 5 × 105  nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC–LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC–MS/MS assay.
Keywords: Methotrexate; 7-Hydroxy methotrexate; DAMPA; Carboxypeptidase-G2; Therapeutic drug monitoring; Mass spectrometry; Turbulent flow liquid chromatography; Method correlation; Dihydrofolate reductase inhibition assay; Fluorescence polarization immunoassay;

Determination of putrescine, cadaverine, spermidine and spermine in different chemical matrices by high performance liquid chromatography–electrospray ionization–ion trap tandem mass spectrometry (HPLC–ESI–ITMS/MS) by Alan Alexander González Ibarra; Katarzyna Wrobel; Alma Rosa Corrales Escobosa; Julio Cesar Torres Elguera; Ma Eugenia Garay-Sevilla; Kazimierz Wrobel (176-184).
The goal of this work was to establish a simple HPLC–ESI–ITMS/MS procedure, suitable for the determination of four common aliphatic polyamines in two different types of biological matrices. To this end, 1,6-diaminohexane was used as the internal standard (IS) and 4-fluoro-3-nitrobenzenotrifluoride (FNBT) as the derivatizing agent. Formation of fully derivatized compounds was confirmed by high resolution ESI–QTOFMS and MS/MS analysis. Reversed phase chromatographic separation was carried out by gradient elution with 0.1% (v/v) formic acid and methanol. In a positive ESI mode, the following pairs of precursor/quantifier ions were used for multiple reaction monitoring: 467.4/261.0 for PUT, 481.2/461.1 for CAD, 713.7/261.0 for SPD, 959.8/507.2 for SPM and 495.3/475.2 for IS. On-column instrumental detection limits of four polyamines were in the range 0.62–2.14 fmol (0.039–0.215 ng/ml). Versatility was demonstrated by analyzing plant extracts and human urine; prior to derivatization, all samples were cleaned-up by dichloromethane extraction. The evaluated signal suppression/enhancement was in the range 82.3–115.4% and the percentage recoveries obtained in the method of standard addition were in the range 83.7–114.4%. Statistically significant differences in polyamines concentrations were found in garden cress exposed to Cd(II) versus control seedlings (t-test, p  < 0.05); results obtained for urine from healthy volunteers and diabetic patients at different clinical conditions suggested possible utility of free polyamines as biomarkers of progressive diabetes.
Keywords: Aliphatic polyamines; 4-fluoro-3-nitrobenzenotrifluoride (FNBT); LC–MS, Multiple reaction monitoring; Plant abiotic stress; Diabetes; Urine;

To explore the brain-targeting of cyclovirobuxine D(CVB-D) after administered intranasally, the pharmacokinetics of CVB-D via three different drug delivery routes: intragastric (i.g.), intranasal (i.n.), and intravenous (i.v.) in rat brain and blood was compared. Firstly, an in vivo microdialysis method for sampling CVB-D in both plasma and brain of the rat was established. Secondly, a liquid chromatography-tandem mass spectrometry method has been developed and validated for determination of CVB-D in microdialysis samples. For plasma and brain microdialysis samples, liquid–liquid extraction was used and donepezil was chosen as internal standard. Both were followed by HPLC separation and positive electrospray ionization tandem mass spectrometry detection (ESI–MS/MS). Chromatographic separation was achieved on a agilent C18 column with a mobile phase of methanol–water (50:50, v/v) (pH 3.2) containing 0.1% formic acid and 5 mM ammonium acetate. Mass spectrometric detection in the positive ion mode was carried out by selected reaction monitoring (MRM) of the transitions at m/z 403.4 → 372.3 for CVB-D and m/z 380.2 → 243.1 for donepezil (IS). Good linearities were obtained in the range of 10–4000 ng/mL in rat microdialysates for CVB-D. The lowest limit of quantitation was 5 ng/mL, with an extraction recovery >75%, and no significant matrix effects. Intra- and inter-day precisions were all <15% with accuracies of 97.26–116.20%. All of which proved that the established method was successfully applied to the pharmacokinetic study of CVB-D. Simultaneously, brain uptake and pharmacokinetic studies were performed by determination of CVB-D concentration in blood and brain respectively for CVB-D i.g., i.n. and i.v.. Results showed that the intranasal CVB-D could improve brain targeting and had advantages for direct nose to brain transport of CVB-D when compared with injection and oral delivery routes, which indicates that intranasal administration of CVB-D could be a promising approach for the treatment of cerebrovascular disease.
Keywords: Microdialysis; LC–MS/MS; Different drug delivery routes; Rat brain and plasma pharmacokinetic;

The aim of the present study is to explore pharmacokinetic and protein binding characteristics of a novel dipeptidylpeptidase-4 (DPP-4) inhibitor, teneligliptin in rats using an ultra high performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC–QTOF-MS). It is required for demonstrating the high protein binding nature of teneligliptin which can be extended for drug repositioning to brain disorders. Sample preparation was accomplished through a protein precipitation procedure using acetonitrile. Separation of teneligliptin and sitagliptin (IS) from endogenous components with high selectivity and sensitivity (0.5 ng/mL) was achieved within 4 min using Poroshell 120 EC-C18 column (100 × 3.0 mm, 2.7 μ). A gradient mobile phase consisting of 10 mM ammonium formate and acetonitrile was applied at a flow rate of 0.45 mL/min. Detection of target ions [M + H]+ at m/z 427.2274 for teneligliptin and m/z 408.1258 for IS was performed in selective ion mode using positive ion electrospray ionization high resolution accurate mass spectrometry. The linearity of the method was found to be in the range of 0.5–1000 ng/mL. The matrix effect was 88.7–94.5% for teneligliptin. Plasma samples were found to stable under different storage conditions. It was successfully applied to pharmacokinetic and plasma protein binding study of drug in rats. Results showed linear dose proportionality of pharmacokinetics at 0.1 and 1 mg/kg and relatively high protein binding of teneligliptin (85.46 ± 0.24 %) compared with other DPP-4 inhibitors.
Keywords: Teneligliptin; DPP-4 inhibitors; Pharmacokinetics; Plasma protein binding; LC–MS; Ultra-filtration;

Dried blood spot analysis without dilution: Application to the LC–MS/MS determination of BMS-986001 in rat dried blood spot by Long Yuan; Alan Schuster; Jim X. Shen; Pamela Garrison-Borowski; Anne-Françoise Aubry (201-209).
Sample dilution is one major challenge in dried blood spot (DBS) bioanalysis. To resolve this issue, we applied a no-dilution strategy for DBS analysis by using a calibration curve with very wide linear range. We developed an LC–MS/MS DBS assay with a linear range of 5 orders of magnitude (50–5000,000 ng/mL) for BMS-986001, an HIV drug under development, by simultaneously monitoring two selective reaction monitoring transitions of different intensity. The assay was validated and successfully applied to the analysis of DBS samples collected in a toxicology study in rats dosed with BMS-986001. All samples were analyzed without any dilution. We also compared the concentration data generated from the DBS method and a validated plasma assay for the same study. The two sets of data agreed well with each other, demonstrating the validity of this strategy for DBS analysis. This approach provides an effective and convenient way to eliminate complicated dilution for DBS and other sample collection techniques.
Keywords: Dried blood spot; Dilution; Extended linear range; Quantitation; LC–MS/MS; Regulated bioanalysis;

Serum metabolomics research of the anti-hypertensive effects of Tengfu Jiangya tablet on spontaneously hypertensive rats by Haiqiang Jiang; Zhenzhen Shen; Yanjun Chu; Yunlun Li; Jihua Li; Xiaoming Wang; Wenqing Yang; Xinya Zhang; Jianqing Ju; Jingwen Xu; Chuanhua Yang (210-217).
A HPLC/TOF-MS-based metabolomic study was conducted to investigate the holistic therapeutic effects of Tengfu Jiangya Tablet (TJT) on spontaneously hypertensive rats (SHRs). The SHRs were divided into valsartan (VST) group, TJT group and model group, in addition, the Wistar-Kyoto rats (WKY) were taken as normal control. Serum samples were separated and identified by HPLC/TOF-MS, while the obtained data was further processed by partial least-squares discriminant analysis (PLS-DA). A clear cluster among the four groups was observed, and we identified thirteen biomarkers involved involved in sphingolipid metabolism (sphinganine, lysosphingomyelin, ceramide), glycerophospholipid metabolism (phosphatidylcholines, phosphatidylethanolamine, lysophosphatidylcholines), arginine and proline metabolism (l-proline, citrulline), tryptophan metabolism (xanthuiulrenic acid, l-kynurenine, l-tryptophan), arachidonic acid metabolism(leukotriene D4), and linoleic acid metabolism (gamma-linolenic acid). Altered metabolic pathways involved in impaired NO production, inflammation and vascular smooth muscle cells (VSMCs) apoptosis and proliferation, which suggesting the possible pathological state in SHRs. TJT as well as VST altered the metabolic state, suggesting a possible anti-hypertension role by improving NO production, and an extra cardiovascular protection role possibly by amelioration of inflammatory state and vascular remodeling.
Keywords: Hypertension; Metabolomics; Traditional chinese medicine; Liquid chromatography; Mass spectrometry;

Study on the determination and chiral inversion of R-salbutamol in human plasma and urine by liquid chromatography–tandem mass spectrometry by Ting Zhou; Jing Zeng; Shan Liu; Ting Zhao; Jie Wu; Wenshi Lai; Mingzhi He; Beining Xu; Shanshan Qu; Ling Xu; Wen Tan (218-227).
The chiral inversion has been a concerned issue during the research and development of a chiral drug. In this study, a sensitive chiral liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for determination of salbutamol enantiomers in human plasma and urine. The chiral inversion mechanism of R-salbutamol was fully investigated for the first time by studying the effects of physicochemical factors, including pH, temperature and time. A fitted model to predict the chiral inversion ratio of R-salbutamol was proposed using a Box–Behnken design. All the samples were separated on an Astec Chirobiotic T column and detected by a tandem mass spectrometer in multiple reaction monitoring mode. Lower limit of quantification of 0.100 ng/mL was achieved under the optimized conditions. The method was fully validated and successfully applied to the clinical pharmacokinetic study of R-salbutamol in healthy volunteers. Chiral inversion of R-salbutamol to S-salbutamol has been detected in urine samples. The results indicated that pH and temperature were two dominant factors that caused the chiral inversion of R-salbutamol, which should be taken into consideration during the analysis of chiral drugs. The chiral inversion of R-salbutamol determined in this study was confirmed resulted from the gastric acid in stomach rather than caused by the analysis conditions. Moreover, the calculated results of the fitted model matched very well with the enantioselective pharmacokinetic study of R-salbutamol, and the individual difference of the chiral inversion ratio of R-salbutamol was related to the individual gastric environment. On the basis of the results, this study provides important and concrete information not only for the chiral analysis but also for the metabolism research of chiral drugs.
Keywords: Stereoselective determination; Chiral inversion; R-salbutamol; LC–MS/MS; Clinical pharmacokinetics;

A method for quantification of new antiepileptic drugs, including lamotrigine (LTG), levetiracetam (LEV), gabapentin (GBP), and topiramate (TPM), in cellular samples, using liquid chromatography/electrospray ionization tandem mass spectrometry was developed to better understand the membrane transport mechanisms of these drugs. Cell lysate was deproteinized by methanol containing LEV-d3 as an internal standard (IS). Chromatographic separation was performed on a C18 column using gradient elution with methanol–water–formic acid (10:90:0.1, v/v/v) and methanol–formic acid (100:0.1, v/v). Analytes were detected in positive ion electrospray mode with selected reaction monitoring (SRM). This method was applicable for a linear range of 5 to 500 pmol for LTG; 5 to 1000 pmol for LEV; 10 to 10,000 pmol for GBP; and 5 to 5000 pmol for TPM. The intra-day precision, inter-day precision, and accuracy data were assessed and found to be acceptable. This developed and validated method was then successfully applied to the investigation of uptake of the new antiepileptic drugs in placental choriocarcinoma BeWo cells. The intracellular concentration of these drugs in BeWo cells, accumulating over 30 min at 37 °C was in the order of GBP > LTG > LEV ≈ TPM. Furthermore, the uptake of GBP at 4 °C was much lower than that at 37 °C. The uptake of GBP was saturated at high concentrations. The kinetic parameters calculated for GBP uptake in BeWo cells were determined as K m of 105.4 ± 6.4 μM and V max at 8153 ± 348 pmol/mg protein/min. The novel method described here should enable investigators to elucidate the transport mechanisms of these antiepileptic drugs in BeWo cells.
Keywords: Cellular uptake experiment; Gabapentin; Lamotrigine; Levetiracetam; Liquid chromatography/tandem mass spectrometry; Topiramate;

Determination of songorine in rat plasma by UPLC–MS/MS: Assay development and application to pharmacokinetic study by Yao-yao Dong; Baiping Mao; Hongguo Guan; Yanfang Bai; Binghuan Chi; Yuan-yuan Shan; Qing-quan Lian; Ren-shan Ge (234-238).
An ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of songorine in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0 min and the elution of songorine was at 1.68 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 358.3 → 340.3 for songorine and m/z 237.2 → 194.3 for carbamazepine (internal standard). The calibration curve was linear over the range of 1–1000 ng/mL with a lower limit of quantitation (LLOQ) of 1.0 ng/mL. Mean recovery of songorine in plasma was in the range of 75.2–87.5%. The intra- and inter-day precision (RSD) was between 3.1–8.5% and 4.3–9.6% and the intra- and inter-day accuracy (RE) ranged from −4.0 to 8.9% and −9.0 to 6.7%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0 mg/kg songorine in rats.
Keywords: Songorine; UPLC–MS/MS; Rat plasma; Pharmacokinetics;

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to simultaneous determine nintedanib and BIBF 1202 in mice plasma and tissue using carbamazepine as the internal standard (IS). Sample preparation was accomplished through a protein precipitation procedure with acetonitrile. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 0.1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 540.3 → 113.1 for nintedanib, m/z 526.3 → 113.1 for BIBF 1202 and m/z 237.1 → 194.1 for IS, respectively. The linearity of this method was found to be within the concentration range of 1–1000 ng/mL with a lower limit of quantification of 1.0 ng/mL for each drug. Only 3.0 min was needed for an analytical run. The inter-day and intra-day precision and accuracy of quality control (QC) samples, evaluated both in plasma and tissue homogenates, were all within 15%. The method was successfully applied to the pharmacokinetic and tissue distribution study of nintedanib and BIBF 1202 in mice after oral administration of nintedanib.
Keywords: Nintedanib; BIBF 1202; UPLC–MS/MS; Plasma; Pharmacokinetics; Tissue distribution;

Pharmacokinetics and tissue distribution of Aucubin, Ajugol and Catalpol in rats using a validated simultaneous LC–ESI-MS/MS assay by Bingyang Xue; Bo Ma; Qi Zhang; Xiaotian Li; Jianwei Zhu; Ming Liu; Xiujuan Wu; Chao Wang; Zimei Wu (245-253).
To enable an investigation of pharmacokinetics and tissue distribution of Aucubin, Ajugol and Catalpol in rats, a high-performance liquid chromatography–electro spray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) method was developed for the simultaneous quantitative determination of the three compounds. Biological samples were prepared by a simple protein precipitation with methanol (containing 0.05% formic acid). The analytes were separated by a C18 reversed phase column and detected with a triple quadrupole tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 364.3 → 149.0 for Aucubin, m/z 366.5 → 151.1 for Ajugol, m/z 380.0 → 183.3 for Catalpol and m/z 530.3 → 183.1 for Picroside-II (IS) in positive ionization. Good linearity of each calibration curve was produced over the concentration range of 1–1000 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL for the three analytes. This method was successfully applied to the pharmacokinetic and tissue distribution studies of Aucubin, Ajugol and Catalpol in rat. The current results revealed pharmacokinetic behaviors of the herb compound and provided novel evidence of the presence of Aucubin and Catalpol in rat brain. The acquired data would be helpful for the clinical application and further studies of Traditional Chinese Medicines (TCM) with active ingredients of Iridoid Glycosides.
Keywords: Aucubin; Ajugol; Catalpol; HPLC–ESI-MS/MS; Pharmacokinetics; Tissue distribution;

A method, using liquid chromatography with tandem mass spectrometric detection (LC–MS/MS), was developed for the determination of suvorexant (MK-4305, Belsomra®), a selective dual orexin receptor antagonist for the treatment insomnia, in human plasma over the concentration range of 1–1000 ng/mL. Stable isotope labeled 13C2H3-suvorexant was used as an internal standard. The sample preparation procedure utilized liquid–liquid extraction, in the 96-well format, of a 100 μL plasma sample with methyl t-butyl ether. The compounds were chromatographed under isocratic conditions on a Waters dC18 (50 × 2.1 mm, 3 μm) column with a mobile phase consisting of 30/70 (v/v %) 10 mM ammonium formate, pH3/acetonitrile at a flow rate of 0.3 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for suvorexant (m/z 451 → 186) and 13C2H3-suvorexant (m/z 455 → 190) on an Applied Biosystems API 4000 tandem mass spectrometer was used for quantitation. Intraday assay precision, assessed in six different lots of control plasma, was within 10% CV at all concentrations, while assay accuracy ranged from 95.6 to 105.0% of nominal. Quality control (QC) samples in plasma were stored at −20 °C. Initial within day analysis of QCs after one freeze-thaw cycle showed accuracy within 9.5% of nominal with precision (CV) of 6.7% or less. The plasma QC samples were demonstrated to be stable for up to 25 months at −20 °C. The method described has been used to support clinical studies during Phase I through III of clinical development.
Keywords: LC–MS/MS; Human plasma; Liquid–liquid extraction; Suvorexant; Bioanalytical;

Comparison of blood plasma sample preparation methods for combined LC–MS lipidomics and metabolomics by Rainey E. Patterson; Antoine J. Ducrocq; Danielle J. McDougall; Timothy J. Garrett; Richard A. Yost (260-266).
The goal of this research was to find the most comprehensive lipid extraction of blood plasma, while also providing adequate aqueous preparation for metabolite analysis. Comparisons have been made previously of the Folch, Bligh–Dyer, and Matyash lipid extractions; furthermore, this paper provides an additional comparison of a phospholipid removal plate for analysis. This plate was used for lipid extraction rather than its intended use in lipid removal for polar analysis, and it proves to be robust for targeted lipid analysis. Folch and Matyash provided reproducible recovery over a range of lipid classes, however the Matyash aqueous layer compared well to a typical methanol preparation for polar metabolite analysis. Thus, the Matyash method is the best choice for an untargeted biphasic extraction for metabolomics and lipidomics in blood plasma.
Keywords: Metabolomics; Lipidomics; Sample preparation; LC–MS;

Determination of endogenous corticosterone in rodent’s blood, brain and hair with LC–APCI–MS/MS by Tian Yu; Hang Xu; Weiwen Wang; Shifei Li; Zheng Chen; Huihua Deng (267-276).
Endogenous corticosterone in rodent’s hair would be a potential biomarker to assess the response of the hypothalamus–pituitary–adrenal axis to chronic stress. However, currently unknown is whether hair corticosterone is associated with endogenous corticosterone in blood and brain. The present study aimed to develop an enhanced assay for determination of endogenous corticosterone in blood, brain and hair, and to examine associations of hair corticosterone with blood and brain corticosterone under basal condition and association with blood corticosterone under chronic stressful condition. Hair at the back and blood samples were collected from non-stressed and stressed rodents, and prefrontal lobe and thalamus from non-stressed rodents. Chronic stress exerted on mice was 30-day repeated social defeat. The analyses were done using high performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization in positive mode. Limits of detection and quantification were 0.2 and 0.5 ng/ml for rat’s blood, and 0.5 and 1.0 pg/mg for rat’s hair and brain, and 1.25 and 2.50 ng/ml (or pg/mg) for mouse’s blood (or hair). Recovery ranged from 84.2 to 108.0%. The intra- and inter-day coefficients of variation were less than 10%. Additionally, correlation of hair corticosterone with blood corticosterone was significant in both mice and rats, but correlations with corticosterone in prefrontal lobe and thalamus were not significant in rats. Both hair and blood corticosterone were significantly higher in stressed mice compared with controls.
Keywords: Hair corticosterone; Blood corticosterone; Brain corticosterone; Repeated social defeat; Atmospheric pressure chemical ionization;

Simultaneous column chromatographic extraction and purification of abscisic acid in peanut plants for direct HPLC analysis by Ya-Wen Zhang; Wei-Wei Fan; Hui Li; He Ni; Han-Bing Han; Hai-Hang Li (277-284).
Abscisic acid (ABA), a universal signaling molecule, plays important roles in regulating plant growth, development and stress responses. The low contents and complex components in plants make it difficult to be accurately analyzed. A novel one-step sample preparation method for ABA in plants was developed. Fresh peanut (Arachis hypogaea) plant materials were fixed by oven-drying, microwave drying, boiling or Carnoy’s fixative, and loaded onto a mini-preparing column. After washed the impurities, ABA was eluted with a small amount of solvent. ABA in plant materials was completely extracted and purified in 2 mL solution and directly analyzed by HPLC, with a 99.3% recovery rate. Multiple samples can be simultaneously prepared. Analyses using this method indicated that the endogenous ABA in oven-dried peanut leaves increased 20.2-fold from 1.01 to 20.37 μg g−1 dry weight within 12 h and then decreased in 30% polyethylene glycol 6000 treated plants, and increased 3.34-fold from 0.85 to 2.84 μg g−1 dry weight in 5 days and then decreased in soil drought treated plants. The method combined the column chromatographic extraction and solid-phase separation technologies in one step and can completely extracted plant endogenous ABA in a purified and highly concentrated form for direct HPLC analysis.
Keywords: Abscisic acid; Phytohormone; Sample preparation; Peanut; Extraction; Purification;

At present, there is a growing concern about human quality of life. In particular, there is an awareness of the impact of volatile organic compounds (VOCs) on the environment and human health, so the monitoring of human exposure to VOCs is an increasingly urgent need. Biomonitoring is theoretically more accurate compared with traditional ambient air monitoring, and it plays an essential role in human environmental exposure assessment. Breath analysis is a biomonitoring method with many advantages, which is applicable to assessments of human exposure to a large number of VOCs. Techniques are being developed to improve the sensitivity and precision of breath analysis based on in-direct and direct measurements which will be reviewed in this paper. This paper briefly reviews the frequently used methods in both of these categories, specifically highlighting some promising new techniques. Furthermore, this review also provides theoretical background knowledge about the use of breath analysis as a biomonitoring tool for human exposure assessment. A review of the application of breath analysis to human exposure monitoring during last two decades is also provided according to occupational/non-occupational exposure. Obstacles and potential challenges in this field are also summarized. Based on the gradual improvements in the theoretical basis and technology reviewed in this paper, breath analysis is an enormous potential approach for the monitoring of human exposure to VOCs.
Keywords: Breath analysis; Biomonitoring; Technology; Occupational exposure; Environmental exposure;

Simultaneous determination of imigliptin and its three metabolites in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry by Yang Liu; Dongyang Liu; Hongzhong Liu; Hanlin Song; Lin Song; Xueting Yao; Frank Wu; Chutian Shu; Xifeng Ma; Huimin Zhou; Ji Jiang; Pei Hu (300-312).
A specific and sensitive method was firstly developed using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) to simultaneously quantify imigliptin (KBP-3853) and its three metabolites (KBP-3926, KBP-3902, KBP-5493) in human plasma and urine. Solid-phase extraction (SPE) and direct dilution were used to extract imigliptin and its three metabolites from plasma and urine, respectively. The extracts were injected onto a SymmetryShield RP8 column with a gradient elution of acetonitrile and water containing 5 mM ammonium acetate (pH 7). Ionization of KBP-3853, KBP-3926, KBP-3902, KBP-5493, and XZP-3244 (internal standard, IS) was performed using an electrospray ionization (ESI) source in positive mode and detection was carried out with multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) of KBP-3853/KBP-3926/KBP-3902/KBP-5493 in human plasma and urine were 0.500/0.500/0.500/0.500 ng/mL and 20.0/20.0/10.0/10.0 ng/mL, respectively. Inter- and intra-batch precision of imigliptin and its three metabolites were less than 15% and the accuracy was within 85–115% for both plasma and urine. The extraction recoveries of all analytes at three concentration levels were consistent. The specificity, matrix effect, linearity and stabilities under various conditions were validated for imigliptin and its three metabolites in human plasma and urine. In conclusion, the validation results showed that this method was robust, specific, and sensitive and it can successfully fulfill the requirement of clinical pharmacokinetic study of imigliptin hydrochloride in Chinese healthy subjects.
Keywords: Imigliptin and metabolites; Human plasma and urine; LC–MS/MS;

Effect of lysozyme solid-phase PEGylation on reaction kinetics and isoform distribution by Benjamin Maiser; Kai Baumgartner; Florian Dismer; Jürgen Hubbuch (313-318).
Display OmittedThe combination of PEG–protein conjugation and chromatographic separation is generally known as solid-phase or on-column PEGylation and can provide advantages compared to commonly applied batch PEGylation. Even though the concept was already applied by several authors, changes in the isoform distribution compared to liquid-phase PEGylation due to sterically hindered PEGylation sites could not be confirmed. In this manuscript, a method for solid-phase PEGylation experiments in a 96-well plate format, using an automated liquid handling station is described. Applying size exclusion chromatography (SEC) and highly sensitive isoform analytics for mono-PEGylated lysozyme, we were able to investigate the differences in reaction kinetics and isoform distribution between adsorber-based PEGylation and modifications in free solution. Accordingly, solid-phase PEGylation with SP Sepharose FF and XL generally showed a reduced PEGylation reaction. In contrast to the predominant N-terminal PEGylation of lysozyme in liquid phase, a main modification of lys 97 and lys 116 was found for solid-phase experiments, which could be explained by binding orientations on corresponding adsorbent materials. Further experiments with varying amounts of bound protein additionally showed an influence on the isoform distribution of mono-PEGylated lysozyme.
Keywords: PEGylation; Lysozyme; On-column; Solid-phase PEGylation; High-throughput screening;

In this study, a strategy based on ultrafiltration-high performance liquid chromatography coupled with diode array detection (UF-HPLC-DAD) was proposed for screening tyrosinase specific inhibitors in Xanthii fructus. The false negatives were distinguished by optimizing the UF-HPLC-DAD parameters to reduce the background noise; the false positives were distinguished by introducing a blocked tyrosinase in the control group for comparison. To obtain the best blocker, the competitive experiments were performed using various known ligands. Using this strategy, three competitive inhibitors (protocatechuic acid; 3,5-di-O-caffeoylquinic acid; and 1,5-di-O-caffeoylquinic acid) and one mixed-type inhibitor (chlorogenic acid) were identified. These results were verified using tyrosinase inhibition assay, kinetic analysis, and structural simulation of the complex. Our experimental results suggest that the proposed strategy could be useful for high-throughput identification of tyrosinase specific inhibitors in natural products.
Keywords: Ultrafiltration; HPLC; Tyrosinase inhibitor; Binding affinity; Xanthium strumarium;

Expeditious discrimination of four species of the Panax genus using direct infusion-MS/MS combined with multivariate statistical analysis by Shinae Kim; Byong-kyu Shin; Dong Kyu Lim; Tae-Jin Yang; Johan Lim; Jeong Hill Park; Sung Won Kwon (329-336).
A practical approach based on direct infusion-MS/MS (DI-MS/MS) was demonstrated for metabolomic classification of four species in the Panax genus. The species Panax ginseng, Panax notoginseng, Panax quinquefolius and Panax vietnamensis were analyzed to develop an efficient tool for authenticating ginseng. Four target ions (m/z 783.5, 945.5, 1107.5 and 1149.2) were selected from LC–MS screening results for DI-MS/MS analysis. The target ions served as classifiers of the four species. As a targeted analysis, DI-MS/MS provided the structural identities of the target ions, clear spectral data and high sensitivity in a shorter time than routine LC–MS analysis. Principal component analysis and partial least squares-discriminant analysis of the DI-MS/MS fingerprinting revealed distinct grouping of the data. The results were validated by cross-validation and a permutation test to examine the utility of the statistical models. The spectral intensities of each species were compared with one another using box plots, which allowed straightforward authentication of the Panax species. The proposed method showed improved efficiency over other current methods for discrimination of large quantities of plant material. Additionally, to the best of our knowledge, this is the first study in which DI-MS/MS has been used to classify plant samples.
Keywords: Panax; Direct infusion MS/MS; LC–MS; Statistical model;

A specific and reliable HPLC-MS/MS method was developed and validated for the simultaneous determination of 2′,3′-dideoxyinosine (ddI) and the active metabolites, 2′,3′-dideoxyadenosine-5′-triphosphate (ddA-TP) in human peripheral-blood mononuclear cell for the first time. The analytes were separated on a HILIC column (100 mm × 2.1 mm, 1.7 μm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The cell homogenates sample was prepared by the solid phase extraction. The calibration curves were linear over a concentration range of 0.5–200.0 ng/mL for ddI and 0.25–100.0 ng/mL for ddA-TP. The intra-day and inter-day precision was less than 15% and the relative error (RE) were all within ±15%. The validated method was successfully applied to assess the disposition characteristics of ddI and support cell pharmacokinetics after the patients with AIDS were orally administrated with ddI and tenofovir disoproxyl fumarate (TDF).
Keywords: HPLC–MS/MS; ddI; dA-TP; Cell pharmacokinetics; Interaction;

Phthalates (dialkyl or alkyl phenyl esters of phthalic acid, benzene-1.2-dicarboxylic acid) are a group of industrial chemicals that have been used for more than 50 years. Phthalates are ubiquitous and can potentially have adverse effects on humans. The present study presents an accurate, sensitive and automated analytical method for measuring 12 phthalate metabolites (free and conjugated) in human urine using on-line solid phase extraction coupled to high performance liquid chromatography – electrospray ionization – tandem mass spectrometry. A small volume of urine sample (300 μL) is required. Glucoronidated phthalate metabolites are deconjugated by incubation with glucoronidase enzyme (Escherihia coli-K 12) and the reaction is stopped by adding formic acid. This is the only sample preparation needed prior to injection into the column switching system. Thus, the method involves minimal sample handling and minimizes possible contaminations from the surroundings. The method was validated by spiking synthetic urine at 5–8 levels in the range of 0.1–500 ng phthalate metabolites/mL synthetic urine. The method is sensitive with limits of detection in the low nanogram range, and rapid with a total run time about 25 min. The accuracy was between 90 and 120 % and the intermediate precision was given as relative standard deviation was below 20% for most of the compounds. The high sensitivity, high throughput and minimal manual handling make the method suitable for large-scale biomonitoring studies. The present method was applied for the determination of phthalate metabolites in urine samples from 116 pregnant women, a subproject within the Norwegian Mother and Child Cohort Study. Concentrations of all the twelve phthalate metabolites was >LOQ in 100% of the samples analysed. Mean urinary concentrations for different phthalate metabolites ranged from 1 to 100 ng/mL, the highest concentrations were observed for di-2-ethylhexyl phthalate (DEHP) metabolites and lowest for di-iso-nonyl phthalate (DiNP) metabolites. The urinary concentrations for most of the phthalate metabolites in the present study were found to be in the same range as found in other studies of pregnant women.
Keywords: Phthalate; Urine; Mass spectrometry; Column switching; Pregnant;

A rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the determination of ribavirin, sofosbuvir and its metabolite GS-331007 in rat plasma was established. The analytes and the internal standard (midazolam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1 mm × 50 mm, 1.7 μm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 245.1 → 113.1 for ribavirin, m/z 530.3 → 243.1 for sofosbuvir, m/z 261.5 → 113.1 for GS-331007 and m/z 326.2 → 291.1 for midazolam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 5–1000 ng/mL for ribavirin, 10–2000 ng/mL for sofosbuvir and 10–2000 ng/mL for GS-331007. Total time for each chromatograph was 3.0 min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) <10.0% and the accuracy values ranged from −10.6% to 11.6%. The method was successfully applied to a pharmacokinetic study of ribavirin, sofosbuvir and GS-331007 in rats.
Keywords: Ribavirin; Sofosbuvir; GS-331007; UPLC–MS/MS; Plasma; Pharmacokinetics;

Macitentan is a newly approved endothelin receptor antagonist (ERA) for the long-term treatment of PAH with superior receptor-binding properties and a longer duration of action compared to other available ERAs. However, analytical methods for simultaneous determination of macitentan and its active metabolite, ACT-132577, in human plasma have not been fully reported in the literature. In this work, a fast, sensitive, and reliable high-performance liquid chromatography-tandem mass spectrometry method (HPLC–MS/MS) was firstly developed and completely validated for simultaneous determination of macitentan and its active metabolite in human plasma. Plasma samples were processed with a protein precipitation using acetonitrile, followed by chromatographic separation using an Inertsil ODS-SP column (100 × 2.1 mm, 3.5 μm) under isocratic elution with a mobile phase consisting of acetonitrile and 0.2% formic acid at a flow rate of 0.3 mL/min. Quantification was operated in multiple reaction monitoring (MRM) mode using the transitions m/z 547.1 → 201.0 for macitentan, m/z 589.0 → 203.0 for ACT-132577, and m/z 380.5 → 243.3 for the IS (donepezil). The assay exhibited a linear range of 1–500 ng/mL for both macitentan and ACT-132577. The accuracy and the intra- and inter-precisions were within acceptable ranges and no significant matrix effect was observed during the method validation. The developed method was successfully utilized to a human pharmacokinetic study of macitentan as well as ACT-132577 after oral administration of 10 mg macitentan tablet in healthy Chinese volunteers.
Keywords: Macitentan; Metabolite; HPLC–MS/MS; Human plasma; Pharmacokinetic;

Vacuum-powered bubble-assisted solvent extraction followed by macroporous resin enrichment for isolation of podophyllotoxin from Sinopodophyllum emodi by Tingting Liu; Lei Yang; Xiaoyu Sui; Jie Zhang; Li Li; Shuang Fu; Wenjing Li; Xin Liang (364-371).
A vacuum-powered bubble-assisted solvent extraction (VBE) technique was used to extract podophyllotoxin from the root of Sinopodophyllum emodi. We optimized the VBE procedure and showed it had the highest efficiency of extraction compared to other conventional extraction techniques. Based upon the results of single-factor experiments, a three-factor, three-level experiment design was developed by application of a Box–Behnken design. The method was validated by stability, repeatability and recovery experiments. The optimal conditions were: solvent, 60% (v/v) ethanol; particle size of the sample, 60–80 mesh; soak time, 2 h; liquid/solid ratio, 21 L/kg; air flow, 32 mL/min; vacuum-powered bubble extraction time, 65 min. The VBE method we developed achieved efficient extraction of podophyllotoxin from S. emodi. The podophyllotoxin extracted can be enriched and separated by an HPD300 macroporous resin adsorption and desorption process. The results indicated that VBE is a convenient, rapid and efficient sample preparation technique.
Keywords: Sinopodophyllum emodi; Podophyllotoxin; Vacuum-powered bubble-assisted solvent extraction; Response surface methodology; Macroporous resin;

Dihydrochalcones are the main active components of Lithocarpus polystachyus Rehd. (Sweet Tea), they are directly related to the sweet tonic beverage and traditional herb. In this work, two runs of preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane/ethyl acetate/ethanol/water (1:4:3:4, v/v) were employed to separate three dihydrochalcones (phloridzin, trilobatin and phloretin) from Sweet Tea. About 6.4 mg of phloridzin, 48.4 mg of trilobatin, and 4.7 mg of phloretin with purities of 96.7%, 98.4% and 98.1% were obtained from 130 mg of the crude Sweet Tea extract. Phloridzin, trilobatin, and phloretin had effective radical scavenging activities, with IC50 values of 866.80, 20.16 and 179.47 μg/mL, respectively, in a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method. The contents of phloridzin, trilobatin and phloretin in dried old leaves and tender leaves of tea were in the range of 10.1–18.0, 113.7–128.8, 3.6–4.3 mg/g and 9.3–9.8, 82.9–103.1, 1.9–2.5 mg/g, respectively. The results indicated that the HPLC had good precision, accuracy and repeatability for the determination of three dihydrochalcones in samples.
Keywords: Dihydrochalcone; Preparative separation; Antioxidant activity; Quantification; Sweet Tea;

Endogenous monoamine neurotransmitters play an essential role in neural communication in mammalians. Many quantitative methods for endogenous monoamines have been developed during recent decades. Yet, matrix effect was usually a challenge in the quantification, in many cases asking for tedious sample preparation or sacrificing sensitivity. In this work, a simple, fast and sensitive method with no matrix effect was developed to simultaneously determine four endogenous monoamines including serotonin, dopamine, epinephrine and norepinephrine in rat brain tissues, using hydrophilic interaction liquid chromatography coupled with atmospheric-pressure chemical ionization tandem mass spectrometry. Various conditions, including columns, chromatographic conditions, ion source, MS/MS conditions, and brain tissue preparation methods, were optimized and validated. Pre-weighed 20 mg brain sample could be effectively and reproducibly homogenized and protein-precipitated by 20 times value of 0.2% formic acid in cold organic solvents (methanol–acetonitrile, 10:90, v/v). This method exhibited excellent linearity for all analytes (regression coefficients > 0.998 or 0.999). The precision, expressed as coefficients of variation, was less than 3.43% for intra-day analyses and ranged from 4.17% to 15.5% for inter-day analyses. Good performance was showed in limit of detection (between 0.3 nM and 3.0 nM for all analytes), recovery (90.8–120%), matrix effect (84.4–107%), accuracy (89.8–100%) and stability (88.3–104%). The validated method was well applied to simultaneously determine the endogenous serotonin, dopamine, epinephrine and norepinephrine in four brain sections of 18 Wistar rats. The quantification of four endogenous monoamines in rat brain performed excellently in the sensitivity, high throughput, simple sample preparation and matrix effect.
Keywords: Monoamine; Serotonin; Dopamine; Epinephrine; Norepinephrine; Hydrophilic interaction liquid chromatography; Tandem mass spectrometry; Brain;

For the first time, electrospun polystyrene nanostructure was used as coating material on a stainless steel wire for solid-phase microextraction. Surface morphology of the coating was studied by scanning electron microscopy which showed the formation of nanofibers on the wire. The coating was stable after conditioning at 250 °C for 2 h. The efficiency of the polystyrene coating was approved by extracting a mixture of seven pesticides (polar and apolar) from head space of honey samples followed by gas chromatography–mass spectrometry. The important parameters affecting extraction efficiency such as, extraction time and temperature, desorption conditions, agitation rate and ionic strength were investigated. Under optimized experimental conditions, detection limits for the investigated pesticides ranged from 0.1–2 μg L−1. The intra- and inter-day precisions of the developed method were 3.5–17.6% and 10.0–25.0%, respectively. Finally, all the investigated pesticides were spiked to honey samples and extracted by the proposed method. The accuracies of determination of all the species were found to be in the range of 81–125%.
Keywords: Polystyrene; Electrospinning; Solid-phase microextraction; Pesticide residues; Honey;

Human nails metabolite analysis: A rapid and simple method for quantification of uric acid in human fingernail by high-performance liquid chromatography with UV-detection by Xi-Ling Li; Gao Li; Ying-Zi Jiang; Dongzhou Kang; Cheng Hua Jin; Qing Shi; Toufeng Jin; Koichi Inoue; Kenichiro Todoroki; Toshimasa Toyo'oka; Jun Zhe Min (394-398).
A rapid and simple analytical method for the quantification of uric acid (UA) in human fingernails by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. UA was extracted from human fingernail samples at 90 °C for 20 min, then separated on an Inertsil ODS-2 column (250 × 4.6 mm I.D., 5.0 μm, GL Sciences) by isocratic elution using methanol: 74 mM phosphate buffer (pH 2.2) 2:98 (v/v). An UV detector was used to monitor at 284 nm. The results indicated that under optimized measurement conditions results were achieved within 8.0 min, and a good linearity was achieved from the calibration curves (r 2  > 0.9999) in the range of 1.0–10000 ng; the limit of detection (S/N = 3) was 2.0 pg, the inter-day and intra-day assay precisions were all less than 0.46% and the mean recoveries (%) of the uric acid spiked in the human fingernail were 101.95%. The amounts of UA in the fingernails of healthy volunteers were determined.
Keywords: Human fingernail; Uric acid; High-performance liquid chromatography with ultraviolet detection;

Bile acids (BAs) are crucial for the diagnosis, follow-up, and prognostics of liver injuries and other BA metabolism related diseases. In particular, rodent unique BAs, α-muricholic acid (α-MCA), β-MCA, ω-MCA, tauro-α-MCA (α-TMCA), and β-TMCA, are valuable biomarkers for preclinical drug development. To the best of our knowledge, however, a simple, selective, sensitive, and robust analytical method for ω-MCA and taurine-conjugated MCAs has never been reported. We have developed a simple, selective, and sensitive analytical method for measurement of 16 BAs including the five rodent unique BAs in rat plasma using an ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) method. Activated charcoal was utilized to prepare BA-free plasma, which served as the surrogate matrix for the preparation of calibration standards and quality control (QC) samples. Results of matrix effects evaluation suggested that the BA-free plasma could be adequate as a surrogate matrix for BAs determination. Three stable isotope labelled internal standards were separated by reverse phase UPLC using gradient elution and were detected by TOF-MS in negative ion mode. The calibration curve was linear for all BAs over a range of 10–25 ng/mL to 1000–10,000 ng/mL, with overall imprecision below 15% and 20% at lower limit of quantification (LLOQ), respectively. This analytical method was used to determine BA concentrations in more than 300 plasma samples from rats with liver injuries induced using α-naphthylisocyanate, carbon tetrachloride, or flutamide. The alteration of BA concentrations was most evident for necrosis, and cholestasis hepatotoxins, with more subtle effects by steatosis and idiosyncratic hepatotoxins. In conclusion, we have developed a simple, selective, and sensitive analytical method to measure plasma 16 BAs including 5 rodent unique BAs, α-MCA, β-MCA, ω-MCA, α-TMCA, and β-TMCA. Our data suggested that α-TMCA and β-TMCA could be useful for identification or prediction of liver injuries, a currently unmet need in preclinical toxicity. Our method using TOF-MS is useful to determine BAs in rat plasma and of use in structural analyses of metabolites in early stage of drug development.
Keywords: Muricholic acid; UPLC-TOF-MS; Biomarker; Liver injury; Rat Plasma; Bile acid;

In the present work, a rapid ionic liquid-based microwave-assisted extraction (ILMAE) method was successfully applied to simultaneous extraction of baicalin, wogonoside, baicalein and wogonin from Scutellaria baicalensis Georgi. A series of 1-alkyl-3-methylirnidazolium ionic liquids with different anions and cations were assessed for extraction efficiency, and 1-octyl-3-methylimidazolium bromide was selected as the optimal solvent. In addition, the parameters of ILMAE procedure for the four flavonoids were optimized, and the optimal ILMAE method was validated in the linearity, stability, precision and recovery. Meanwhile, the microstructures of S. baicalensis powders were observed before and after extraction with the help of a scanning electron microscope (SEM) in order to explore the extraction mechanism, and the activity of the crude enzyme solution from S. baicalensis was determined through the hydrolysis of baicalin. Finally, the extraction yields and extraction time of WaterHRE, WaterMAE, ILHRE and Chp were 5.18% (30 min), 8.77% (90 s), 16.94% (30 min) and 18.58% (3 h), respectively. The results indicated that compared with the conventional extraction approaches, ILMAE possessed great advantages in extracting flavonoids, such as the highest extraction yield (22.28%), the shortest extraction time (90 s), etc.
Keywords: Ionic liquids; Microwave-assisted extraction (MAE); Flavonoids; Scutellaria baicalensis Georgi; Scanning electron microscopy (SEM);