Journal of Chromatography B (v.1046, #C)

Display OmittedA rapid, sensitive and specific ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated to simultaneously determine the twelve major bioactive ingredients (neochlorogenic acid, chlorogenic acid, caffeic acid, cynarin, scopoletin, scutellarin, isochlorogenic acid A, apigenin-7-o-glucuronide, isochlorogenic acid C, scutellarein, luteolin, and apigenin) in rat plasma. Gallic acid and wogonoside were used as internal standards (IS1 and IS2). The plasma samples were pretreated and extracted by liquid–liquid extraction and protein precipitation with ethyl acetate–acetonitrile (95:5, v/v). Chromatographic separation was accomplished on Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 μm) utilizing 0.1% formic acid aqueous solution and acetonitrile as mobile phase under gradient conditions at a flow rate of 0.3 mL·min−1. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in positive and negative mode. The whole intra- and inter-day precision (as relative standard deviation) of all analytes were less than 11.03%, and the accuracy (as relative error) were in the range from −10.43% to 9.76% and from −10.14% to 10.33%. The lower limits of quantification (LLOQ) were 20, 3.0, 100, 7.0, 0.30, 2.0, 70, 1.0, 20, 30, 10, and 2.0 ng mL−1 for neochlorogenic acid, chlorogenic acid, caffeic acid, cynarin, scopoletin, scutellarin, isochlorogenic acid A, apigenin-7-o-glucuronide, isochlorogenic acid C, scutellarein, luteolin, and apigenin, respectively. Extraction recovery, matrix effect and stability were found to be the required limits. This method was selective and sensitive for the investigation of the pharmacokinetics of twelve constituents following oral administration to research study about in Erigeron breviscapus of clinical practices for separately analytes on rats.
Keywords: Erigeron breviscapus; UHPLC–MS/MS; Bio active ingredient; Pharmacokinetics; Rat plasma;

Quantification of meropenem in human plasma by HILIC – tandem mass spectrometry by Jens Martens-Lobenhoffer; Stefanie M. Bode-Böger (13-17).
Meropenem is a substance from the carbapenem class of antibiotics. It is widely used in intensive care units to treat a broad range of severe bacterial infections. In this critically ill patient group therapeutic drug monitoring (TDM) is highly advisable in order to avoid under- as well as over-dosing. Here, we describe a HILIC-MS/MS method for the quantification of meropenem in human plasma which is suitable for TDM. After simple protein precipitation, the polar analyte was chromatographically separated by the HILIC mechanism on a silica based stationary phase with polyethylenimide coating. The calibration was linear over the range of 1–50 μg/ml. The stable isotope labelled internal standard led to matrix independency of the quantification and to very good precision (relative standard deviations less than 5%) and accuracy (deviations from the expected values less than 4%). A cross-validation with a validated and established HPLC-UV method confirmed the accuracy of the HILIC-MS/MS method in real patient samples.
Keywords: Meropenem; Human plasma; Therapeutic drug monitoring; Hydrophilic interaction liquid chromatography; Tandem mass spectrometry;

The pharmacokinetics and tissue distribution of coumaroylspinosin in rat: A novel flavone C-glycoside derived from Zizyphi Spinosi Semen by Xiaotong Zhao; Junjun Liu; Zhiyou Wen; Yanqing Zhang; Mingxin Yu; Bingcheng Pan; Jun Zeng; Junbo Xie (18-25).
Zizyphi Spinosi Semen (ZSS) has a long history of sedative-hypnotic use in China. As a novel flavone C-glycoside, coumaroylspinosin is a main flavonoid only found in ZSS. Up to now, its pharmacokinetic information and tissue distribution in vivo are not available yet. With a simple, rapid and sensitive HPLC–MS/MS method, the pharmacokinetics and tissue distribution of coumaroylspinosin were investigated in Sprague-Dawley (SD) rats after its intravenous administration. Puerarin was used as the internal standard (IS). The samples were extracted by a simple protein precipitation method with methanol. The MS analysis was performed with multiple reaction monitoring (MRM), and the transitions were set at m/z 753.3 → 427.0 for coumaroylspinosin and m/z 415.3 → 295.3 for IS, respectively. The method was successfully applied for investigating the pharmacokinetics and tissue distribution of coumaroylspinosin in Sprague Dawley (SD) rats after tail vein injection with 4.0 mg/kg of the flavonoid. The calibration curves covered over the range of 0.02–10 μg/mL in plasma and various tissues samples with good linearity(r ≥ 0.9956). The lower limit of quantification (LLOQ) in all samples was less than 20 ng/mL. The intra- and inter-day precisions were below 15% and accuracy was from −3.78% to 4.68%. No significant matrix effect was observed, and the average extraction recovery was acceptable. Coumaroylspinosin could be cleared quickly from the rat plasma with the half-life (t1/2) of 1.86 ± 0.15 h. It was distribute widely in vivo, and the main tissue depots of coumaroylspinosin in rats were found to be intestine, muscle and lung. With the method, the pharmacokinetic parameters and tissue distribution of coumaroylspinosin in SD rats were investigated for the first time. The results demonstrated that coumaroylspinosin was distributed widely and rapidly in various rat tissues after intravenous administration.
Keywords: Zizyphi Spinosi Semen; Coumaroylspinosin; HPLC–MS/MS; Pharmacokinetics; Tissue distribution;

An ultra performance liquid chromatography-tandem mass spectrometry method for the therapeutic drug monitoring of isavuconazole and seven other antifungal compounds in plasma samples by Balthazar Toussaint; Fanny Lanternier; Christian Woloch; Denis Fournier; Manon Launay; Eliane Billaud; Eric Dannaoui; Olivier Lortholary; Vincent Jullien (26-33).
A new analytical method was developed for the routine Therapeutic Drug Monitoring of 8 antifungals compounds in 50 μL of plasma: isavuconazole (ISZ), voriconazole (VRZ), posaconazole (PSZ), fluconazole (FCZ), caspofungin (CSF), flucytosine (5FC), itraconazole (ITZ) and its metabolite OH-itraconazole (OH-ITZ). After adding 50 μL of the internal standard, which consisted in a mixture of the deuterated isotopes of the quantified compounds, the sample treatment consisted in a simple protein precipitation with 400 μL of acetonitrile. Five microliters of the supernatant were directly injected into the chromatographic system. The chromatographic separation was performed with a Waters C18-BEH column and a mobile phase consisting in a mixture of water and acetonitrile, both containing 0.1% of formic acid. The total run time was 3 min and the detection of the analytes was performed by electrospray ionization in a positive mode using selected reaction monitoring. Intra and inter-day precision and inaccuracy were < 15% over the calibration ranges that were determined according to their clinical relevance: 0.20–20.0 mg/L for ISZ, VRZ, PSZ, ITZ, and OH-ITZ; 0.50–50.0 mg/L for FCZ and CSF; 2.00–200 mg/L for 5FC. This simple and fast method was found suitable for routine therapeutic drug monitoring.
Keywords: Antifungal drugs; Therapeutic drug monitoring; Liquid chromatography; Tandem mass spectrometry;

In this work pH-responsive neutral and cationic polyacrylamide molecularly imprinted polymers (nMIP and cMIP, respectively) were prepared for separation of recombinant and wild type human serum albumin (HSA, pI 4.7) using mixture of polymerization initiators. The effect of pH during preparation and adsorption stages at pI(HSA)  ± 2.0 on binding capacity and selectivity; imprinting factor (IF) was thoroughly investigated. SE-HPLC and RP-HPLC were employed for thorough evaluation of the stability of HSA at the studied experimental conditions and for simultaneous determination of HSA and erythropoietin (EPO) in their mixtures, respectively. Results showed that nMIP were generally superior to cMIP, where nMIP prepared at pH 2.7 and tested at pH 6.7 showed superior binding characteristics (IF 42.91). The pH at the preparation stage imposed minimal effect on the stability of HSA owing to entrapment of HSA within the polymer network. Adsorption experiments carried out at pH 2.7, regardless of polymer type and pH of preparation revealed poor selectivity. Adsorption of HSA onto MIP followed Sips model with pseudo second-order kinetics. Scanning electron microscopy (SEM) revealed a rough surface for MIP and a smooth one with wider pore diameter for non-imprinted polymer (NIP). Successful separation of recombinant HSA from its binary mixture with EPO and wild type HSA from crude plasma was demonstrated using RP-HPLC. This suggested that MIP should be applicable for downstream purification of therapeutic grade HSA at scale either from plasma or recombinant sources and isolation of HSA from plasma for diagnostic purposes.
Keywords: Molecular imprinted polymer; pH-responsive polymer; Human serum albumin; Erythropoietin; Biopharmaceuticals;

All experiments were performed on an Hitachi LaChrom Elite® HPLC system. The method was validated according to International Conference on Harmonisation (ICH) guidelines for serum samples. Additionally, other biological matrices were tested, namely whole blood, urine, B16 F-10 melanoma cell line, growth medium conditioned with the same cell line, bacterial and yeast suspensions.From all the protocols tested, the best results were obtained using 0.5% CH3COOH:MeOH:H2O (15:15:70) as mobile phase, with detection at wavelengths 215, 276 and 356 nm, at 25 °C, and using a flow rate of 1 mL min−1. By using this protocol, it was possible to obtain a linear calibration curve, limits of detection and quantification in the order of μg L−1, and a short analysis time (<15 min per sample). The developed protocol allowed the successful detection and quantification of 3-NT in all biological matrices tested, with detection at 356 nm.This method, successfully developed and validated for 3-NT quantification, is simple, cheap and fast. These features render this method a suitable option for analysis of a wide range of biological matrices, being a promising useful tool for both research and diagnosis activities.
Keywords: 3-Nitrotyrosine; Nitrosative stress; HPLC-DAD; Quantification methods;

Graphene oxide-SiO2 nanocomposite as the adsorbent for extraction and preconcentration of plant hormones for HPLC analysis by Xiaona Zhang; Jiahua Niu; Xiaoting Zhang; Rui Xiao; Minghua Lu; Zongwei Cai (58-64).
In this research, a modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method based on graphene oxide@SiO2(SiO2@GO) nanocomposite as adsorbent of dispersive solid-phase extraction (dSPE) combined with high performance liquid chromatography (HPLC) for the analysis of four plant hormones in different plants was established. The as-prepared SiO2@GO was characterized by scanning electron microscopy, transmission electron microscopy and infrared spectroscopy. The experimental conditions for dSPE, including the ratio of material to liquid, pH of sample, adsorption and desorption time, desorption temperature as well as desorption solution, were investigated. The detection limits for the analysis of indole-3-acetic acid, indole-3-butyric acid, 1-naphthylacetic acid and abscisic acid were achieved below 0.05 μg mL−1. The established method was applied to the analysis of the plant hormones in fruits, vegetables and other food samples. The obtained results indicated that the method was sensitive, accurate, convenient and quick, which provided an alternative analytical approach for plant hormones in complex matrices.
Keywords: Graphene oxide-SiO2 nanocomposite; Dispersive solid-phase extraction; Plant hormones; HPLC; QuEChERS;

In this study, a dual-template molecularly imprinted polymer-coated stir bar capable of recognizing nine fluoroquinolone drugs was prepared. Its recognition abilities for fluoroquinolones and other drugs were studied by using computational simulation method. Results showed that the shape and volume of the imprinted cavities were responsible for the polymer’s recognition performance. After optimization of several important parameters, a stir-bar-sorptive-extraction method was developed that was combined with high performance liquid chromatography for determination of the nine drugs in meat. The stir bar showed high enrichment factors (33–47 folds), high capture capacities (4640–4950 ng) and high recoveries (>90%) for the nine drugs, and could be reused for thirty times. The limits of detection for the nine drugs were in the range of 0.1–0.3 ng g−1, and the recoveries from the fortified blank meat were in the range of 67.4%–99.0%. Therefore, this method could be used as a simple, rapid and specific tool for routine detection of residual fluoroquinolones in meat.
Keywords: Fluoroquinolones; Dual-template molecularly imprinted polymer; Stir bar; Computational simulation; High performance liquid chromatography; Meat;

Ionic liquid-impregnated agarose film two-phase micro-electrodriven membrane extraction (IL-AF-μ-EME) for the analysis of antidepressants in water samples by Nor Suhaila Mohamad Hanapi; Mohd Marsin Sanagi; Abd Khamim Ismail; Wan Aini Wan Ibrahim; Nor’ashikin Saim; Wan Nazihah Wan Ibrahim (73-80).
Display OmittedThe aim of this study was to investigate and apply supported ionic liquid membrane (SILM) in two-phase micro-electrodriven membrane extraction combined with high performance liquid chromatography-ultraviolet detection (HPLC-UV) for pre-concentration and determination of three selected antidepressant drugs in water samples. A thin agarose film impregnated with 1-hexyl-3-methylimidazolium hexafluorophosphate, [C6MIM] [PF6], was prepared and used as supported ionic liquid membrane between aqueous sample solution and acceptor phase for extraction of imipramine, amitriptyline and chlorpromazine. Under the optimized extraction conditions, the method provided good linearity in the range of 1.0–1000 μg L−1, good coefficients of determination (r2  = 0.9974–0.9992) and low limits of detection (0.1–0.4 μg L−1). The method showed high enrichment factors in the range of 110–150 and high relative recoveries in the range of 88.2–111.4% and 90.9–107.0%, for river water and tap water samples, respectively with RSDs of ≤7.6 (n=  3). This method was successfully applied to the determination of the drugs in river and tap water samples. It is envisaged that the SILM improved the perm-selectivity by providing a pathway for targeted analytes which resulted in rapid extraction with high degree of selectivity and high enrichment factor.
Keywords: Micro-electrodriven membrane extraction; Supported ionic liquid membrane; Agarose film; Antidepressant drugs; Water samples;

Display OmittedTraditional Tibetan medicine is important for discovery of drug precursors. However, information about the chemical composition of traditional Tibetan medicine is very limited due to the lack of appropriate chromatographic purification methods. In the present work, A. kansuensis was taken as an example and a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography(HILIC) method based on on-line HPLC-DPPH bioactivity-guided assay was developed for the purification of analogue antioxidant compounds with high purity from the extract of A. kansuensis. Based on the separation results of many different chromatographic stationary phases, the first-dimensional (1D) preparation was carried on a RP-C18HCE prep column, and 2 antioxidant fractions were obtained from the 800 mg crude sample with a recovery of 56.7%. A HILIC-XAmide prep column was selected for the second-dimensional (2D) preparation. Finally, a novel antioxidant β-carboline Alkaloids (Glusodichotomine AK) and 4 known compounds (Tricin, Homoeriodictyol, Luteolin, Glucodichotomine B) were purified from A. kansuensis. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single analogue antioxidant compounds of high purity from the extract of A. kansuensis. Additionally, this method showed great potential in the preparation of analogue structure antioxidant compounds and can serve as a good example for the purification of analogue structure antioxidant carboline alkaloids and flavonoids from other plant materials.
Keywords: Two-dimensional separation; Arenaria kansuensis; HPLC-DPPH-DAD; Antioxidant;

Simultaneous quantification of loxapine, loxapine N-oxide, amoxapine, 8-hydroxyloxapine and 7-hydroxyloxapine in human plasma using LC–MS/MS by Min Meng; Nan Zhao; Carrie C. Pederson; Edward Harrison; Charisse Green; Steven H. Gorman; Scott Reuschel (87-97).
Loxapine is an antipsychotic medication used for the treatment of schizophrenia. In vivo, loxapine is metabolized to multiple metabolites. A high performance liquid chromatographic-tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of loxapine and 4 of its metabolites, loxapine N-oxide, amoxapine (N‐desmethyl loxapine), 8-hydroxyloxapine and 7-hydroxyloxapine, in human plasma to support regulated clinical development. During method development, several technical challenges such as poor chromatography, separation of structural isomers, and inadequate sensitivity were met and overcome. The final method utilized micro-elution solid phase extraction (SPE) to extract plasma samples (100 μL), and the resulting extracts were analyzed using reversed phase LC–MS/MS using a turbo-ionspray interface in positive ionization mode with selected reaction monitoring (SRM). The method was fully validated according to the current regulatory guidance for bioanalysis over the calibration curve range 0.0500–50.0 ng/mL for all analytes using 1/x2-weighted linear regression analysis. Based on three separate runs, the between-run precision and inter-day precision for all five analytes at all concentrations, including the LLOQ (lower limit of quantitation) quality control at 0.0500 ng/mL, varied from 0.0% to 13.8%, while the accuracy ranged from 86.4% to 109.3% of nominal. The extraction recoveries of loxapine and the four metabolites were above 80%. Various forms of short-term and long-term stability were established in both solutions and matrix, including the stability of loxapine and the four metabolites in human plasma for up to 260 days of storage at −20 °C. This method has been used to support a regulated clinical study, which included the successful execution of incurred sample reanalysis (ISR) testing. To the best of our knowledge, this is the first published methodology in which these five analytes were quantified with a single extraction and injection.
Keywords: Loxapine; Human plasma; High performance liquid chromatography and tandem mass spectrometer (HPLC–MS/MS); Selected reaction monitoring (SRM);

Liquid to liquid extraction and liquid chromatography-tandem mass spectrometry determination of hainanmycin in feed by Ze Ping Wang; Jian Zhong Shen; Robert J. Linhardt; Hui Jiang; Lin Li Cheng (98-101).
Hainanmycin is a new veterinary polyether antibiotic and has few sensitive analytical method in present days. In this study, a liquid chromatography-tandem mass spectrometry (LC–MS/MS) relying on multiple reaction monitoring (MRM) detection was developed for analysis of hainanmycin in animal feed. Feed samples were extracted with ethyl acetate and purified by two steps of liquid-liquid extraction (LLE) to get rid of water solvable matrix and lipids one by one. The final simple was analyzed by LC–MS/MS. The LC mobile phase was composed of 0.1% aqueous formic acid and 0.1% formic acidified acetonitrile by gradient elution. Average recoveries ranged from 74.22% to 87.85%, as determined by spiking with 2.0 (LOQ) ∼2500 μg kg−1 of hainanmycin. The inter-day and intra-day coefficient of variation was 9.21% to 11.77% and 7.67% to 13.49%, respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.36 μg kg−1 and 2.0 μg kg−1, respectively.
Keywords: Hainanmycin; Feed; Liquid chromatography–tandem mass spectrometry; Sample preparation;

Physalin A, one of the major active components isolated from the calyces of Physalis alkekengi var. franchetii is considered to be a promising natural product due to its anti-inflammatory and excellent antitumor activities. Until now, only one paper is available from our group concerning identification of two sulfonate metabolites from rat feces after physalin A treatment. All the other researches related to physalin A were focused on its extraction, separation and biological activities. In this research, a rapid and reliable ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q–TOF–MS/MS) method was developed and employed for the comprehensive study of the metabolism of physalin A in vivo for the first time. A total of 24 proposed metabolites were identified in plasma, bile, urine and feces of rats after oral administration of physalin A. The results indicated that sulfonation, reduction and hydroxylation were the major metabolic pathways of physalin A in vivo. Furthermore, this research provides scientific and reliable support for full understanding of the metabolism of physalin A and the results could help to elucidate the safety and efficacy of physalin A, as well as other physalins.
Keywords: Physalin A; Metabolites; UPLC/Q–TOF–MS/MS; Rats;

Screening and identification of the main metabolites of 2-amino-9H-pyrido[2,3-b]indole (AαC) in liver microsomes and rat urine by using UPLC-Q-TOF-MS/MS by Kai Hu; Ge Zhao; Yufeng Fu; Sheng Wang; Hang Yuan; Fuwei Xie; Shusheng Zhang; Huimin Liu; Minying Liu (110-121).
2-Amino-9H-pyrido[2,3-b]indole (AαC), which has been reported to be 40–258 ng per cigarette, was regarded as a probable human carcinogen (Group 2B) and harmful composition in Hoffman list. Thus, it is of great significance to develop an effective method for the accurate identification of AαC and its metabolites. In the present study, we have investigated for the first time the in vivo and in vitro metabolites of AαC using ultra performance liquid chromatography combined with diode array detector and time-of-flight mass spectrometry (UPLC-DAD and UPLC-Q-TOF–MS/MS). A comparative study showed that the metabolic patterns of AαC in beagle, mouse, rat and human liver microsomes were of significant difference with these in rat urine. For the metabolism of AαC in liver microsomes, nine metabolites of AαC, including five hydroxy metabolites, two quinone metabolites and two N-dimer metabolites, have been found. However, metabolism of AαC in rats is a phase II process with complex enzyme catalysis, 23 metabolites including C- and N-oxidation, O- and N-glycosylation, O- and N-sulfonation, and N-acetylation were identified in rat urine. In addition, five new N-acetyl-AαC-OH metabolites were identified for the first time, indicating a possible new pathway for the metabolism. This study significantly enriched our knowledge about the metabolism of AαC, and will be useful for a better understanding of its harmfulness and toxicity.
Keywords: :UPLC-Q-TOF-MS/MS; 2-Amino-9H-pyrido[2,3-b]indole; Metabolites; Liver microsomes; Rat urine;

Automated 2D-HPLC method for characterization of protein aggregation with in-line fraction collection device by Abasha Williams; Erik K. Read; Cyrus D. Agarabi; Scott Lute; Kurt A. Brorson (122-130).
Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module.
Keywords: 2D-HPLC; Protein aggregation; PAT; QBD; Heart-cutting; Monoclonal antibody purification;

Determination of topiramate in dried blood spots using single-quadrupole gas chromatography–mass spectrometry after flash methylation with trimethylanilinium hydroxide by Roberta Zilles Hahn; Marina Venzon Antunes; Priscila Costa Arnhold; Natalia Bordin Andriguetti; Simone Gasparin Verza; Rafael Linden (131-137).
Dried blood spots (DBS) sampling obtained from fingerpricks is a promising and patient friendly alternative for obtaining samples for drug quantification, that could be of interest for topiramate (TOP) therapeutic drug monitoring. The aim of this study was to develop and validate a simple and fast GC–MS assay for TOP measurement in dried blood spots (DBS). The method uses a liquid extraction of one 8 mm DBS, followed by a flash methylation with TMAH, and separation in a DB–5 ms capillary column. Total analytical run time was 15 min. Precision assays presented CV% lower than 9.1% and accuracy was in the range of 94.5–115%. TOP was stable at 25 and 45 °C up to 21 days. TOP presents saturable binding to red blood cells, resulting in a fraction in plasma (fp) of 0.09–0.03 at 0.8 μg ml−1 and 0.71–0.45 at 20 μg ml−1 (both at 25–50 Hct% range). The method was applied to DBS samples obtained after phlebotomy and fingerpicks from an adult individual after oral intake of 100 mg TOP (0.25–96 h post dose). Plasma and DBS concentrations were moderately correlated (r  = 0.61), with estimated fp values in the range of 0.06–0.18. Translation of TOP DBS to plasma concentrations is challenging due to its concentration-dependent binding to erythrocytes. Thus, the use of whole blood concentrations for patients monitoring should be considered, which favors to the use of DBS in the clinical context.
Keywords: Topiramate; Dried blood spots; Gas chromatography–mass spectrometry; Therapeutic drug monitoring;

Between 2008 and 2011, four polar bears (Ursus maritimus) from the Greenland population swam and/or drifted on ice to Iceland where they arrived in very poor body condition. Body fat resources in these animals were only between 0% and 10% of the body weight (usually 25%). Here we studied the lipid composition in different tissues (adipose tissue if available, liver, kidney and muscle). Lipid classes were determined by thin layer chromatography (TLC) and on-column gas chromatography with mass spectrometry (GC/MS). The fatty acid pattern of total lipids and free fatty acids was analyzed by GC/MS in selected ion monitoring (SIM) mode. Additionally, cholesteryl esters and native fatty acid methyl esters, initially detected as zones in thin layer chromatograms, were enriched by solid phase extraction and quantified by GC/MS. The ratio of free fatty acids to native fatty acid methyl esters could be correlated with the remained body lipids in the polar bears and thus may also serve as a marker for other starving animals or even for humans.
Keywords: Polar bear; Lipid class; Thin layer chromatography; GC/MS; Fatty acid; Fatty acid methyl ester;

Metabolomic study on the antihypertensive effect of S-1-propenylcysteine in spontaneously hypertensive rats using liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry by Toshiaki Matsutomo; Mitsuyasu Ushijima; Yukihiro Kodera; Masashi Nakamoto; Miyuki Takashima; Naoaki Morihara; Koichi Tamura (147-155).
Aged garlic extract (AGE) has been shown to improve hypertension in both clinical trials and experimental animal models. However, the active ingredient of AGE remains unknown. In the present study, we investigated the antihypertensive effects of AGE and its major constituents including S-1-propenylcysteine (S1PC) and S-allylcysteine (SAC) using spontaneously hypertensive rats (SHR) and found that S1PC is an active substance to lower blood pressure in SHR. In addition, the metabolomics approach was used to investigate the potential mechanism of the antihypertensive action of S1PC in SHR. Treatment with AGE (2 g/kg body weight) or S1PC (6.5 mg/kg body weight; equivalent to AGE 2 g/kg body weight) significantly decreased the systolic blood pressure (SBP) of SHR after the repeated administration for 10 weeks, whereas treatment with SAC (7.9 mg/kg body weight; equivalent to AGE 2 g/kg body weight) did not decrease the SBP. After the treatment for 10 weeks, the plasma samples obtained from Wistar Kyoto (WKY) rats and SHR were analyzed by means of ultra high performance liquid chromatography coupled with high-resolution quadrupole-Orbitrap mass spectrometry. Multivariate statistical analysis of LC–MS data showed a clear difference in the metabolite profiles between WKY rats and SHR. The results indicated that 30 endogenous metabolites significantly contributed to the difference and 7 of 30 metabolites were changed by the S1PC treatment. Furthermore, regression analysis showed correlation between SBP and the plasma levels of betaine, tryptophan and 3 LysoPCs. This metabolomics approach suggested that S1PC could exert its antihypertensive effect by affecting glycine, serine and threonine metabolism, tryptophan metabolism and glycerophospholipid metabolism.
Keywords: S-1-Propenylcysteine; Hypertension; LC–MS; Metabolomics; Plasma;

New process for purifying high purity α1-antitrypsin from Cohn Fraction IV by chromatography: A promising method for the better utilization of plasma by Chaoji Huangfu; Jinchao Zhang; Yuyuan Ma; Junting Jia; Maomin Lv; Xiong Zhao; Jingang Zhang (156-164).
α1-antitrypsin (AAT) is a 52 kDa serine protease inhibitor that is abundant in plasma. It is synthesized mainly by hepatic cells, and widely used to treat patients with emphysema due to congenital deficiency of AAT. A new isolation method for the purification of AAT from Cohn Fraction IV (Cohn F IV) is described. Cohn F IV is usually discarded as a byproduct from Cohn process. Using Cohn F IV as starting material does not interfere with the production of other plasma proteins and the cost of purification could be reduced greatly. Parameters of each step during purification were optimized, 15% polyethyleneglycol (PEG) concentration and pH 5.2 for PEG precipitation, elution with 0.05 M sodium acetate and pH 4.7 for ion-exchange chromatography, and two steps blue sepharose affinity chromatography were chosen for AAT purification. The final protein with purity of 98.17%, specific activity of 3893.29 IU/mg, and yield of 28.35%, was achieved. Western blotting was applied for qualitative identification of final product, which specifically reacted with goat anti-human AAT antibody. LC-ESI-MS/MS was also employed to confirm the final protein. High performance liquid chromatography was used to analyze the composition of purified protein suggesting that pure protein was achieved. The molecular weight of AAT is 51062.77 Da which was identified by LC–MS-MS. The manufacturing process described here may make better use of human plasma with Cohn F IV as starting material. The simple process described in this study is simple and inexpensive, it has a potential value for large scale production.
Keywords: α1-antitrypsin; Ion-exchange chromatography; Blue sepharose affinity chromatography; Cohn Fraction IV; High performance liquid chromatography;

A liquid chromatography-tandem mass spectroscopy (LC–MS/MS) assay was developed and validated to simultaneously quantify anticancer drugs reparixin and paclitaxel in this study. The compounds were extracted from plasma and urine samples by protein precipitation with acetone (supplemented with 0.1% formic acid). Chromatographic separation was achieved using a C18 column, and drug molecules were ionized using dual ion source electrospray and atmospheric pressure chemical ionization (DUIS: ESI-APCI). Reparixin and paclitaxel were quantified using negative and positive multiple reaction monitoring (MRM) mode, respectively. Stable isotope palcitaxel-D5 was used as the internal standard (IS). The assay was validated for specificity, recovery, carryover and sample stability under various storage conditions; it was also successfully applied to measure drug concentrations collected from a pharmacokinetic study in rats. The results confirmed that the assay was accurate and simple in quantifying both reparixin and paclitaxel in plasma and urine with minimal sample pretreatment.
Keywords: Reparixin; Paclitaxel; UHPLC–MS/MS; Pharmacokinetics;

Bee pollen collected by honeybees, which is in powdered form, is a good nutritional supplement. Nitrofuran antibiotics are assumed not to be present in bee pollen, which is important as the level of antibiotics in bee pollen is strongly regulated in many countries. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to detect nitrofurans in honey has been developed, but this method is not suitable for bee pollen because of it being in powdered form. During preparation of bee pollen samples, the dispersal of powder particles in an aqueous solution often makes them susceptible to forming an emulsion with solvent components such as hexane and ethyl acetate. This may reduce the reproducibility and sensitivity of analyses of nitrofuran levels in bee pollen. Therefore, we attempted to optimize the sample preparation conditions to detect nitrofurans in bee pollen by determining three nitrofuran residues, namely, 3-amino-2-oxazolidinone (AOZ), 3-amino-5-methyl-morpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), using LC–MS/MS. The optimized method prevented the formation of powder-induced emulsion. To verify the reproducibility and sensitivity of this method, it was validated using nitrofuran-free bee pollen spiked with analytes with different side chains at 1.0, 2.0, and 5.0 μg kg−1. The accuracy levels were 94.1%–104.0% and the coefficients of variation were less than 12%. The limits of detection for AOZ, AMOZ, and AHD were 0.18, 0.25, and 0.30 μg kg−1, respectively, while their limits of quantitation were 0.59, 0.83, and 1.00 μg kg−1. The LC–MS/MS method developed to analyze nitrofuran in bee pollen should contribute to the quality control of bee pollen and food safety.
Keywords: Nitrofurans; Bee pollen; LC–MS/MS; Quality control; Food safety;

Recreational drugs have large impact on public health and security, and to monitor them is of urgent demand. In the present study, ultrasound-assisted dispersive liquid-liquid microextraction combined with the detection of gas chromatography-mass spectrometry was applied to the determination of seven common recreational drugs, including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, meperidine, methadone and ketamine in 200 μL of human whole blood. A series of factors which would affect the extraction efficiency were systematically investigated, including the nature and the volume of extraction and dispersing solvents, ultrasonication time, salting-out effect and pH value. The method consumed small amount of sample. The limits of detection and limits of quantification for each analyte were 10 and 40 ng/mL, respectively, and the linearity was in the range of 0.04–25 μg/mL (R2 higher than 0.99). Good specificity, precision (1.5–8.2% for the intra-day study and 2.6–12.8% for the inter-day study), satisfactory accuracy (85.0–117.1%) and extraction recovery (77.0–92.4%) were obtained, which makes it a high performance method for the determination of recreational drugs in human whole blood samples.
Keywords: Ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME); Gas chromatography–mass spectrometry (GC–MS); Human whole blood; Recreational drugs;

Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although 1H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with 1H NMR, but with a more than 100-fold lower detection limit for absolute quantification.
Keywords: Glutathione S-conjugates; Alkaline hydrolysis; O-phthaldialdehyde; Diclofenac; Clozapine; Acetaminophen;

Determination of methylmalonyl coenzyme A by ultra high-performance liquid chromatography tandem mass spectrometry for measuring propionyl coenzyme A carboxylase activity in patients with propionic acidemia by Kana Gotoh; Yoko Nakajima; Go Tajima; Yoriko Watanabe; Yuji Hotta; Tomoya Kataoka; Yoshihiro Kawade; Naruji Sugiyama; Tetsuya Ito; Kazunori Kimura; Yasuhiro Maeda (195-199).
Propionic acidemia (PA) is an inherited metabolic disease caused by low activity of propionyl coenzyme A (CoA) carboxylase (PCC), which metabolizes propionyl-CoA into methylmalonyl-CoA. Although many patients with PA have been identified by tandem mass spectrometry since the test was first included in neonatal mass screening in the 1990s, the disease severity varies. Thus, determining the specific level of PCC activity is considered to be helpful to grasp the severity of PA. We developed a new PCC assay method by the determination of methylmalonyl-CoA, which is formed by an enzyme reaction using peripheral lymphocytes, based on ultra high-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). With methylmalonyl-CoA concentrations of 0.05, 0.5, and 5 μmol/L, the intra-assay coefficients of variation (CVs) were 8.2%, 8.7%, and 5.1%, respectively, and the inter-assay CVs were 13.6%, 10.5%, and 5.9%, respectively. The PCC activities of 20 healthy individuals and 6 PA patients were investigated with this assay. Methylmalonyl-CoA was not detected in one PA patient with a severe form of the disease, but the remaining PA patients with mild disease showed residual activities (3.3–7.8%). These results demonstrate that determination of PCC activity with this assay would be useful to distinguish between mild and severe cases of PA to help choose an appropriate treatment plan.
Keywords: Propionic acidemia; Methylmalonyl coenzyme A; Propionyl coenzyme A carboxylase; UPLC–MS/MS;

Display OmittedFelodipine (FLD), a calcium channel antagonist, is commonly prescribed for the treatment of hypertension either with Metoprolol (MET) or Ramipril (RAM) in two different drug combinations. FLD has high plasma protein binding ability affecting its extraction recoveries from plasma samples. Hence, a specific ultrasound assisted dispersive liquid-liquid microextraction (UA-DLLME) method coupled with HPLC using photodiode array detector was developed and validated for the simultaneous determination of FLD, MET and RAM in rat plasma after oral administration of these combinations. The factors affecting UA-DLLME were carefully optimized. In this study, UA-DLLME method could provide simple and efficient plasma extraction procedures with superior recovery results. Under optimum condition, all target drugs were separated within 13 min. The validation procedures was carried out in agreement with US-FDA guidelines and shown to be suitable for anticipated purposes. Linear calibration ranges were obtained in the range 0.05–2.0 μg mL−1 for FLD and MET and 0.1–2.0 μg mL−1 for RAM with detection limits of 0.013–0.031 μg mL−1 for all the studied drug combinations. The%RSD for inter-day and intra-day precisions was in range of 0.63–3.85% and the accuracy results were in the range of 92.13–100.5%. The validated UA-DLLME-HPLC method was successfully applied for the bioavailability studies of FLD, MET and RAM. The pharmacokinetic parameters were calculated for all the investigated drugs in rats after single-dose administrations of two different drug combinations. Although FLD was bioequivalent in the two formulations, a small increase in plasma levels of MET and RAM was found in the presence of FLD.
Keywords: Felodipine; Ultrasound; Dispersive extraction; Therapeutic drug monitoring; Bioavailability study;

Two-step high speed countercurrent chromatography method, following normal phase and elution-extrusion mode of operation by using selected solvent systems, was introduced for phenolic compounds separation. Phenolic compounds including gallic acid, ethyl gallate, ethyl digallate and ellagic acid were separated from the ethanol extract of mango (Mangifera indica L.) flowers for the first time. In the first step, gallic acid of 3.7 mg and ethyl gallate of 3.9 mg with the purities of 98.87% and 99.55%, respectively, were isolated by using hexane-ethylacetate-methanol-water (4:6:4:6, v/v) in normal phase high speed countercurrent chromatography from 200 mg of crude extract, while ethyl digallate and ellagic acid were collected in the form of mixture fraction. In the second step, further purification of the mixture was carried out with the help of another selected solvent system of dichloromethane-methanol-water (4:3:2, v/v) following elusion-extrusion mode of operation. Ethyl digallate of 3.8 mg and ellagic acid of 5.7 mg were separated well with high purities of 98.68% and 99.71%, respectively. The separated phenolic compounds were identified and confirmed by HPLC, UPLC-QTOF/ESI–MS, 1H and 13C NMR spectrometric analysis.
Keywords: Phenolic compounds; Mangifera indica L. flowers; HSCCC; UPLC-QTOF/ESI–MS; 1HNMR; 13C NMR;

A validated LC–MS/MS method for the estimation of glimepiride and pitavastatin in rat plasma: Application to drug interaction studies by Shruti Surendran; David Paul; Ratna Sushmita; Lavanya Krishna; Nirbhay Kumar Tiwari; Sanjeev Giri; Nanjappan Satheeshkumar (218-225).
Glimepiride (GLI) is prescribed for the management of type-2 diabetes where as pitavastatin (PIT) for the treatment of diabetes associated dyslipidemia. Both the drugs are metabolized by CYP2C9 and have the potential of altering the enzyme through either inhibition or induction. In this respect, we present a simple, fast and validated bioanalytical LC–MS/MS method for the simultaneous estimation of GLI and PIT from rat plasma. Waters XTerra RP HPLC column (4.6 × 100 mm, 5 μm) with mobile phase consisting of acetonitrile and 10 mM ammonium acetate (pH-6.0) in the ratio 85:15 (v/v) at a flow rate of 1 mL/min was used for the chromatographic separation. The negative ionization mode with MRM transitions: m/z 420.17 → 288.13 for PIT, m/z 489.59 → 350.12 for GLI and m/z 380.08 → 316.31for celecoxib as internal standard (IS). A total run time of 3 min and LLOQ was found to be 5 ng/mL for both PIT and GLI. The method was applied to study the drug interaction between GLI and PIT in rat liver microsomes. In vivo rat pharmacokinetics study showed there was a 1.29-fold increase in AUC0-∞ and 1.2-fold decrease in the clearance of PIT in presence of GLI. No notable difference in the pharmacokinetic profile of GLI was observed upon the intravenous co-administration of PIT.
Keywords: Glimepiride; Pitavastatin; LC–MS/MS; Drug interaction; Rat liver microsomes;

Application of time-of-flight mass spectrometry for screening of crude glycerins for toxic phorbol ester contaminants by Kithsiri Herath; Lauren Girard; Renate Reimschuessel; Hiranthi Jayasuriya (226-234).
Since 2007, the U.S. Food and Drug Administration (FDA) has received numerous complaints of pet illnesses that may be related to the consumption of jerky pet treats. Many of those treats include glycerin as an ingredient. Glycerin can be made directly from oils such as palm seed oil, but can also be derived from the seed oil of toxic Jatropha plant during biodiesel production. If crude glycerin from biodiesel production from Jatropha curcas is used in the manufacture of animal feed, toxic tigliane diterpene phorbol esters (PEs), namely Jatropha factors (JFs), may be present and could lead to animal illnesses. Considering the numerous uses of glycerin in consumer products there is a need for a rapid method to screen crude glycerin for JF toxins and other PE contaminants. We describe the development of an ultra-high pressure liquid chromatography/quadrupole time of flight (UHPLC/Q-TOF) method for screening crude glycerin for PEs. An exact mass database, developed in-house, of previously identified PEs from Jatropha curcas as well as putative compounds was used to identify possible contaminants.
Keywords: Jatropha factors; Glycerin; Jatropha curcas; UHPLC/Q-TOF;

Yucca schidigera Roezl. (yucca) is one of the major industrial sources of steroid saponins used as animal and human food additives. This work describes a new, systematic and reproducible three-step method by medium and high-pressure liquid chromatography (under RP, NP and RP conditions), for the isolation and purification of three groups of saponins, which were further purified in six sub-fractions, and finally into twelve individual steroid saponins previously reported in Y. schidigera. In accordance to the increasing applications of yucca extracts, further analytical, biological and physicochemical studies are still required. The presented method is applicable to the preparation of steroids saponins previously reported in commercial extracts of Y. schidigera, both as highly purified mixtures of defined composition, including twelve pure components.
Keywords: Yucca schidigera; Steroid saponins; HPLC-ELSD; Purification; Chromatography; Qualitative analysis;

Development and validation of a LC–MS/MS assay for quantification of cisplatin in rat plasma and urine by Abdul Naveed Shaik; Deborah A. Altomare; Lawrence J. Lesko; Mirjam N. Trame (243-249).
Till date, no analytical method published to detect Cisplatin has been validated according to the U.S. Food and Drug Administration (FDA) guidance using liquid chromatography mass spectrometry (LC–MS/MS). We report, a validated LC–MS/MS method for quantitative determination of cisplatin in rat plasma and urine according to FDA guidlines. Cisplatin is a platinum containing compound used for the treatment of different types of cancers. Quantitative determination of cisplatin has been carried out using atomic absorption spectroscopy, high pressure liquid chromatography with phosphorescence, ultra-violet detection, or with inductively coupled plasma mass spectrometry. Few LC–MS/MS methods have been reported for the analysis of cisplatin either for direct quantification or indirect by derivatizing with organic compounds but none of the reported methods have validated the method. The developed and validated assay presented here is a highly sensitive LC–MS/MS method developed and validated for the quantitative determination of cisplatin following derivatization with diethyldithiocarbamate (DDTC) in order to detect platinum (Pt) of cisplatin, suitable for pharmacokinetic studies in rats and to further use it to study human toxicology. Chromatographic separation was achieved using a Poroshell 120 EC-C18 column (3 × 50 mm, 2.7 μm) with a binary gradient mobile phase. Quantification was performed on a triple quadruple with electrospray ionization and detection was performed using multiple reaction monitoring. The method has a limit of detection of 1 ng/mL, and the quantifiable range was 3–3000 ng/mL in rat plasma and urine. The method was accurate and precise with an accuracy and precision for intra-day and inter-day of ±20% for lower limit of quantitation and of ±15% for low, mid and high quality control samples. This method was successfully applied to study the pharmacokinetic profile of cisplatin in rat plasma and urine given a range of doses from 0.5 to 3.5 mg/kg.
Keywords: Cisplatin; Cisplatin rat pharmacokinetics; Non-compartmental analysis of cisplatin; Cisplatin derivatization with DDTC; FDA method validation; LC–MS/MS;