Journal of Chromatography B (v.1025, #C)

Metabolomic study of aging in mouse plasma by gas chromatography–mass spectrometry by Chan Seo; Yun-Ho Hwang; Youngbae Kim; Bo Sun Joo; Sung-Tae Yee; Cheol Min Kim; Man-Jeong Paik (1-6).
Metabolomic analysis of aging was performed in plasma samples of young (8 weeks) and old (72 weeks) mice as ethoxycarbonyl/methoxime/tert-butyldimethylsilyl derivatives by gas chromatography–mass spectrometry (GC–MS). As new approaches, study of altered metabolism from aging was attempted by simultaneous profiling analysis of amino acids (AAs), organic acids (OAs) and fatty acids (FAs) by GC–MS in a single run combined with pattern analysis. As a result, 27 amino acids (AAs), 17 organic acids (OAs) and 24 fatty acids (FAs) were positively screened with large variations in plasma samples. Among altered metabolites, levels of six AAs (proline, methionine, 4-hydroxyproline, pipecolic acid, glutamic acid, α-aminoadipic acid) as neurotransmetters and nutrients, five OAs (2-hydroxybutyric acid, 2-hydroxyglutaric acid, cis-aconitic acid citric acid, isocitric acid) including intermediate metabolites in the TCA cycle, and three n-3 polyunsaturated FAs (PUFAs) of α-octadecatrienoic acid, eicosapentaenoic acid and docosahexaenoic acid as potential biomarkers were significantly different between young and old groups. Their levels were normalized to the corresponding mean values of the young group and then plotted into star symbol patterns, which were clearly distinct compared with numerical data and readily distinguishable for young and old groups. Thus, the present metabolomic screening and the star pattern recognition method might be useful for understanding the complexity of biochemical events in aging.
Keywords: Metabolomics; Aging; Amino acid; Organic acid; Fatty acid;

Buddleoside (also known as linarin) as the major flavonoid in Chrysanthemum morifolium Ramat., has been reported to possess a wide range of pharmacological activities. The human intestinal microbiota might have an important impact on drug metabolism and ultimately on the drug oral bioavailability. However, the interaction of the buddleoside with human intestinal bacteria remains unknown. In this study, the conversion of buddleoside by different bacteria from human feces was firstly investigated. A reliable, sensitive and rapid analytical method, ultra performance liquid chromatography was established and successfully applied to investigate the metabolites and metabolic profile of buddleoside by human intestinal bacteria. Among the isolated bacteria, four strains including Escherichia sp. 4, Escherichia sp. 34, Enterococcus sp. 45 and Bacillus sp. 46 showed more powerful conversion capability. Based on the accurate mass data and the characteristic MSn product ions, the parent and six metabolites were detected and tentatively identified compared with blank samples. The metabolites were produced by four main metabolic pathways including deglycosylation, acetylation, methylation and hydroxylation. Buddleoside could be firstly converted to its aglycon acacetin (M2) by the majority of the isolated intestinal bacteria. Subsequently, M2 was further metabolize to its methylated (M3), acetylated (M4), hydroxylated (M5) and hydrogenated product (M6). However, acacetin-7-glucosid (M1) was obtained only from the minor bacterial samples like Bacillus sp. 46. To further explain the metabolism of buddleoside, the β-d-glucosidase and α-l-rhamnosidase activities of four strains were analyzed. Bacillus sp. 46 could only produce α-l-rhamnosidase, while the other three strains showed two kinds of enzyme activities. Furthermore, the activities of α-l-rhamnosidase and β-d-glucosidase reached the highest level at 12–18 h and 10–12 h, respectively. The metabolic routes and metabolites of buddleoside produced by human intestinal microflora were firstly reported in this paper. The results will be very helpful for the further investigation of the pharmacokinetic research of buddleoside and to unravel how it works in vivo.
Keywords: Buddleoside; UPLC-LTQ/Orbitrap/MS/MS; Intestinal microflora; Enzyme activity;

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate) by Thiago Pajeú Nascimento; Amanda Emmanuelle Sales; Camila Souza Porto; Romero Marcos Pedrosa Brandão; Galba Maria de Campos-Takaki; José Antônio Couto Teixeira; Tatiana Souza Porto; Ana Lúcia Figueiredo Porto; Attilio Converti (16-24).
A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 23 full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000 g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000 g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS–PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97 kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37° C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150 min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.
Keywords: Mucor subtilissimus; Fibrinolytic protease; ATPS;

Simultaneous determination of nine coumarins in rat plasma by HPLC–MS/MS for pharmacokinetics studies following oral administration of Fraxini Cortex extract by Minmin Zhao; Weijing Ding; Shuang Wang; Chunying Wang; Yingfeng Du; Huijun Xu; Qiao Wang; Shumin Jin (25-32).
A high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed for the simultaneous determination of nine coumarins including aesculin, aesculetin, fraxin, fraxetin, scopoletin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin, 8-hydroxy-6,7-dimethoxy coumarin and umbelliferone in rat plasma using nodakenin as the internal standard (IS). The plasma samples were pretreated by a one-step direct protein precipitation with methanol. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of methanol and water (containing 0.05% acetic acid). All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. This method was fully validated in terms of the sensitivity, specificity, accuracy, precision (intra- and inter-day), matrix effect, recovery as well as the stability of the analyte under various conditions, and the results satisfied the requirements of biological sample measurement. The validated method was successfully applied to pharmacokinetic study of the nine coumarins in rat plasma after oral administration of Fraxini Cortex aqueous extract, among which the pharmacokinetics of four coumarins including fraxetin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin and 8-hydroxy-6,7-dimethoxy coumarin were studied for the first time.
Keywords: Fraxini cortex; Pharmacokinetics study; HPLC–MS/MS; Coumarins;

Microchip electrophoresis (MCE) is particularly attractive as it provides high sensitivity and selectivity, short analysis time and low sample consumption. An on-line preconcentration strategy combining field-amplified stacking (FASS) and reversed-field stacking (RFS) was developed for efficient and sensitive analysis of neurotransmitters in real urine samples by MCE with laser induced fluorescence (LIF) detection. In this study, the multiple-preconcentration strategy greatly improves the sensitivity enhancement and surpass other conventional analytical methods for neurotransmitters detection. Under optimal conditions, the separation of three neurotransmitters (dopamine, norepinephrine and serotonin), was achieved within 3 min with limits of detection (S/N = 3) of 1.69, 2.35, and 2.73 nM, respectively. The detection sensitivities were improved by 201-, 182-, and 292-fold enhancement, for the three neurotransmitters respectively. Other evaluation parameters such as linear correlation coefficients were considered as satisfactory. A real urine sample was analyzed with recoveries of 101.8–106.4%. The proposed FASS-RFS-MCE method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of three neurotransmitters in human urine.
Keywords: Microchip electrophoresis; Neurotransmitters; Field-amplified stacking; Reversed-field stacking;

Application of metabolomics to investigate the antitumor mechanism of flavopiridol in MCF-7 breast cancer cells by Xiaojian Shao; Dan Gao; Yini Wang; Feng Jin; Qin Wu; Hongxia Liu (40-47).
Flavopiridol is reported to have potent antitumor effects by inhibition of cyclin-dependent kinases (CDKs). However, most studies of flavopiridol focus on specific genes and kinases, so the antitumor mechanism needs further elucidation at the metabolic level. In the present study, an UPLC/Q-TOF MS metabolomics approach was used to investigate its antiproliferative effects on MCF-7 breast cancer cells. Comparing flavopiridol-treated MCF-7 cells with vehicle control, 21 potential biomarkers involved in five metabolism pathways were identified. Two pathways involving glutathione metabolism and glycerophospholipid metabolism showed that glutathione (GSH) and phosphatidylcholines (PCs) levels were reduced while their oxidized products oxidized glutathione (GSSG) and lysophosphatidylcholines (LysoPCs) were greatly increased. Further investigation showed an apparent accumulation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential (MMP). Thus, we suggest that oxidative stress was provoked in MCF-7 cells to reduce the GSH and PCs levels and cause mitochondria lesions. Moreover, cell cycle analysis showed that flavopiridol blocked cells at G1 stage, which was consistent with the depletion of spermidine and spermine that are believed to promote cancer progression. Taking these together, we concluded that flavopiridol could induce oxidative stress and cell cycle arrest, which finally lead to cell apoptosis in MCF-7 cells. This study provides a new strategy for studying the antitumor mechanism of flavopiridol, which could be used for its further improvement and application.
Keywords: Metabolomics; Flavopiridol; Antitumor mechanism; Oxidative stress; Cell cycle arrest;

This article describes a full analytical method for the clean-up and detection of benzylpenicillin in chicken and beef meat samples using a previously developed molecularly imprinted polymer in a solid-phase extraction cleanup step followed by analysis using LC–MS/MS. The method was validated based on the criteria and the requirements of the European Commission Decision 2002/657/EC. This validation revealed method performance characteristics that meet all the criteria in the Decision with limits of detection for chicken and beef meat samples of 6.2 and 14.4 μg/kg, respectively, 8 and 3.5 times lower than the MRL. This method holds strong potential when a specific cleanup of benzylpenicillin is required, because with slight modifications it is also applicable in a milk matrix [1].
Keywords: Molecularly imprinted polymer; Benzylpenicillin; MISPE; Meat; Validation;

Liquid chromatography mass spectrometry (LC–MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The “middle-up” mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars.
Keywords: Monoclonal antibody; Biosimilar; Antibody variants; Mass spectrometry; Process development; Comparative analysis;

As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI–MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50 °C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource.
Keywords: α2-macroglobulin; Cohn fraction IV; Plasma derivatives; Better utilization of plasma; Immobilized metal affinity chromatography;

Determination of 7α-OH cholesterol by LC–MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs by Changhong Yun; Taijun Yin; Katherine Shatzer; Douglas G. Burrin; Liwei Cui; Yifan Tu; Ming Hu (76-82).
An LC–MS/MS method was developed and validated to determine 7α-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50 × 4.6 mm i.d., 3 μm). The mobile phase (consisting of 0.1% HCOOH solution and acetonitrile) eluted in gradient at a flow rate of 1 ml/min. MS detection was operated on APCI (+) mode; the MRM transitions for 7α-OH cholesterol and D7-cholesterol (I.S.) were 385.1 ≥ 159.1 and 376.4 ≥ 266.3, respectively. The linear response range of 7α-OH cholesterol was covered from 1.563 to 100.0 ng/ml. All of the validation items meet the requirement of FDA guidance for bioanalytical method validation. This method was applied to enzymatic studies for determination of cholesterol 7alpha-hydroxylation activity catalyzed by CYP7A1 in the cholestatic minipigs liver microsomes.
Keywords: LC–MS/MS; CYP7A1; Cholesterol 7α-hydroxylase activity; 7α-OH cholesterol; Liver microsome;

A sequential solid-phase extraction (SPE) method was developed and validated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) for the detection and quantification of salbutamol enantiomers in porcine urine. Porcine urine samples were hydrolysed with β-glucuronidase/arylsulfatase from Helix pomatia and then subjected to a double solid-phase extraction (SPE) first using the Abs-Elut Nexus SPE and then followed by the Bond Elut Phenylboronic Acid (PBA) SPE. The salbutamol enantiomers were separated using the Astec CHIROBIOTIC™ T HPLC column (3.0 mm × 100 mm; 5 μm) maintained at 15 °C with a 15 min isocratic run at a flow rate of 0.4 mL/min. The mobile phase constituted of 5 mM ammonium formate in methanol. Salbutamol and salbutamol-tert-butyl-d9 (internal standard, IS) was monitored and quantified with the multiple reaction monitoring (MRM) mode. The method showed good linearity for the range of 0.1–10 ng/mL with limit of quantification at 0.3 ng/mL. Analysis of the QC samples showed intra- and inter-assay precisions to be less than 5.04%, and recovery ranging from 83.82 to 102.33%.
Keywords: Solid-phase-extraction (SPE); Salbutamol enantiomer; Liquid chromatography–mass spectrometry (LC–MS/MS); Method validation; Porcine urine;

A single method for detecting 11 organophosphate pesticides in human plasma and breastmilk using GC-FPD by Warangkana Naksen; Tippawan Prapamontol; Ampica Mangklabruks; Somporn Chantara; Prasak Thavornyutikarn; Mark G. Robson; P.Barry Ryan; Dana Boyd Barr; Parinya Panuwet (92-104).
Organophosphate (OP) pesticides are widely used for crop protection in many countries including Thailand. Aside from causing environmental contamination, they affect human health especially by over-stimulating of the neurotransmission system. OP pesticides, as with other non-persistent pesticides, degrade quickly in the environment as well as are metabolized quite rapidly in humans. Assessing human exposures to these compounds requires analytical methods that are sensitive, robust, and most importantly, suitable for specific laboratory settings. The aim of this study was to develop and validate an analytical method for measuring 11 OP pesticide residues in human plasma and breast milk. Analytes in both plasma and breast milk samples were extracted with acetone and methylene chloride, cleaned-up using aminopropyl solid phase extraction cartridges, and analyzed by gas chromatography with flame photometric detection. The optimized method exhibited good linearity, with the coefficients of determination of 0.996–0.999 and <7% error about the slope. Extraction recoveries from spiked plasma and breast milk samples at low and medium concentrations (0.8–5.0 and 1.6–10 ng mL−1, respectively) ranged from 59.4% (ethion) to 94.0% (chlorpyrifos). Intra-batch and inter-batch precisions ranged from 2.3–18.9% and 5.8–19.5%, respectively. Method detection limits of plasma and breast milk ranged from 0.18–1.36 and 0.09–2.66 ng mL−1, respectively. We analyzed 63 plasma and 30 breastmilk samples collected from farmworkers in Chiang Mai Province to determine the suitability of this method for occupational exposure assessment. Of the 11 pesticides measured, seven were detected in plasma samples and five were detected in breast milk samples. Mass spectrometry was used to confirm results. Overall, this method is rapid and reliable. It offers the laboratories with limited access to mass spectrometry a capacity to investigate levels OP pesticides in plasma and breastmilk in those occupationally exposed for health risk assessment.
Keywords: Organophosphate pesticides; Plasma; Breast milk; GC-FPD;

Simultaneous determination of three azo dyes in food product by ion mobility spectrometry by Jiandong Jiao; Jinfeng Wang; Mingfeng Li; Junqing Li; Qihong Li; Qinbo Quan; Jinquan Chen (105-109).
Color is an important property for food evaluation. Synthetic azo dyes are usually used in food product to obtain better appearance because of their stability and low cost. However, such dyes should be strictly controlled because of their potential threat to human health. A simple, rapid and sensitive method has been developed to determine orange II, allura red, and para red simultaneously by ion mobility spectrometry. The three dyes could be separated at the same time and the migration time of orange II, allura red, and para red are 12.070 ± 0.010, 8.180 ± 0.015, and 11.037 ± 0.016 ms, respectively. The effects of different parameters, such as pH, solvent, percentage of water, were investigated to establish the optimal condition. The detection limits were 0.1, 0.05, and 0.2 μg/mL for orange II, allura red, and para red, respectively. The recoveries of the three azo dyes from jellies were all higher than 81%. The developed method is fast and accurate for the detection of the three synthetic dyes.
Keywords: Ion mobility spectrometry; Azo dye; Orange II; Allura red; Para red;

Etravirine (ETR), maraviroc (MVC) and raltegravir (RAL) are promising antiretroviral drugs being used in HIV treatment and may be interesting for prevention applications such as oral or topical pre-exposure prophylaxis. Here we describe a sensitive and accurate method for the simultaneous detection of ETR, MVC and RAL from pigtail macaque plasma, vaginal secretions, and vaginal tissue. This method is characterized by a straightforward precipitation extraction method, a limit of quantification <0.5 ng mL−1 for all three antiretrovirals bolstered by a corresponding internal standard for each drug analyte, and short run time. Quantification is performed using positive ion electrospray triple quadrupole mass spectrometry. This method was validated over clinically relevant ranges for the three ARV drugs in all three matrices: 0.1–100 ng mL−1 for ETR, 0.05–100 ng mL−1 for MVC and 1–100 ng mL−1 for RAL. Our method is accurate and precise, with measured mean inter-assay precision (%CV) and accuracy (% bias) of 5.08% and 1.96%, respectively, while the mean intra-assay precision and accuracy were 3.44% and 1.08%. The overall post-extraction recovery for ETR, MVC and RAL was >94% in all cases. We also show that extracted biological samples are stable after storage at room temperature or 4 °C and after three freeze/thaw cycles. This is the first analytical method capable of quantifying ETR, MVC and RAL in biological matrices relevant for pre-clinical testing of oral or topical HIV prevention methods in pigtailed macaques.
Keywords: Etravirine; Maraviroc; Raltegravir; Plasma; Vaginal tissue; Vaginal secretions;

To support a novel anthracycline agent – Sabarubicin’s pharmacokinetics study in Chinese small cell lung cancer patients, a rapid, sensitive, and high throughput ultra-performance liquid chromatography tandem mass spectrometry method using Doxorubicin hydrochloride as internal standard (IS) was developed and validated for simultaneously quantifying Sabarubicin and its alcohol metabolite M3 in human plasma. Plasma samples were pre-extracted with n-hexane to remove hydrophobic interferences and the target compounds were extracted into a 1 ml mixture of chloroform and isopropanol (1:1, v/v) and separated on an ACQUITY UPLC BEH Shield RP18 (100mm × 2.1 mm, 1.7 μm) column with gradient mobile phase composed of acetonitrile and water containing 0.1% formic acid. Detection was performed by electrospray ionization in the positive ionization mode under multiple reaction monitoring of the transitions at m/z 644 → 130 for Sabarubicin, m/z 646 → 333.2 for M3, and m/z 544 → 360 for IS. For Sabarubicin and M3, calibration curves over 2–400 ng/ml and 0.5–100 ng/ml could achieve excellent linearity respectively(r > 0.99). Intra- and inter-day precisions were 1.5%–9.1% and 2.2%–12.8%, and accuracy were −9.6% to 0.7% and −4.8% to 5.9% for Sabarubicin and M3 respectively at four concentration levels. The mean recovery for Sabarubicin was 62.4%, 71.9% for M3, and 58.8% for IS. This method was completely validated and successfully applied in the pharmacokinetics study of Sabarubicin and M3 in Chinese small cell lung cancer patients.
Keywords: Sabarubicin; Alcohol metabolite; UHPLC–MS/MS; Bioanalysis;

Corrigendum to “Evaluation and comparison of three different separation techniques for analysis of retroamide enantiomers and their biological evaluation against h-P2X7 receptor” [J. Chromatogr. B 986–987 (2015) 35–43] by Davy Baudelet; Christophe Furman; Alina Ghinet; Xavier Dezitter; Sahil Adriouch; Frédéric Capet; Tiphaine Rogez-Florent; Philippe Gautret; Benoit Rigo; Régis Millet; Claude Vaccher; Emmanuelle Lipka (126-127).