Journal of Chromatography B (v.1000, #C)

Quantitative profiling of polar primary metabolites of two chickpea cultivars with contrasting responses to salinity by Daniel Anthony Dias; Camilla Beate Hill; Nirupama Samanmalie Jayasinghe; Judith Atieno; Tim Sutton; Ute Roessner (1-13).
This study reports a GC–QqQ–MS method for the quantification of forty-eight primary metabolites from four major classes (sugars, sugar acids, sugar phosphates, and organic acids) which can be applied to a number of biological systems. The method was validated in terms of linearity, reproducibility and recovery, using both calibration standards and real samples. Additionally, twenty-eight biogenic amines and amino acids were quantified using an established LC–QqQ–MS method. Both GC–QqQ–MS and LC–QqQ–MS quantitative methods were applied to plant extracts from flower and pod tissue of two chickpea (Cicer arietinum L.) cultivars differing in their ability to tolerate salinity, which were grown under control and salt-treated conditions. Statistical analysis was applied to the data sets using the absolute concentrations of metabolites to investigate the differences in metabolite profiles between the different cultivars, plant tissues, and treatments. The method is a significant improvement of present methodology for quantitative GC–MS metabolite profiling of organic acids and sugars, and provides new insights of chickpea metabolic responses to salinity stress. It is applicable to the analysis of dynamic changes in endogenous concentrations of polar primary metabolites to study metabolic responses to environmental stresses in complex biological tissues.
Keywords: Quantitative profiling; Primary metabolites; GC–QqQ–MS; LC–QqQ–MS; Chickpea; Salinity;

Lipidomic differentiation between human kidney tumors and surrounding normal tissues using HILIC-HPLC/ESI–MS and multivariate data analysis by Eva Cífková; Michal Holčapek; Miroslav Lísa; David Vrána; Bohuslav Melichar; Vladimír Študent (14-21).
The characterization of differences among polar lipid classes in tumors and surrounding normal tissues of 20 kidney cancer patients is performed by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI–MS). The detailed analysis of identified lipid classes using relative abundances of characteristic ions in negative- and positive-ion modes is used for the determination of more than 120 individual lipid species containing attached fatty acyls of different chain length and double bond number. Lipid species are described using relative abundances, providing a better visualization of lipidomic differences between tumor and normal tissues. The multivariate data analysis methods using unsupervised principal component analysis (PCA) and supervised orthogonal partial least square (OPLS) are used for the characterization of statistically significant differences in identified lipid species. Ten most significant up- and down-regulated lipids in OPLS score plots are also displayed by box plots. A notable increase of relative abundances of lipids containing four and more double bonds is detected in tumor compared to normal tissues.
Keywords: Lipidomics; Kidney cancer; Glycerophospholipids; HILIC-HPLC/ESI–MS; Multivariate data analysis;

Ipragliflozin is a highly potent and selective sodium-dependent glucose co-transporter-2 (SGLT2) inhibitor, a novel class of hypoglycemic agents. The aim of the present study was to establish a new highly sensitive and rapid liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the quantitative analysis of ipragliflozin in rat plasma and apply this method to a pharmacokinetic study in rats. Empagliflozin was used as an internal standard (I.S.) and liquid–liquid extraction was conducted using tert-butyl methyl ether. Chromatographic separation was accomplished on a Quicksorb ODS (2.1 mm i.d. × 150 mm, 5 μm in size) with acetonitrile/0.1% formic acid (90:10, v/v) at a flow rate of 0.2 mL/min. An API 3200 triple quadrupole mass spectrometer operating in the positive electrospray ionization mode with multiple reaction monitoring was used to detect ipragliflozin and I.S. transitions: m/z 422.0 [M + NH4]+  → 151.0 for ipragliflozin and m/z 451.2 [M + H]+  → 71.0 for I.S. Inter- and intra-day accuracies and precisions were within ±15%. This validated method was successfully applied to a pharmacokinetic study of ipragliflozin in rats. This assay method may contribute to assessment of novel SGLT2 inhibitors using the rat as an animal model.
Keywords: SGLT2; Ipragliflozin; Empagliflozin; Liquid chromatography-tandem mass spectrometry;

Challenges and solutions in the bioanalysis of BMS-986094 and its metabolites including a highly polar, active nucleoside triphosphate in plasma and tissues using LC–MS/MS by Ang Liu; John Lute; Huidong Gu; Bonnie Wang; Kevin J Trouba; Mark E Arnold; Anne-Françoise Aubry; Jian Wang (29-40).
BMS-986094, a nucleotide polymerase inhibitor of the hepatitis C virus, was withdrawn from clinical trials because of a serious safety issue. To investigate a potential association between drug/metabolite exposure and toxicity in evaluations conducted after the termination of the BMS-986094 development program, it was essential to determine the levels of BMS-986094 and its major metabolites INX-08032, INX-08144 and INX-09054 in circulation and the active nucleoside triphosphate INX-09114 in target and non-target tissues. However, there were many challenges in the bioanalysis of these compounds. The chromatography challenge for the extremely polar nucleoside triphosphate was solved by applying mixed-mode chromatography which combined anion exchange and reversed-phase interactions. The LC conditions provided adequate retention and good peak shape of the analyte and showed good robustness. A strategy using simultaneous extraction but separate LC analysis of the prodrug BMS-986094 and its major circulating metabolites was used to overcome a carryover issue of the hydrophobic prodrug while still achieving good chromatography of the polar metabolites. In addition, the nucleotide analytes were not stable in the presence of endogenous enzymes. Low pH and low temperature were required for blood collection and plasma sample processing. However, the use of phosphatase inhibitor and immediate homogenization and extraction were critical for the quantitative analysis of the active triphosphate, INX-09114, in tissue samples. To alleviate the bioanalytical complexity caused by multiple analytes, different matrices, and various species, a fit-for-purpose approach to assay validation was implemented based on the needs of drug safety assessment in non-clinical (GLP or non-GLP) studies. The assay for INX-08032 was fully validated in plasma of toxicology species. The lower limit of quantification was 1.00 ng/mL and the linear curve range was 1.00–500.00 ng/mL using a weighted (1/x 2) linear regression model. Intra-assay and inter-assay precision (CV, %) ranged from 2.3% to 5.5% and accuracy within ±2.2% from nominal. INX-08032 was found to be stable in acidified mouse plasma for at least 24 h in wet ice bath, 125 days at −70 °C and following at least three freeze–thaw cycles. No endogenous components in plasma were found to interfere with the measurement. The extraction recovery was between 90% and 95%. The assays for BMS-986094, INX-08144, INX-09054 and INX-09114 were qualified with wider acceptance criteria for accuracy and precision. Analyte stability was also evaluated to guide sample collection, storage, and processing. These assays were successfully applied to an investigative toxicokinetic and tissue metabolite profiling study described in the article.
Keywords: Nucleoside triphosphate; Mixed-mode chromatography; Stability; Method validation; Tissue analysis; LC–MS/MS;

Conventional microfluidic devices are typically complex and expensive. The devices require the use of pneumatic control systems or highly precise pumps to control the flow in the devices. This work investigates an alternative method using paper based microfluidic devices to replace conventional microfluidic devices. Size based separation and extraction experiments conducted were able to separate free dye from a mixed protein and dye solution. Experimental results showed that pure fluorescein isothiocyanate could be separated from a solution of mixed fluorescein isothiocyanate and fluorescein isothiocyanate labeled bovine serum albumin. The analysis readings obtained from a spectrophotometer clearly show that the extracted tartrazine sample did not contain any amount of Blue-BSA, because its absorbance value was 0.000 measured at a wavelength of 590 nm, which correlated to Blue-BSA. These demonstrate that paper based microfluidic devices, which are inexpensive and easy to implement, can potentially replace their conventional counterparts by the use of simple geometry designs and the capillary action. These findings will potentially help in future developments of paper based microfluidic devices.
Keywords: Microfluidic; Paper based devices; Separation; Extraction; Protein; Fluorescein Isothiocyanate;

A new method was developed to determine twelve intact-glucosinolates (GLSs) (glucoiberin, GIB; glucoraphanin, GRA; glucoerucin GER; gluconapin, GNA; glucotropaeolin, GTL; glucobrassicin, GBC; gluconasturtiin, NAS; glucoalyssin, ALY; 4-hydroxyglucobrassicin, 4OH; 4-methoxyglucobrassicin, 4ME; neoglucobrassicin, NEO; sinigrin, SIN) in bee pollen, by means of liquid chromatography tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI). An efficient extraction procedure was proposed (average analyte recoveries were between 85% and 96%); this involved a solid–liquid extraction (SLE) with heated water, followed by a solid phase extraction (SPE) with a weak anion exchange (NH2) sorbent. Chromatography was performed on a Gemini® C18 analytical column with a mobile phase of formic acid in water (0.5%, v/v) and formic acid in acetonitrile (0.5%, v/v), in gradient elution mode at 1 mL/min, resulted in baseline-separated peaks and a run time of 30 min. The method was fully validated in terms of selectivity, limits of detection (LOD) and quantification (LOQ), linearity, carry-over effect, reinjection reproducibility, precision and accuracy. A good selectivity, low LODs and LOQs, ranging from 1 to 16 μg/kg, wide linear ranges from LOQ to 1000 μg/kg, and satisfactory reinjection reproducibility, precision and accuracy with relative standard deviation and relative error values lower than or equal to 9%; meanwhile, results indicates a negligible carry-over effect. The proposed method was applied to analyze intact-GLSs in bee pollen. Nine of the GLSs studied were identified in certain samples analyzed over a wide concentration range (LOQ-2226 μg/kg), and significant differences in GLS content were observed among the samples.
Keywords: Keywords: Bee pollen; Glucosinolates; LC–MS/MS; Solid–liquid extraction; Solid phase extraction;

High-throughput screening for new psychoactive substances (NPS) in whole blood by DLLME extraction and UHPLC–MS/MS analysis by Sara Odoardi; Marco Fisichella; Francesco Saverio Romolo; Sabina Strano-Rossi (57-68).
The increasing number of new psychoactive substances (NPS) present in the illicit market render their identification in biological fluids/tissues of great concern for clinical and forensic toxicology. Analytical methods able to detect the huge number of substances that can be used are sought, considering also that many NPS are not detected by the standard immunoassays generally used for routine drug screening. The aim of this work was to develop a method for the screening of different classes of NPS (a total of 78 analytes including cathinones, synthetic cannabinoids, phenethylamines, piperazines, ketamine and analogues, benzofurans, tryptamines) from blood samples. The simultaneous extraction of analytes was performed by Dispersive Liquid/Liquid Microextraction DLLME, a very rapid, cheap and efficient extraction technique that employs microliters amounts of organic solvents. Analyses were performed by a target Ultrahigh Performance Liquid Chromatography tandem Mass Spectrometry (UHPLC–MS/MS) method in multiple reaction monitoring (MRM). The method allowed the detection of the studied analytes with limits of detection (LODs) ranging from 0.2 to 2 ng/mL. The proposed DLLME method can be used as an alternative to classical liquid/liquid or solid-phase extraction techniques due to its rapidity, necessity to use only microliters amounts of organic solvents, cheapness, and to its ability to extract simultaneously a huge number of analytes also from different chemical classes.The method was then applied to 60 authentic real samples from forensic cases, demonstrating its suitability for the screening of a wide number of NPS.
Keywords: NPS; UHPLC–MS/MS; Blood; DLLME; Forensic toxicology; Clinical toxicology;

Activated sludge consumes a large amount of energy to degrade a xenobiotic organic compound. By tracking the energy inventory of activated sludge biomass during the sludge’s degradation of a xenobiotic, any disadvantageous effect on the sludge’s performance caused by energy deficiency can be observed. The purpose of this study was to develop a reliable and accurate method for measuring the ATP contents of activated sludge cells that were to degrade a xenobiotic organic. Cell disruption and cellular ATP extraction were performed by a protocol with which xenobiotic degrading activated sludge biomass was washed with SDS, treated by Tris and TCA, and followed by bead blasting. The suspension of disrupted cells was filtered before the filtrate was injected into HPLC that was set at optimal conditions to measure the ATP concentration therein. This extraction protocol and HPLC measurement of ATP was evaluated for its linearity, limits of detection, and reproducibility. Evaluation test results reported a R 2 of 0.999 of linear fit of ATP concentration versus activated sludge concentration, a LOD = 0.00045 mg/L, a LOQ = 0.0015 mg/L for HPLC measurement of ATP, a MDL = 0.46 mg/g SS for ATP extraction protocol, and a recovery efficiency of 96.4 ± 2%. This method of ATP measurement was simple, rapid, reliable, and was unburdened of some limitations other methods may have.
Keywords: ATP measurement; Cellular extraction; Bead blasting; Activated sludge; Xenobiotic treatment;

Quantification of pyridoxal-5′-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC–MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20 μL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC–MS/MS system. Calibration curves were linear and recovery was >92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17 h or at −80 °C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC–MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.
Keywords: Pyridoxal-5′-phosphate; LC–MS/MS; Method development; Method validation; Endogenous compounds; Bioanalysis;

Evaluation of the matrix effect of different sample matrices for 33 pharmaceuticals by post-column infusion by Julia Rossmann; Robert Gurke; Lars David Renner; Reinhard Oertel; Wilhelm Kirch (84-94).
Matrix effects that occur during quantitative measurement by liquid chromatography mass spectrometry specifically when using electrospray ionization are a widely recognized phenomenon. Sample matrix compounds affect the ionization process of the target analytes, lead to a low signal response, and flawed analytical results. How these matrix compounds directly influence the ionization process has not yet been completely understood. In the present study, we determined the matrix effect for 33 pharmaceutical substances in sample extracts of urine, plasma and wastewater. Most of the investigated substances were subject to a signal suppression effect. Only for a small subset of the compounds we detected a signal enhancement effect. We investigated the matrix effect profiles in detail to disentangle the influence of different matrices and to correlate the impact of specific components and groups of the analyzed extract in suppressing or enhancing effects in the profile.Most signal suppression effects were detected in the first half of the chromatographic run-time for the matrix extracts of urine and wastewater. The observed effects are caused by high mass flow of salts and other diverse matrix components that were contained in high concentrations in those biological matrices. We also found signal suppression in the matrix effect profile of plasma samples over a wide time range during the chromatographic separation that were associated with a high content of triglycerides of diverse carbohydrate chain lengths. Here, we provide a broader picture of how 33 substances were influenced during analysis. Our results imply that a high number of the investigated substances had comparable effects of matrix compounds, despite differences in their chemical structure.
Keywords: LC-(ESI+)MS/MS; Matrix effect; Post-column infusion; Signal suppression; Signal enhancement; Pharmaceuticals;

Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines by Lucas Willmann; Manuel Schlimpert; Sebastian Halbach; Thalia Erbes; Elmar Stickeler; Bernd Kammerer (95-104).
Although the concept of aerobic glycolysis in cancer was already reported in the 1930s by Otto Warburg, the understanding of metabolic pathways remains challenging especially due to the heterogeneity of cancer.In consideration of four different time points (1, 2, 4, and 7 days of incubation), GC–MS profiling of metabolites was performed on cell extracts and supernatants of breast cancer cell lines (MDA-MB-231, -453, BT-474) with different sub classification and the breast epithelial cell line MCF-10A. To the exclusion of trypsinization, direct methanolic extraction, cell scraping and cell disruption was executed to obtain central metabolites.Major differences in biochemical pathways have been observed in the breast cancer cell lines compared to the breast epithelial cell line, as well as between the breast cancer cell lines themselves. Characteristics of breast cancer subtypes could be correlated to their individual metabolic profiles. PLS-DA revealed the discrimination of breast cancer cell lines from MCF-10A based on elevated amino acid levels.The observed metabolic signatures have great potential as biomarker for breast cancer as well as an improved understanding of subtype specific phenomenons of breast cancer.
Keywords: GC–EI–MS; Metabolomics; Metabolic profiling; Breast cancer;

Quantitative hydrophilic interaction chromatography–mass spectrometry analysis of N-acetylneuraminic acid and N-acetylmannosamine in human plasma by Yifan Shi; Xin Xu; Meng Fang; Michael Zhang; Yinghe Li; Brad Gillespie; Selwyn Yorke; Nora Yang; John C. McKew; William A. Gahl; Marjan Huizing; Nuria Carrillo-Carrasco; Amy Qiu Wang (105-111).
N-acetylneuraminic acid (Neu5Ac or NANA) is the most predominant sialic acid in mammals. As a terminal component in many glycoproteins and glycolipids, sialic acid is believed to be an important biomarker related to various diseases. Its precursor, N-acetylmannosamine (ManNAc), is being investigated as a potential treatment for GNE myopathy. In this work, we developed two highly sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods for the quantitation of ManNAc and free Neu5Ac in human plasma. A fit-for-purpose approach was adopted during method validation and sample analysis. To measure the endogenous compounds and overcome the interference from plasma samples, a surrogate matrix that contained 5% bovine serum albumin (BSA) was used for the preparation of calibration standards and certain levels of quality control (QC) samples. QC samples at higher concentrations were prepared in the authentic matrix (human plasma) to best mimic incurred samples. For both methods, an Ostro 96-well phospholipid removal plate was used for sample extraction, which efficiently removed the phospholipids from the plasma samples prior to LC injection, eliminated matrix effect, and improved sensitivity. Chromatographic separation was achieved using hydrophilic interaction chromatography (HILIC) and gradient elution in order to retain the two polar compounds. The lower limit of quantitation (LLOQ) for ManNAc and Neu5Ac was 10.0 and 25.0 ng/mL, respectively. The overall accuracy of the two assays was within 100% ± 8.3% based on three levels of QC samples. Inter- and intra-run precision (coefficient of variation (%CV)) across three analytical runs was less than 6.7% for ManNAc and less than 10.8% for Neu5Ac. These methods have been validated to support clinical studies.
Keywords: N-acetylmannosamine; N-acetylneuraminic acid; HILIC; LC–MS/MS; Phospholipid removal; Validation;

A sensitive, specific and rapid liquid chromatography–mass spectrometry (LC–MS) method was developed and validated for analysis of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rat plasma using sulfamethoxazole as an internal standard (IS). Separation was conducted out on an Agilent Eclipse XDB C18 column with liner gradient elution using acetonitrile (A) and 0.1% aqueous acetic acid (B). A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. A novel multi-determination-periods program was executed to achieve a higher sensitivity by setting three scanning periods. All analytes exhibited good linearity within the concentration range (r  > 0.9973). The lower limits of quantitation (LLOQ) of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin were 2.64, 4.32, 2.32 and 1.56 ng/mL, respectively. Intra-day and inter-day precisions of the investigated components exhibited an RSD within 8.3%, and the accuracy (RE) ranged from −8.6% to 6.0% at all quality control levels. The developed method was successfully applied to a pharmacokinetic study of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rats after oral administration of a Weifuchun tablet.
Keywords: LC–MS/MS; Ginsenoside Rb1; Naringin; Ginsenoside Rb2; Oridonin; Pharmacokinetics;

Determination of alkylresorcinols and their metabolites in biological samples by gas chromatography–mass spectrometry by Roksana Wierzbicka; Huaxing Wu; Milan Franek; Afaf Kamal-Eldin; Rikard Landberg (120-129).
High throughput GC–MS methods for quantification of alkylresorcinols (AR), biomarkers of whole grain wheat and rye intake, in plasma and adipose tissue and their metabolites in urine were developed and optimised. Alkylresorcinols in plasma (200 μL) and adipose tissues (10–50 mg) were extracted with diethyl ether, whereas main AR metabolites such as DHBA and DHPPA and newly identified metabolites in urine (50 μL) were extracted with ethyl acetate after enzymatic deconjugation. All extracts were purified on OASIS-MAX solid phase extraction cartridges. Plasma and adipose tissue sample extracts were then derivatised with trifluoroacetic anhydride and reconstituted in undecane, whereas AR metabolites in urine samples were derivatised with BSTFA + TMCS (99:1, v/v, 100 μL). Prepared samples were quantified by GC–MS (EI-SIM). Analysis of all compounds in the different matrices showed good selectivity, sensitivity, linearity, precision (<15% within and between batches), adequate recovery (75–108%), and short total run time (10–12 min). The methods developed are applicable to large-scale sample sets such as epidemiological studies.
Keywords: Alkylresorcinol; Alkylresorcinols metabolites; Whole grain; Biomarker; Gas chromatography–mass spectrometry;

Chiral analysis of methorphan in opiate-overdose related deaths by using capillary electrophoresis by Anna Bertaso; Giacomo Musile; Rossella Gottardo; Catia Seri; Franco Tagliaro (130-135).
An enantioselective CE-based determination of methorphan and its main metabolites in blood is described. Enantiomeric separations were carried out in 50 cm × 50 μm (ID) uncoated fused silica capillaries, using a background electrolyte composed of 150 mM sodium phosphate pH 4.4 added with 5 mM 2-(hydroxypropyl)-β-cyclodextrin and methanol 20% (v/v), at a constant voltage of 25 kV. Sample injections were performed under field amplified sample stacking conditions. Detection was by recording UV absorbance at the wavelength of 200 nm. Linearity of response was assessed within a concentration range from 25 to 500 ng/mL for dextrometorhan, levomethorphan and their main metabolites (namely dextrorphan and levorphanol, respectively). Folcodine was used as internal standard. Under these conditions, the limit of quantification resulted 25 ng/mL for each one of the analytes. The intra-day and inter-day precision, in terms of coefficient of variation (CV) were below 3.7% and 14.9 % for migration times and peak areas, respectively. The present method was successfully applied to the analysis of post-mortem blood samples from ten subjects died for heroin overdoses. Among the samples “positive” for methorphan (n  = 4), the d-enantiomer was found in concentrations ranging from 214 to 1282 ng/mL. The concentration of its main metabolite dextrorphan in the same samples ranged from 49 to 389 ng/mL.
Keywords: Dextromethorphan; Levomethorphan; Opiate overdose; Chiral analysis; Capillary electrophoresis;

Simultaneous determination of ten compounds in rat plasma by UPLC-MS/MS: Application in the pharmacokinetic study of Ma-Zi-Ren-Wan by Dong-Dong Hu; Quan-Bin Han; Linda Li-Dan Zhong; Yan-Hong Li; Cheng-Yuan Lin; Hing-Man Ho; Man Zhang; Shu-Hai Lin; Ling Zhao; Tao Huang; Hong Mi; Hong-Sheng Tan; Hong-Xi Xu; Zhao-Xiang Bian (136-146).
Ma-Zi-Ren-Wan (MZRW) is a classic Chinese formula which has been used to treat human constipation in China for over 2000 years. In order to make good and rational use of this formula in the future, this paper presents the first attempt to track the pharmacokinetic features of MZRW in rat using rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Ten chemical components of MZRW, namely, rhein, emodin, aloe emodin, hesperidin, naringin, amygdalin, albiflorin, paeoniflorin, magnolol and honokiol, were simultaneously determined in rat plasma after a single oral administration (10 g/kg body weight) of MZRW to rats. Geniposide and liquiritin were used as internal standards. The separation was performed on a Waters ACQUITY BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The detection was conducted by multiple-reaction monitoring (MRM) in negative ionization mode. Two highest abundant MRM transitions without interference were optimized for each analyte. This method was well validated in terms of linearity, precision, accuracy, recovery, matrix effect and stability. All calibration curves had good linearity (r 2  > 0.995) over the concentration range from 3.9 to 125.0 ng/mL for emodin, 3.9–500.0 ng/mL for amygdalin, 2.0–4000.0 ng/mL for naringin and hesperidin, 3.9–2000.0 ng/mL for magnolol, 7.8–2000.0 ng/mL for rhein and 3.9–4000.0 ng/mL for albiflorin, paeoniflorin, aloe emodin and honokiol. The intra-day and inter-day precision (relative standard deviation) was within 15%, the accuracy (relative error) ranged from −13.6% to 15.1%, and the lower limit of quantification in plasma ranged between 2.0 ng/mL and 7.8 ng/mL. Extraction recovery, matrix effect and stability were satisfactory. The validated method was successfully applied to a pharmacokinetic study of these ten compounds after oral administration of MZRW to rats. The pharmacokinetic parameters of each compound can facilitate clinical studies in the future.
Keywords: Ma-Zi-Ren-Wan; UPLC-MS/MS; Active ingredient; Pharmacokinetics; Rat plasma;

Metabolism of nitazoxanide in rats, pigs, and chickens: Application of liquid chromatography coupled to hybrid linear ion trap/Orbitrap mass spectrometer by Xianhui Huang; Chunna Guo; Zhangliu Chen; Yahong Liu; Limin He; Zhenling Zeng; Chaoqun Yan; Guangfang Pan; Shuaipeng Li (147-154).
Nitazoxanide (NTZ) is a nitrothiazole benzamide compound with a broad activity spectrum against parasites, Gram-positive and Gram-negative anaerobic bacteria, and viruses. In this study, hybrid linear ion trap/Orbitrap mass spectrometer providing a high mass resolution and accuracy was used to investigate the metabolism of NTZ in rats, pigs, and chickens. The results revealed that acetylation and glucuronidation were the main metabolic pathways in rats and pigs, whereas acetylation and sulfation were the major metabolic pathways in chickens, which indicated interspecies variations in drug metabolism and elimination. With the accurate mass data and the characteristic MSn product ions, we identified six metabolites in which tizoxanide and hydroxylated tizoxanide were phase I metabolites and tizoxanide glucuronide, tizoxanide glucose, tizoxanide sulfate and hydroxyl tizoxanide sulfate were phase II metabolites. Hydroxylated tizoxanide and tizoxanide glucose were identified for the first time. All the comprehensive data were provided to make out the metabolism of NTZ in rats, pigs and chickens more clearly.
Keywords: Nitazoxanide; Hybrid linear ion trap/Orbitrap mass spectrometer; Animals; Metabolites;

Determination and validation of psammaplin A and its derivatives in rat plasma by liquid chromatography–tandem mass spectrometry and its application in pharmacokinetic study by Jae-Young Lee; Mee Yeon Lee; Min Woo Ha; Tae Hyung Won; Hyun-Jong Cho; Jongheon Shin; Hyeung-geun Park; Dae-Duk Kim (155-162).
A liquid chromatography–tandem mass (LC–MS/MS) method was developed for the determination of psammaplin A (PsA) and its newly synthesized derivatives (PsA 107, PsA 109, and PsA 123) in rat plasma using bupropion as an internal standard (IS). The plasma samples were deproteinized with acetonitrile. Chromatographic separation was performed on hydro-RP column (75 × 2.0 mm, 80 Å, 4 μm) with isocratic elution using 5 mM ammonium formate buffer/acetonitrile (30:70, v/v) at a flow rate of 0.4 mL/min and the total run time was 5 min. Mass spectrometric detection was performed with positive electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The ion transitions monitored were m/z 663.2 → 331.0, 687.2 → 343.1, 587.3 → 293.1, 563.3 → 281.0, and 240.0 → 184.0 for PsA, PsA 107, PsA 109, PsA 123, and IS, respectively. All analytes showed good linearity over the concentration range of 5.00–5000 ng/mL (r 2  ≥ 0.994). The lower limit of quantification was 5 ng/mL for PsA and its three PsA derivatives. Within- and between-run precisions (relative standard deviation, RSD) were less than 9.66% and accuracy (relative error, RE) ranged from −9.34% to 7.25%. Established method was successfully applied to the investigation of pharmacokinetic properties of PsA and its derivatives in rats after intravenous administration at a dose of 2 mg/kg.
Keywords: Psammaplin A; Psammaplin A derivatives; Validation; LC–MS/MS; Pharmacokinetics;

Trantinterol is a novel β2-adrenoceptor agonist, currently undergoing clinical trials for the treatment of asthma. We developed and validated an liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of trantinterol and its major metabolite, 1-carbonyl trantinterol (SPFF―COOH), in rat plasma. Aliquots (100 μL) of heparinized plasma samples were processed by protein precipitation with acetonitrile. Chromatographic separation used an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) and acetonitrile-0.1% formic acid (20:80, v/v) as mobile phase, at a flow rate of 0.25 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple-reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The precursor-to-product ion transitions m/z 310.9 →  m/z 237.9 for trantinterol, m/z 324.9 →  m/z 251.9 for SPFF―COOH and m/z 368.0 →  m/z 294.0 for bambuterol (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentration of 0.25–100 ng/mL for both trantinterol and SPFF―COOH. The intra- and inter-day precision (relative standard deviations, RSD) values were below 15% and accuracy (relative error, RE) was from −4.3% to 6.6% at all quality control (QC) levels. The method was successfully applied to compare the pharmacokinetics of trantinterol and SPFF―COOH in male and female Wistar rats after a single oral administration of trantinterol.
Keywords: Trantinterol 1-carbonyl trantinterol; LC–MS/MS; Determination; Comparative pharmacokinetics;

Determination of amphetamines in biological samples using electro enhanced solid-phase microextraction-gas chromatography by Jingbin Zeng; Jingjing Chen; Min Li; Fazle Subhan; Fayun Chong; Chongying Wen; Jianfeng Yu; Bingwen Cui; Xi Chen (169-175).
In this work, an ordered mesoporous carbon (OMC)/Nafion coated fiber for solid-phase microextraction (SPME) was prepared and used as the working electrode for electro-enhanced SPME (EE-SPME) of amphetamines. The EE-SPME strategy is primarily based on the electro-migration and complementary charge interaction between fiber coating and ionic compounds. Compared with traditional SPME, EE-SPME exhibited excellent extraction efficiency for amphetamine (AP) and methamphetamine (MA) with an enhancement factor of 7.8 and 12.1, respectively. The present strategy exhibited good linearity for the determination of AP and MA in urine samples in the range of 10–1000 ng mL−1 and 20–1000 ng mL−1, respectively. The detection limits were found to be 1.2 ng mL−1 for AP and 4.8 ng mL−1 for MA. The relative standard deviations were calculated to be 6.2% and 8.5% for AP and MA, respectively. Moreover, the practical application of the proposed method was demonstrated by analyzing the amphetamines in urine and serum samples with satisfactory results.
Keywords: Electro-enhanced solid-phase microextraction; Ordered mesoporous carbon/Nafion coated fiber; Amphetamines; Urine and serum samples; Gas chromatography-flame thermionic detection;

Misoprostol is a widely used alternative of prostaglandin for labor induction. Based on previous studies, we envision that small and frequent oral dosage of misoprostol is an effective method for labor induction. To monitor the misoprostol content during labor induction, a rapid, sensitive, and selective microElution solid phase extraction (μElution SPE) combined with liquid chromatography tandem mass spectrometry (LC–MS/MS) was developed. Using μElution SPE could minimize the sample consumption and elution volume in order to maximize the sample enrichment and throughput. The misoprostol acid, a metabolite of misoprostol, was gradient separated in a Bidentate C18 column, then quantified by highly-selective reaction monitoring (H-SRM) in a total run time of 6 min. The developed method was optimized and validated in human plasma, and showed linear range of 0.01–10 ng/mL. The limit of detection (LOD) was 0.001 ng/mL. The recovery ranged from 89.0 to 96.0%, and no significant matrix effect or carryover was observed. The precision, accuracy and stability were met with the criteria of U.S. FDA guidance. The developed method was successfully applied to evaluate misoprostol concentration during labor induction in pregnant women. The concentration-time profiles approves that hourly oral administration of misoprostol is a safe and effective method without drug accumulation for labor induction.
Keywords: Labor induction; Misoprostol acid; Liquid chromatography tandem mass spectrometry; highly-selective reaction monitoring; microElution solid phase extraction;

Chidamide (epidaza), a new oral isotype-selective histone deacetylase inhibitor (HDACi), which is just approved in China for the treatment of recurrent or refractory peripheral T-cell lymphoma (PTCL) in December 2014, is the first listed benzamide class of HDACi in the world, and is currently undergoing global clinical trials for solid tumor treatments. Here, we report a sensitive, rapid and robust HPLC–MS/MS method for determination of chidamide in human plasma. Plasma sample was subjected to a simple acetonitrile protein precipitation containing MS-275 used as an internal standard (IS). Chromatography was performed on a Hypersil GOLD C18 analytical column, using a gradient methanol/water mobile phase containing 0.1% formic acid. A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive-ion mode. Selected reaction monitoring (SRM) using the precursor/ product transitions (m/z) of 391.1/265.1 for chidamide and 377.1/359.2 for IS were used for quantification, respectively. Good linearity was obtained in the range of 1–1000 ng/mL. The method gave R.S.D.% values for precision always lower than 13.8% and R.E.% values for accuracy between −3.7 and 9.1%. In addition, the specificity, recovery, stability and matrix effect were satisfactory too. The method is now being successfully applied to plasma samples as part of an ongoing chidamide phase Ib clinical trial in patients with solid tumors, and had demonstrated consistent AUClast and t 1/2 results with the published phase I pharmacokinetic data, which was also analyzed by this method, thus further confirming the reproducibility and accuracy during its clinical application. Considering the excellent performance of this method, it will continue being utilized for future clinical developments of chidamide and for routine monitoring of plasma exposure of chidamide during its clinical therapy.
Keywords: HPLC–MS/MS; Chidamide; Histone deacetylase inhibitor; Benzamide; Pharmacokinetics;

Polycyclic aromatic hydrocarbons (PAHs) are formed from the incomplete combustion or pyrolysis of organic matter during industrial processing and various human activities, but human exposure to PAHs has not yet been elucidated in detail. To assess long-term exposure to PAHs, we developed a simple and sensitive method for measuring PAHs in hair by online in-tube solid-phase microextraction using a CP-Sil 19CB capillary column as an extraction device, followed by high-performance liquid chromatography using a Zorbax Eclipse PAH column and fluorescence detection. Seventeen PAHs could be analyzed simultaneously, with good linearity from 20 to 1000 pg/mL each as determined using stable isotope-labeled PAH internal standards. The detection limits of PAHs were 0.5–20.4 pg/mL. PAHs in human hair samples were extracted by ultrasonication in 50 mM NaOH in methanol, and successfully analyzed without any interference peaks, with good recovery rates above 70% in spiked hair samples. Using this method, we evaluated the suitability of using hair PAHs as biomarkers for long-term exposure.
Keywords: Polycyclic aromatic hydrocarbons; Hair; Exposure biomarkers; In-tube solid-phase microextraction; High-performance liquid chromatography-fluorescence detection;

Application of microwave-assisted micro-solid-phase extraction for determination of parabens in human ovarian cancer tissues by Muhammad Sajid; Chanbasha Basheer; Kothandaraman Narasimhan; Mahesh Choolani; Hian Kee Lee (192-198).
Parabens (alkyl esters of p-hydroxybenzoic acid) are widely used as preservatives in food, cosmetics and pharmaceutical products. However, weak estrogenicity of some parabens has been reported in several studies, which provided the impetus for this work. Here, a simple and efficient analytical method for quantifying parabens in cancer tissues has been developed. This technique involves the simultaneous use of microwave-assisted solvent extraction (MASE) and micro-solid phase extraction (μ-SPE), in tandem with high performance liquid chromatography (HPLC/UV) analysis for the determination of parabens. The pollutants studied included four parabens (methyl, ethyl, propyl and butyl parabens). Optimization of the experimental parameters for MASE and μ-SPE was performed. Good relative standard deviation (%RSD) ranged from 0.09 to 2.81% and high enrichment factors (27–314) were obtained. Coefficients of determination (r 2) up to 0.9962 were obtained across a concentration range of 5.0–200 ng g−1. The method detection limits for parabens ranged from 0.005 to 0.0244 ng g−1. The procedure was initially tested on prawn samples to demonstrate its feasibility on a complex biological matrix. Preliminary studies on human ovarian cancer (OC) tissues showed presence of parabens. Higher levels of parabens were detected in malignant ovarian tumor tissues compared to benign tumor tissue samples.
Keywords: Human cancer tissues; Microextraction; Liquid-chromatography; Bioaccumulation; Environmental applications;