Journal of Chromatography B (v.898, #C)

Display Omitted► Validated methods for the vascular targeted cancer drug combretastatin A1 in human plasma and urine by HPLC–MS. ► Validated methods for the phosphate pro-drug and three glucuronides found in human urine by HPLC–MS. ► Application to the analysis of patient samples following a Phase I clinical trial of combretastatin A1 phosphate.Validated methods for the determination of CA1, the active agent derived from the prodrug CA1P, in human plasma and urine, and of CA1P and three glucuronides CA1G1, CA1G2 and CA1DG in human urine were developed using LC–MS. Plasma CA1 was extracted using solid phase extraction and validated over the range 5–1000 nM. Urine samples were analysed without extraction, and the assays validated over the range 50–2000 nM (CA1P), 25–2000 nM (CA1), 50–40,000 nM (CA1G1 and CA1G2) and 25–4000 nM (CA1DG). The mean correlation coefficient (r 2) was ≥0.997 for all assays. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical methods (<15%). Mean recovery of CA1 from plasma was 101%, and 97% from urine. Mean urine recovery of CA1P was 98%, CA1G1 96%, CA1G2 93% and CA1DG 93%. The method was applied to plasma and urine samples from a recently completed clinical trial of the prodrug. Peak plasma concentrations of up to 470 nM CA1 were seen. The majority of drug-related material measured in urine comprised of the two monoglucuronides; CA1 and the diglucuronide were about 10-fold lower. No CA1P was detectable in urine.
Keywords: OXi4503; CA1P; Plasma; Urine; HPLC–MS; Glucuronide metabolites;

► HBsAg protein, when over expressed in P. patoris gets associated with membranes. ► The membrane association of HBsAg can be disrupted by 2% Tween-20. ► A centrifugation step followed by lysis ensures removal of supernatant impurities. ► Phenyl-600M Toyopearl resin can purify HBsAg to homogeneity. ► Membrane purified HBsAg elicits protective antibody responses in mice.The recombinant hepatitis B surface antigen (HBsAg) vaccine provides excellent protection against hepatitis B virus (HBV). However, high costs of its production prevents many underdeveloped and developing nations from implementing HBsAg vaccination. This in turn increases the risk of contracting HBV related diseases. Majority of the commercial HBV vaccines are derived from purified HBsAg expressed in recombinant yeasts. Most of the cost in production of the vaccine is incurred during the downstream processing. The costs associated with HBsAg purification can be decreased by optimizing the pre-chromatography steps and by reducing the impurity burden on chromatography operations. Here in this work we present a novel strategy for the enriched extraction of recombinant HBsAg from Pichia pastoris membranes. We have also developed a simple, easy to operate process for the purification of HBsAg VLPs from the membranes of P. pastoris. This novel strategy, while utilizing a single column chromatographic step in the purification scheme results in the highest recovery of HBsAg VLPs reported in the literature. The yield of HBsAg at the end of purification was nearly 5% (85 μg/g of induced wet cell biomass). The HBsAg purified from this process has shown the presence of VLPs. The immunization of these VLPs in BALB/c mice with alhydrogel adjuvant has shown good titers of neutralizing antibodies.
Keywords: HBsAg; Pichia pastoris; Virus like particle; Purification; Membrane; Vaccine;

A synthetic Protein G adsorbent based on the multi-component Ugi reaction for the purification of mammalian immunoglobulins by Jianing Qian; Graziella El Khoury; Hamzah Issa; Khaled Al-Qaoud; Penelope Shihab; Christopher R. Lowe (15-23).
De novo design and synthesis by the Ugi multicomponent reaction of a ligand mimicking Protein G binding to immunoglobulins (IgGs). ► Purification of IgGs from various mammalian species. ► Effective elution at neutral pH conditions. ► Comparison of the Ugi adsorbent with Protein A and Protein G adsorbents. ► Purification of camelid IgGs including heavy-chain only antibodies from camel milk.Numerous efforts have been devoted to develop synthetic affinity ligands mimicking natural immunoglobulin-binding proteins, such as Proteins A and L, in order to overcome intrinsic drawbacks involving their high cost and acidic pH elution. However, few reports have focused on a Protein G mimic. This work describes the use of the solid phase multi-component Ugi reaction to generate a low cost, rationally designed, affinity ligand to mimic Protein G for the purification of mammalian immunoglobulins, including the heavy-chain only camelid IgGs, with effective elution at neutral pH. An aldehyde-functionalised Sepharose™ resin constituted one component (aldehyde) of the four-component Ugi reaction, whilst the other three components (a primary or secondary amine, a carboxylic acid and an isonitrile) were varied to generate a tri-substituted Ugi scaffold, with a wide range of functionality, suitable for mimicking peptides for immunoglobulin purification. Ligand A2C11I1 was designed to mimic Asn35 and Trp43 of Protein G (PDB: 1FCC) and in silico docking into the Fc domain showed a key binding interface closely resembling native Protein G. This candidate ligand demonstrated affinity towards IgGs derived from human, cow, goat, mouse, sheep, pig, rabbit and rat serum, chicken IgY and recombinant camelid Fc domain, out of which cow and sheep IgG demonstrated 100% binding under the conditions selected. Preparative chromatography of IgG from human serum under a standardised buffer regime eluted IgG of ∼65% purity, compared to ∼62% with Protein G. This adsorbent achieved highest elution of IgG at neutral pH (0.1 M sodium phosphate pH 7.0, 30%, v/v, ethylene glycol), an advantage for purifying antibodies sensitive to extremes of pH. The ligand demonstrated a static binding capacity of 24.6 mg IgG ml−1 resin and a dissociation constant (K d) of 4.78 × 10−6  M. The solid phase Ugi scaffold provides a strategy to develop pseudo-biospecific ligands to purify immunoglobulins and other potentially high-value biotherapeutic proteins.
Keywords: Synthetic affinity ligand; Ugi multi-component reaction; Mammalian immunoglobulin purification; Camelid IgG;

Computational design and synthesis of molecular imprinted polymers for selective extraction of allopurinol from human plasma by Mehrdad Tabandeh; Soheila Ghassamipour; Heydar Aqababa; Meisam Tabatabaei; Meisam Hasheminejad (24-31).
► Rational development of polymers for selective extraction of allopurinol from human plasma. ► Computational modeling combined with molecular imprinting technology to obtain polymers. ► Production of MIPs capable of extracting allopurinol from human plasma.The present study was focused on the rational development of polymers for selective extraction of allopurinol (ALP) from human plasma. Therefore, a computational modeling approach was combined with the molecular imprinting technology to obtain the polymers. The computational approach was used in order to screen the functional monomers as well as the polymerization solvents for rational design of molecular imprinted polymers (MIPs). It was based on the comparison of the binding energy (ΔE) of the formed complexes between the template molecule and different functional monomers. In the design, the effect of the polymerization solvent was also included using the polarizable continuum model. The theoretical calculation results showed that among virtual solvents tested, acrylamide (AAM) gave the largest ΔE while acrylonitrile (ACN) gave the smallest ΔE in acetone. Therefore, the MIP prepared using AAM as functional monomer in acetone was desired. To examine the validity of this approach, three MIPs were synthesized with different functional monomers i.e. AAM, acrylic acid (AA), and ACN, and then evaluated using Langmuir–Freundlich (LF) isotherm. The results obtained from this experiment confirmed the computational results that the MIP prepared by AAM was the most appropriate adsorbent. Subsequently, the MIP was used to develop a molecular imprinted solid-phase extraction (MISPE) procedure. Finally, the MISPE procedure followed by HPLC was developed for selective extraction and determination of allopurinol in human plasma. For the proposed MISPE method, the linearity between peak area and concentration was found in the range of 0.100–25.000 μM with a linear regression coefficient (R 2) of 0.995. The limit of detection (LOD) and quantification (LOQ) in plasma were 0.028 and 0.093 μM, respectively. The results of this study indicated the possibility of using computer aided design for rational selection of functional monomers and solvents for preparation of the MIPs capable of extracting allopurinol from human plasma.
Keywords: Molecular imprinted polymers (MIP); Allopurinol; Bonding energy; Computational approach;

► Published analytic methods for tegafur are suboptimal for pharmacokinetic studies. ► An analytic method was developed for the anticancer drug tegafur and its metabolite. ► Accuracy, precision and recovery of the analytes from plasma were acceptable. ► The method was used to successfully determine human pharmacokinetic parameters.Tegafur is a 5-fluorouracil (5-FU) prodrug widely used outside the United States to treat colorectal cancer as well as cancers of the head and neck. The resulting plasma concentrations of tegafur are much higher than those of 5-FU; thus, analytical methods are needed that are sensitive enough to detect low plasma concentrations of 5-FU and robust enough to simultaneously analyze tegafur. Previous LC–MS/MS methods have either failed to demonstrate the ability to simultaneously measure low 5-FU and high tegafur plasma levels, or failed to be applicable in clinical studies. Our goal was to develop a method capable of measuring low concentrations of 5-FU (8–200 ng/ml) and high concentrations of tegafur (800–20,000 ng/ml) in human plasma and to subsequently evaluate the utility of the method in patient samples collected during a phase I clinical study where oral doses of either 200 mg or 300 mg UFT®/LV (uracil and tegafur in a 4:1 molar ratio plus leucovorin) were administered. A combined LC–MS/MS and LC–UV method was developed utilizing negative ion atmospheric pressure ionization (API). The method provides an accuracy and precision of <10% and <6%, respectively, for both analytes. Material recoveries from the liquid–liquid extraction technique were 97–110% and 86–91% for tegafur and 5-FU, respectively. Utilization of this method to determine tegafur and 5-FU plasma concentrations followed by noncompartmental pharmacokinetic analyses successfully estimated pharmacokinetic parameters (C MAX, t MAX and AUC0–10 h) in the clinical study patients. Overall, this method is ideal for the simultaneous bioanalysis of low levels of 5-FU and relatively higher levels of its prodrug, tegafur, in human plasma for clinical pharmacokinetic analysis.
Keywords: 5-Fluorouracil; Pharmacokinetics; Phase I studies; Esophageal cancer;

► We established a GC–MS/MS method for determination of 284 analytes in adipose sample. ► We conducted systematic method optimisation, including extraction and cleanup. ► Method validation showed performance characteristics for each analyte were satisfied. ► The method has been successfully applied for human adipose tissue samples. ► It was an efficient tool for evaluating human exposure extent of contaminants.This paper describes a method for the simultaneous determination of 284 environmental contaminants, including 57 pesticides, 15 polycyclic aromatic hydrocarbons (PAHs), 209 polychlorinated biphenyls (PCBs) and 3 phthalate esters (PAEs), in adipose tissue samples. For the first time, a gas chromatography–tandem mass spectrometry (GC–MS/MS) method following a homogenised extraction using acetonitrile and purification by gel permeation chromatography (GPC) was used. Various performance characteristics, such as the limit of detection (LOD), limit of quantification (LOQ), linear range, recovery and precision, were determined for each analyte. The LOD for most analytes was below 0.01 mg/kg. The recoveries and relative standard deviations (RSDs) were determined by spiking untreated samples with the analytes at the LOQ, 2 × LOQ and 4 × LOQ levels. The average recovery for most pesticides was between 70% and 120% and the precision values, expressed as RSD, were all below 20.4% (n  = 6). This method may provide an efficient tool for evaluating the extent of exposure to organic contaminants using human adipose tissue.
Keywords: Pesticides; Polycyclic aromatic hydrocarbons (PAHs); Polychlorinated biphenyls (PCBs); Phthalate esters (PAEs); GC–MS/MS;

Determination of reboxetine in rat brain microdialysates and plasma samples using liquid chromatography coupled to fluorescence detection by Naser Shraim; Ralph Clinckers; Sophie Sarre; Yvette Michotte; Ann Van Eeckhaut (53-61).
► Reboxetine is a selective noradrenalin reuptake inhibitor. ► We developed a liquid chromatographic method with fluorescence detection. ► Validation was performed according to the existing guidelines. ► Reboxetine was quantified in rat brain microdialysis and plasma samples. ► The method was applicable for pharmacokinetic profiling.A liquid chromatographic method with fluorescence detection was developed and validated for the quantification of the antidepressant reboxetine (RBX), a selective noradrenalin reuptake inhibitor, in rat brain microdialysates. After modification of the method in terms of sample preparation and sensitivity, it was also validated for the quantification of RBX in rat plasma samples. To enable fluorescence detection, a pre-column derivatization step with 9-fluorenylmethyl chloroformate was included. Separations were performed on a reversed phase C18 column using gradient elution. The retention time for RBX was found to be 8.8 min. The assay of RBX in brain microdialysis samples showed a linear relationship in the calibration curve from 2 to 200 ng/mL, with a correlation coefficient ≥0.999. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 0.6 and 2.0 ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.5% and 11.7% with an average recovery of 101.2 ± 8.2% (mean ± SD, n  = 40). For the analysis of plasma samples, the calibration curve was linear between 20 and 700 ng/mL with a correlation coefficient ≥0.999. LOD and LLOQ were 6 and 20 ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.7% and 11.5% with an average recovery of 98.5 ± 7.3% (mean ± SD, n  = 40). We demonstrated the applicability of the method to determine the concentration–time profiles of RBX in brain and plasma following systemic administration.
Keywords: Reboxetine; Liquid chromatography; Fluorescence detection; Intracerebral microdialysis; Plasma; Rat;

A determinative and confirmatory method for ceftiofur metabolite desfuroylceftiofur cysteine disulfide in bovine kidney by LC–MS/MS by Shixia Feng; Chaitali Chattopadhaya; Philip Kijak; Oscar A. Chiesa; Elizabeth A. Tall (62-68).
► We developed a new LC–MS/MS method for ceftiofur metabolite in bovine kidney. The new method is both determinative and confirmatory. The method utilizes a simple and effective extraction procedure. A deuterated internal standard was synthesized and used for quantitation.Ceftiofur is a cephalosporin β-lactam antibiotic widely used for treating certain bacterial infections in beef and dairy cattle. The regulatory HPLC–UV method for ceftiofur residues in animal tissues is time consuming and non-specific. Additionally, because the regulatory method involves chemical reactions to convert the metabolites into a single moiety, it is virtually impossible to incorporate the procedure into a multi-residue method. Ceftiofur residue violations in beef and dairy cattle have been frequently reported and therefore an improved method is needed. Herein we report a rapid and sensitive LC–MS/MS method for the determination and confirmation of ceftiofur metabolite, desfuroylceftiofur cysteine disulfide (DCCD), in bovine kidney tissue. The new method utilizes a simple extraction with phosphate buffer followed by SPE cleanup. A deuterated internal standard was synthesized and used for quantitation. The matrix-based calibration curve was linear from 25 to 2000 ng/g. The average accuracy for control kidney samples from six different sources fortified at 50–1000 ng/g was 97.7–100.2% with CV ≤ 10.1%. The limit of confirmation was 50 ng/g.
Keywords: Ceftiofur; Desfuroylceftiofur cysteine disulfide; Bovine kidney; LC–MS/MS;

Uptake and metabolism of olive oil polyphenols in human breast cancer cells using nano-liquid chromatography coupled to electrospray ionization–time of flight-mass spectrometry by Rocío García-Villalba; Alegría Carrasco-Pancorbo; Cristina Oliveras-Ferraros; Javier A. Menéndez; Antonio Segura-Carretero; Alberto Fernández-Gutiérrez (69-77).
► Evaluation of cellular uptake/metabolism of olive oil phenols in breast cancer cells. ► An important application of nano-LC MS in a field where not extensively used. ► Some compounds are absorbed, metabolized and excreted to the extra cellular medium.Polyphenols from extra virgin olive oil (EVOO), a main component of the Mediterranean diet, have demonstrated repeatedly anti-tumor activity in several in vitro and in vivo studies. However, little is known about the efficiency of the absorption process and metabolic conversion of these compounds at cellular level. In this study, a nano liquid chromatography–electrospray ionization–time of flight mass spectrometry (nanoLC–ESI–TOF MS) method was developed to study the cellular uptake and metabolism of olive oil phenols in JIMT-1 human breast cancer cells. After incubation for different time periods with EVOO-derived phenolic extracts, culture media, cytosolic fraction and solid particles fraction were separated and analyzed. Most of the free phenols, mainly hydroxytyrosol, its secoiridoid derivatives, and the flavonoid luteolin, disappeared in the culture media in different ways and at different times. Besides, several metabolites were detected in the culture media, fact that may indicate absorption and intracellular metabolism followed by rapid cellular export. Low intracellular accumulation was observed with only traces of some compounds detected in the cytosolic and solid particles fractions. Methylated conjugates were the major metabolites detected, suggesting a catalytic action of catechol-O-methyl transferase (COMT) in cancer cells.
Keywords: Olive oil; Phenolic compounds; Cancer cells; Metabolites; Nano-liquid chromatography; Electrospray–time of flight-mass spectrometry;

RETRACTED: Pilot production of recombinant human clotting factor IX from transgenic sow milk by Yu-ling Sun; Yuo-sheng Chang; Yin-shen Lin; Chon-ho Yen (78-89).
Available online 26 April 2012This article has been retracted at the request of the Authors.The authors would like to apologise as they unwittingly used certain proprietary information that they were not entitled to and so have found that they have no choice but to request a retraction of the article. The mistake was made in good faith.

► New method for the analysis of bisphenol A (BPA) and BPA–glucuronide is proposed. ► Use of d6-labelled BPA–glucuronide internal standard to quantify conjugated BPA. ► New method applied to BPA determination in maternal serum and umbilical cord serum.Bisphenol A is widely used as a monomer in the manufacture of polycarbonates and epoxy resins, as an antioxidant in polyvinyl chloride (PVC) plastics and as an inhibitor of end polymerisation in PVC. Several different methods have been used to quantify total BPA in biological specimens. However, quantification of both free and conjugated BPA continues to present challenges. Moreover, there is limited data concerning fetal exposure. Therefore, the objective of this study was to develop a new method for the analysis of both free and conjugated BPA in human maternal and umbilical cord blood serum. For the analysis of free BPA, the method consisted of a liquid–liquid extraction followed by a two-step solid-phase extraction sample cleanup on Florisil and Oasis HLB sorbents, derivatization of the extract using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analysis by gas chromatography/tandem mass spectrometry (GC/EI-MS/MS). To determine the amount of conjugated BPA in serum samples, bisphenol A-d6 β-glucuronide (4-[1-(4-hydroxyphenyl)-1-methylethyl-d6]phenyl β-d-glucopyranosiduronic acid) was added to each sample prior to enzymatic deconjugation. The MDL and LOQ for BPA were 0.026 ng/mL and 0.087 ng/mL, respectively. The observed recoveries ranged between 65% and 88%. The new method was applied to the determination of paired human maternal and umbilical cord blood serum samples. The results demonstrated that total BPA concentrations in human maternal serum at mid-pregnancy and at delivery ranged from <0.026 ng/mL to 10.425 ng/mL (median 0.548 ng/mL, n  = 12) and <0.026 ng/mL to 3.048 ng/mL (median 1.461 ng/mL), respectively. Results for matching umbilical cord blood serum BPA concentrations were in the range of <0.026–2.569 ng/mL (median 1.823 ng/mL). The concentrations measured in this study agreed well with BPA levels in human serum reported internationally. Only 2 mid-pregnancy serum samples out of 12 contained quantifiable amounts of conjugated BPA, indicating that BPA–glucuronide is not abundant in either human maternal or umbilical cord blood serum.
Keywords: Bisphenol A; Bisphenol A-d6 β-glucuronide; Human maternal serum; Umbilical cord blood serum; Derivatization; GC/EI-MS/MS;

Identification of rutin deglycosylated metabolites produced by human intestinal bacteria using UPLC–Q-TOF/MS by Jing Yang; Dawei Qian; Shu Jiang; Er-xin Shang; Jianming Guo; Jin-ao Duan (95-100).
► Quercetin 3-O-glucoside and leucocyanidin are deglycosylated metabolites of rutin. ► The deglycosylated pathway of rutin by human intestinal bacteria. ► α-l-rhamnosidase and β-d-glucosidase activities of five human intestinal bacteria. ► UPLC–Q-TOF/MS was used for identifying the metabolites of rutin.In this paper, rutin was metabolized by human intestinal bacteria and five isolated strains including Bacillus sp. 52, Bacteroides sp. 45, 42, 22 and Veillonella sp. 32, the metabolites were identified using ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC–Q-TOF/MS). As a result, Bacillus sp. 52 and Bacteroides sp. 45 could metabolize rutin to quercetin 3-O-glucoside and leucocyanidin. Bacteroides sp. 42 and Veillonella sp. 32 could convert rutin to leucocyanidin. Bacteroides sp. 22 could hydrolyze rutin to quercetin-3-O-glucoside. In order to further explain the metabolism pathway of rutin, the β-d-glucosidase and α-l-rhamnosidase activities of five strains were determined. Bacteroides sp. 22 could produce α-l-rhamnosidase but did not produce β-d-glucosidase or β-d-glucosidase activity was too low to be detected. The other four strains all demonstrated α-l-rhamnosidase and β-d-glucosidase activities. Furthermore, α-l-rhamnosidase and β-d-glucosidase activities of Veillonella sp. 32 and Bacteroides sp. 42 were higher than those of Bacteroides sp. 45 and Bacillus sp. 52. Based on these results, we can propose the deglycosylated rout of rutin: rutin was metabolized to be quercetin-3-O-glucoside by α-l-rhamnosidase produced from these bacteria, thereafter, quercetin-3-O-glucoside was further metabolized by β-d-glucosidase to form leucocyanidin. Because of the higher enzyme activity in Veillonella sp. 32 and Bacteroides sp. 42, quercetin-3-O-glucoside was completely metabolized to leucocyanidin by these two bacteria. Due to the lack of β-d-glucosidase activity, Bacteroides sp. 22 could not further metabolize quercetin-3-O-glucoside to leucocyanidin. This study will be helpful for understanding the deglycosylated rout of rutin and the role of different intestinal bacteria on the metabolism of natural compounds.
Keywords: Rutin; Intestinal bacteria; UPLC–Q-TOF/MS; Deglycosylation; Enzymic activity;

Simultaneous extraction and determination of HBCD isomers and TBBPA by ASE and LC–MSMS in fish by Guillaume ten Dam; Olga Pardo; Wim Traag; Martijn van der Lee; Ruud Peters (101-110).
► The simultaneous determination of HBD isomers and TBBPA. ► TBBPA adsorbs by hydrogen bonding to analytical materials. ► α- and γ-HBCD are not fully resolved from δ-, ɛ-HBCD in LC separation. ► Identification of HBCD by LC–MS/MS does not meet the EU criteria (2002/657/EC). ► HBCD has been found up to 134 ng/g in eel.Since the EFSA enquired a call for data for TBBPA and HBCD in 2009, the analytical determination of these compounds in food became of regulatory interest. Therefore, a method for the simultaneous determination of TBBPA and the three major HBCD stereoisomers was developed. Conventional techniques like soxhlet, ASE, GPC, sulphuric acid digestion, and acidified silica SPE are generally used in sample pre-treatment while detection is mostly performed by LC–MSMS. A combined analysis of HBCD and TBBPA is problematic due to the hydroxyl groups in the TBBPA molecule. However, using a specific mesh-size sodium sulphate in ASE extraction and an acid silica column combined with a Sep-pack Plus silica cartridge for purification resulted in recoveries between 80% and 110% for all compounds. The accuracy and reproducibility determined using proficiency test samples were 104% and 4% for the sum of the HBCD isomers. Typical limits of detection were 0.01 ng/g product or 0.004 ng on column, while the linear dynamic range is between 0.01 ng and 10 ng on column. Levels of TBBPA and HBCD isomers were determined in eel samples. TBBPA was occasionally detected and only marginally above the quantification limit of 0.05 ng/g, whereas total amounts of HBCD were between 0.2 and 150 ng/g with α-HBCD being the dominant HBCD isomer.
Keywords: HBCD; TBBPA; LC–MS/MS; Extraction; Clean-up; Eel;

Intracellular metabolite profiling of platelets: Evaluation of extraction processes and chromatographic strategies by Giuseppe Paglia; Manuela Magnúsdóttir; Steinunn Thorlacius; Ólafur E. Sigurjónsson; Sveinn Guðmundsson; Bernhard Ø. Palsson; Ines Thiele (111-120).
► This is the first metabolomic study reporting analytical protocols for platelets. ► Seven different extraction methods and two different UPLC–MS methods were tested. ► MeOH–H2O extraction coupled with HILIC–MS method identified 107 metabolites.An extraction method for intracellular metabolite profiling should ideally be able to recover the broadest possible range of metabolites present in a sample. However, the development of such methods is hampered by the diversity of the physico-chemical properties of metabolites as well as by the specific characteristics of samples and cells. In this study, we report the optimization of an UPLC–MS method for the metabolite analysis of platelet samples. The optimal analytical protocol was determined by testing seven different extraction methods as well as by employing two different LC–MS methods, in which the metabolites were separated by using hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC). The optimal conditions were selected using the coverage of the platelets’ metabolome, the response of the identified metabolites, the reproducibility of the analytical method, and the time of the analysis as main evaluation criteria. Our results show that methanol–water (7:3) extraction coupled with HILIC–MS method provides the best compromise, allowing identification of 107 metabolites in a platelet cell extract sample, 91% of them with a RSD% lower than 20. A higher number of metabolites could be detected when analyzing the platelet samples with two different LC–MS methods or when using complementary extraction methods in parallel.
Keywords: Metabolomics; UPLC–MS; Platelets; Extraction; HILIC; Reversed phase liquid chromatography;

► A rapid SPE/LCMS method assaying kynurenine metabolites has been developed. ► LCMS detection was performed in positive and negative electrospray ionization. ► The method had a flow rate of 0.2 mL/min and a total run time of 12 min. ► The assay method was within acceptance range of all the validation parameters. ► Noteworthy changes in plasma tryptophan metabolism in SIR rats were observed.It is highly beneficial to monitor the activity of the kynurenine pathway in a large series of samples with high accuracy and reliability in a single experimental protocol. We have developed a rapid specific solid-phase extraction (SPE)–liquid chromatography–electrospray ionization tandem mass spectrometry method for assaying tryptophan, kynurenine, kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid and quinolinic acid (QA) in rat plasma. We also evaluated picolinic acid (PA) in this method, but it presented with unacceptable validation parameters. The assay involves pre-purification by SPE followed by chromatographic separation by C18 reversed phase chromatography. Mass spectrometric detection was performed using a mass spectrometer in positive and negative electrospray ionization; with a flow rate of 0.2 mL/min and an injection volume of 10 μL. Total run time including sample clean-up was 12 min. The assay method was found to be linear (R 2  > 0.95) and all the validation parameters were within acceptance range. The developed technique also demonstrated a significant elevation in plasma tryptophan, kynurenine, anthranilic acid and QA, and a significant decrease in KYNA, in rats subjected to post-weaning social isolation rearing, a putative animal model of relevance for depression and schizophrenia. This method can therefore be applied to measure metabolites of the kynurenine pathway in plasma accurately and precisely by LC–MS/MS, thereby helping to realize new opportunities in pharmacological and diagnostic research.
Keywords: Kynurenine pathway; Plasma; Solid phase extraction; Liquid chromatography; Mass spectrometry;

► The safety of co-administration of SHL and levofloxacin injection was studied. ► An LC–MS method was developed and validated for pharmacokinetic study in rat. ► There were obvious differences in the pharmacokinetics after combination. ► This study provides a rational basis for the secure use of SHL injection in clinic.A sensitive and specific high performance liquid chromatography coupled with mass spectrometric (LC–MS) method was developed and validated for the simultaneous determination of three main active constituents of Shuang-huang-lian injection with and without the combination use of levofloxacin injection in rat plasma. After addition of the internal standard rutin, plasma samples were protein precipitated with acetonitrile, the chromatographic separation was achieved on a Kromasil C18 column (250 mm × 4.6 mm, 5 μm), using a gradient mobile phase system of acetonitrile–water containing 0.05% formic acid. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 0.04–20 μg/mL for chlorogenic acid, 0.8–400 μg/mL for baicalin and 0.01–5.0 μg/mL for phillyrin, respectively. The accuracy (relative error, R.E.%) were between −2.7 and 3.4%, while the intra-day and inter-day precisions were less than 9.2 and 9.6% for the three analytes, respectively. This method was successfully applied to the drug interaction study of Shuang-huang-lian freeze-dried powder combined with levofloxacin injection after intravenous administration to rats. The results indicated that there were obvious differences in the pharmacokinetic behaviors after combination compared with only administration of Shuang-huang-lian injection.
Keywords: Shuang-huang-lian injection; Levofloxacin injection; Pharmacokinetics; Drug interaction; LC–MS;

A stable-isotope HPLC–MS/MS method to simplify storage of human whole blood samples for glutathione assay by R.L.G. Norris; M. Paul; R. George; A. Moore; R. Pinkerton; A. Haywood; B. Charles (136-140).
► A n HPLC–MS/MS assay for determination of reduced glutathione is presented. ► Deuterated internal standard with precipitation at sample collection allows storage for 99 days. ► Improved storage compared to EDTA/perchloric acid/serine borate pre-treatment.Glutathione is the principal non-protein tripeptide thiol present in most mammalian cells and plays an important role in the redox status of biological systems. The accurate assessment of reduced glutathione (GSH) status as a reliable index of oxidative stress is of research and clinical significance. GSH undergoes rapid oxidation after sample collection and this presents a challenge.Validation of an HPLC–MS/MS assay is reported. Storage stability using four variants of a methanolic precipitation with addition of stable isotope internal standard at collection is compared to l-serine borate/EDTA with perchloric acid precipitation (SBPE).Precipitation with methanol and addition of stable isotope on sample collection, combined with storage in solution at −70 °C showed superior storage stability to SBPE and other variants of the methanolic precipitation method up to 99 days.The combination of stable isotope with methanolic precipitation at collection, with assay by HPLC–MS/MS provides superior results after storage of whole blood samples for at least 99 days.
Keywords: Glutathione; GSH; Assay; Stability; HPLC–MS/MS;