Journal of Chromatography B (v.1017-1018, #C)

UPLC–MS/MS assay for the simultaneous determination of ethinyl estradiol, norgestimate and 17-Desacetyl norgestimate at low pg/mL in human plasma by Mike-Qingtao Huang; Lijuan Kang; Weimin Wang; Donna Skee; Mu Chen; Zhongping John Lin; Tom Verhaeghe; Naidong Weng (1-9).
Previously, because of the difficulty of measuring very low levels (pg/mL) of norgestimate (NGM) due to rapid metabolism to its active and major metabolite, 17-Desacetyl norgestimate (DNGM), only DNGM and the co-administered ethinyl estradiol (EE2) were required to be analyzed in bioequivalence (BE) studies for oral contraceptive NGM/EE2 tablets. Recently, with more sensitive assays available, health authorities have requested that bioequivalence of NGM be also demonstrated in addition to DNGM and EE2. Therefore, it was important to establish a 3-in-1 method for the quantitation of EE2, NGM and DNGM in human plasma. Here a UPLC–MS/MS method for the simultaneous quantitation of EE2, NGM and DNGM in human plasma at low pg/mL range is described. EE2, NGM, DNGM and their isotopic labeled internal standards (IS) EE2-d4, NGM-d6 and DNGM-d6 in 0.4 mL of human plasma were extracted with hexane/ethyl acetate. The extracts were evaporated to dryness and derivatized with dansyl chloride, to enhance the mass spec response. The derivatives were reconstituted with methanol and analyzed by UPLC–MS/MS. In order to minimize the ex-vivo conversion of NGM to DNGM, sodium fluoride/potassium oxalate was used as anti-coagulant. To achieve desirable throughput for sample analysis, UPLC–MS/MS with a run time of 4.4 min was utilized. The calibration curve ranges were 5–500 pg/mL for EE2 and NGM, and 25–2500 pg/mL for DNGM, respectively. The chromatographic separation was achieved on a Waters Acquity UPLC HSS T3 (100 × 2.1 mm, 1.8 μm) column with a gradient elution. Assay accuracy, precision, linearity, selectivity, sensitivity and analyte stability covering sample storage and analysis were established. This validated UPLC–MS/MS method has been applied to a BE study for the determination of EE2, NGM and DNGM concentrations in human plasma.
Keywords: Ethinyl estradiol; Norgestimate; 17-Desacetyl norgestimate; Derivatization; Human plasma; UPLC–MS/MS;

Quantification of flavonol glycosides in Camellia sinensis by MRM mode of UPLC-QQQ-MS/MS by Yahui Wu; Xiaolan Jiang; Shuxiang Zhang; Xinlong Dai; Yajun Liu; Huarong Tan; Liping Gao; Tao Xia (10-17).
Phenolic compounds are major components of tea flavour, in which catechins and flavonol glycosides play important roles in the astringent taste of tea infusion. However, the flavonol glycosides are difficult to quantify because of the large variety, as well as the inefficient seperation on chromatography. In this paper, a total of 15 flavonol glycosides in the tea plant (Camellia sinensis) were identified by the high performance liquid chromatography (HPLC) coupled to a time-of-flight mass spectrometer (TOF-MS), and a quantitative method was established based on multiple reaction monitoring (MRM) mode of ultra-high performance liquid chromatography (UPLC) coupled to a triple quadrupole mass spectrometer (QQQ-MS/MS). It provided the limit of detection and quantification to the order of picogram, which was more sensitive than the HPLC detection of the order of nanogram. The relative standard deviations of the intra- and inter-day variations in retention time and signal intensity (peak area) of six analytes were less than 0.26% and 4%, respectively. The flavonol glycosides of four tea cultivars were relatively quantified using the signal intensity (peak area) of product ion, in which six flavonol glycosides were quantified by the authentic standards. The results showed that the flavonol mono-, di- and tri-glycoside mostly accumulated in young leaves of the four tea cultivars. Notably, the myricetin 3-O-galactoside was the major component among the six flavonol glycosides detected.
Keywords: Quantification; Camellia sinensis; Flavonol glycosides; HPLC-TOF-MS; UPLC-QQQ-MS/MS;

A new kind of selective magnetic dispersive solid-phase sorbent based on multiple Fe3O4 nanospheres as the core structure and molecular imprinted material as the shell structure was synthesized with tert-butylamine and 2-chloroaniline as the templates. The obtained multicore–shell-structured sorbent was spherical (diameter distribution 25–90 μm) with porous morphologies, thus incorporating strong magnetic properties and specific molecular recognition coupled with rapid adsorption and dynamic equilibrium. The sorbent was applied for rapid and selective screening of clenbuterol (CLB) in bovine urine samples. Good linearity was obtained in the range 1.25–200 ng mL−1 with the average recovery at three spiked levels ranging from 91.4% to 105.3%. The proposed method significantly improved the purification and extraction efficiency of CLB in urine samples and eliminated the effect of template leakage during quantitative analysis.
Keywords: Magnetic dispersive sorbent; Molecular imprinting; Dispersive solid-phase extraction; Clenbuterol; Urine;

An ultra fast liquid chromatography-tandem mass sepectrometry (UFLC-MS/MS) method was developed for simultaneous determination of seven active alkaloid components (tetrahydropalmatine, corydaline, α-allocryptopine, tetrahydroberberine, tetrahydrocoptisine, tetrahydrocolumbamine and dehydrocorydaline) in rat plasma after oral administration of Cerebralcare Granule. Plasma samples were pretreated by protein precipitation with acetronitrile containing the internal standard diazepam. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 μm) with gradient elution using mobile phase consisting of acetonitrile −0.1% formic acid in water at a flow rate of 0.3 mL/min. The detection was performed on an electrospray ionization triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM) with positive ionization mode. The established method was fully validated and proved to be sensitive and specific with lower limits of quantification (LLOQs) all less than 0.0265 ng/mL in rat plasma. Good linearities of seven alkaloids were obtained in respective concentration ranges (r  > 0.9923). The intra- and inter-day precisions were below of 15% for all the seven alkaloids in terms of relative standard deviation (RSD), and the accuracies were ranged from −2.7% to 8.3% in terms of relative error (RE). Extraction recovery, matrix effect and stability were within the required limits in rat plasma. The validated method was successfully applied to investigate the pharmacokinetics of the seven alkaloids in rat plasma after oral administration of Cerebralcare Granule (CG).
Keywords: Cerebralcare Granule; Alkaloids; UFLC-MS/MS; Pharmacokinetics;

Profiling post-translational modifications of histones in neural differentiation of embryonic stem cells using liquid chromatography–mass spectrometry by Shuzhen Zheng; Ming Sun; Kai Zhang; Junjie Gu; Zhenchang Guo; Shanshan Tian; Guijin Zhai; Xiwen He; Ying Jin; Yukui Zhang (36-44).
The neural differentiation of embryonic stem cells (ESCs) is of great significance for understanding of the mechanism of diseases. Histone post-translational modifications (HPTMs) play a key role in the regulation of ESCs differentiation. Here, we combined the stable isotope chemical derivatization with nano-HPLC-mass spectrometry (MS) for comprehensive analysis and quantification of histone post-translational modifications (HPTMs) in mouse embryonic stem cells (mESCs) and neural progenitor cells (mNPCs) that was derived from ESCs. We identified 85 core HPTM sites in ESCs and 78HPTM sites in NPCs including some novel lysine modifications. Our quantitative analysis results further revealed the changes of HPTMs from ESCs to NPCs and suggested effect of combinational HPTMs in the differentiation. This study demonstrates that HPLC–MS-based quantitative proteomics has a considerable advantage on quantification of combinational PTMs and expands our understanding of HPTMs in the differentiation.
Keywords: HPLC–MS/MS; Derivatization; Proteomics; Quantitative analysis; Cell differentiation;

A novel method for analysing key corticosteroids in polar bear (Ursus maritimus) hair using liquid chromatography tandem mass spectrometry by Johan J. Weisser; Martin Hansen; Erland Björklund; Christian Sonne; Rune Dietz; Bjarne Styrishave (45-51).
This paper presents the development and evaluation of a methodology for extraction, clean-up and analysis of three key corticosteroids (aldosterone, cortisol and corticosterone) in polar bear hair. Such a methodology can be used to monitor stress biomarkers in polar bears and may provide as a useful tool for long-term and retrospective information. We developed a combined pressurized liquid extraction (PLE)–solid phase extraction (SPE) procedure for corticosteroid extraction and clean-up followed by high pressure liquid chromatography tandem mass spectrometry (HPLC–MS/MS) analysis. This procedure allows for the simultaneous determination of multiple steroids, which is in contrast to previous polar bear studies based on ELISA techniques. Absolute method recoveries were 81%, 75% and 60% for cortisol, corticosterone and aldosterone, respectively. We applied the developed method on a hair sample pooled from four East Greenland polar bears. Herein cortisol and corticosterone were successfully determined in levels of 0.32 ± 0.02 ng/g hair and 0.13 ± 0.02 ng/g hair, respectively. Aldosterone was below limit of detection (LOD < 0.17 ng/g). The cortisol hair concentration found in these East Greenland polar bears was consistent with cortisol levels previously determined in the Southern Hudson Bay and James Bay in Canada using ELISA kits.
Keywords: PLE; SPE; LC–MS/MS; Polar bear hair; Fur; Cortisol; Corticosterone; Aldosterone;

In the present work the plasma levels of PZA and its two active metabolites, pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OH PA) were determined by a sensitive and rapid LC–MS/MS method. The analytes and their labeled internal standards were extracted from 200 μL plasma samples by liquid-liquid extraction with methyl tert-butyl ether: diethyl ether (90:10, v/v) under acidic conditions. Their separation was achieved on a Zorbax Eclipse XDB C18 (100 × 4.6 mm, 3.5 μm) column using methanol and 0.1% acetic acid (65:35, v/v) as the mobile phase within 4.0 min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions, m/z 124.1 → 81.1,  m/z 125.0 → 80.9 and m/z 141.0 → 81.0 for PZA, PA and 5-OH PA respectively in the positive ionization mode. All the analytes were baseline resolved with a resolution factor of 3.3 and 6.4 between PZA and its metabolites, PA and 5-OH PA respectively. The calibration curves were linear from 0.100–30.0 μg/mL, 0.03–9.00 μg/mL and 0.002–0.600 μg/mL for PZA, PA and 5-OH PA respectively with r2  ≥ 0.9980 for all the analytes. The intra-batch and inter-batch accuracy and precision (% CV) across quality controls varied from 93.5–106.7% and 1.10–4.57 respectively for all the analytes. The mean extraction recovery of PZA, PA and 5-OH PA was 83.7%, 89.2% and 80.8% respectively, which was consistent at higher as well as lower concentration levels. The% change in the stability of analytes under different storage conditions ranged −6.7 to 7.1 for all the analytes. The method was applied to assess the comparative bioavailability of a 500 mg PZA test and reference formulation in healthy subjects. The assay reproducibility was also tested by reanalysis of 22 incurred subject samples.
Keywords: Pyrazinamide; Pyrazinoic acid; 5-hydroxy pyrazinoic acid; LC–MS/MS; Human plasma; Pharmacokinetic study;

A simple, sensitive, and efficient method has been developed for simultaneous estimation of valsartan and atorvastatin in human plasma by combination of solid-based dispersive liquid–liquid microextraction and high performance liquid chromatography–diode array detection. In the proposed method, 1,2-dibromoethane (extraction solvent) is added on a sugar cube (as a solid disperser) and it is introduced into plasma sample containing the analytes. After manual shaking and centrifugation, the resultant sedimented phase is subjected to back extraction into a small volume of sodium hydrogen carbonate solution using air-assisted liquid–liquid microextraction. Then the cloudy solution is centrifuged and the obtained aqueous phase is transferred into a microtube and analyzed by the separation system. Under the optimal conditions, extraction recoveries are obtained in the range of 81–90%. Calibration curves plotted in drug-free plasma sample are linear in the ranges of 5–5000 μg L−1 for valsartan and 10–5000 μg L−1 for atorvastatin with the coefficients of determination higher than 0.997. Limits of detection and quantification of the studied analytes in plasma sample are 0.30–2.6 and 1.0–8.2 μg L−1, respectively. Intra-day (n = 6) and inter-days (n = 4) precisions of the method are satisfactory with relative standard deviations less than 7.4% (at three levels of 10, 500, and 2000 μg L−1, each analyte). These data suggest that the method can be successfully applied to determine trace amounts of valsartan and atorvastatin in human plasma samples.
Keywords: Atorvastatin; Biological samples; Diode array detection; High-performance liquid chromatography; Solid disperser; Valsartan;

For the first time, a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of fraxin and its metabolite, fraxetin, in rat plasma, using esculin as the internal standard (IS). The plasma samples were precipitated with methanol before separation on an Nova-Pak C18 column (150 mm × 3.9 mm, 3 μm) using a mobile phase consisting of 0.1% formic acid and methanol (55:45) at a flow rate of 0.8 mL/min. The analytes were detected by multiple reaction monitoring in the negative ion mode with the mass transitions at m/z 368.9→ m/z 191.9 (fraxin), m/z 206.9→ m/z 191.8 (fraxetin) and m/z 339.0→ m/z 176.9 (esculin, IS). The results demonstrated that the calibration curves for both analytes have good linearity (r ≥ 0.995) over a concentration range of 5.00–3000 ng/mL. The assay was validated according to the regulatory bioanalytical guidelines and proved acceptable. The intra- and inter-day precisions (R.S.D.%) were within 10.9% for both analytes, whereas the deviation of assay accuracies (R.E.%) ranged from −5.3 to 1.0%. The method was successfully applied to a pharmacokinetic study after a single oral dose of fraxin at 50 mg/kg to rats.
Keywords: Fraxin; Fraxetin; Metabolite; LC–MS/MS; Plasma;

An ion chromatographic method with conductivity detection for the precise and accurate analysis of lithium ions in phosphate-buffered saline, used as a cervicovaginal lavage (CVL) fluid, was developed and validated. The lithium ion dilution factor during the CVL is used to calculate the volume of cervicovaginal fluid (CVF) collected. Initial CVL Li+ concentrations of 1 mM and 10 mM were evaluated. The method is robust, practical, and afforded an accurate measurement (5% of the measurement, or better) at 24 μL of vaginal fluid simulant collected per mL of CVL fluid, as low as 5 μL mL−1 using 10 mM Li+ with a measurement accuracy of 6.7%. Ion chromatograms of real-world CVL samples collected in vivo from common animal models (sheep and pig-tailed macaque) and a human volunteer demonstrate that the analysis is interference-free. The method is readily transferrable and should enable the accurate measurement of CVF volume collected during CVLs benefitting a broad range of research disciplines, including pharmacokinetic, pharmacodynamic, metabolomic, and microbiome studies.
Keywords: Cervicovaginal fluid dilution; Cervicovaginal lavage; Ion chromatography; Lithium;

Qualitative screening of veterinary anti-microbial agents in tissues, milk, and eggs of food-producing animals using liquid chromatography coupled with tandem mass spectrometry by Dongmei Chen; Jie Yu; Yanfei Tao; Yuanhu Pan; Shuyu Xie; Lingli Huang; Dapeng Peng; Xu Wang; Yulian Wang; Zhenli Liu; Zonghui Yuan (82-88).
A method for the analysis of 120 drugs in animal derived food was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These analytes belong to 12 families of veterinary anti-microbial agents (quinolones, macrolides, β-lactams, nitroimidazoles, sulfonamides, lincomycines, chloramphenicols, quinoxalines, tetracyclines, polypeptides, and antibacterial synergists) as well as other compounds not assigned to a particular drug family. The animal derived food samples include muscle and liver of swine, bovine, sheep, and chicken, as well as hen eggs and dairy milk. The sample preparation included ultrasound-assisted extraction (UAE) with acetonitrile-water and a final clean-up with auto solid-phase extraction (SPE) on HLB cartridges. The detection and quantification of 120 anti-microbial agents was performed using LC-MS/MS in positive and negative ion mode. The chromatographic separation was performed on a C18 column using acetonitrile and 0.1% formic acid as the mobile phase. The limit of detection (LOD) and limit of quantification (LOQ) of all drugs in food-producing animals were 0.5–3.0 μg/kg and 1.5–10.0 μg/kg, respectively. The developed method was successfully utilized to monitor real samples, which demonstrated that it is a simple, fast, and robust method, and could be used as a regulatory to screen for the presence of residues from veterinary anti-microbial drugs in animal-derived foods.
Keywords: Veterinary anti-microbial agent; LC-MS/MS; Screening; Multi-residue; Animal derived food; Ultrasound-assisted extraction;

Preparation, characterization and usage of molecularly imprinted polymer for the isolation of quercetin from hydrolyzed nettle extract by Şeyda Karaman Ersoy; Esma Tütem; Kevser Sözgen Başkan; Reşat Apak; Cevdet Nergiz (89-100).
Quercetin (3,3′,4′,5,7-pentahydroxyflavone, QC) is a health-beneficial flavonoid, widely occurring in leaves, fruits, and flowers of various plants. In this work aiming isolation, purification and pre-concentration of QC, QC imprinted polymers (QC-MIPs) in different molar ratios {template:monomer:cross-linker (1:4:20, 1:5:30, 1:8:40, 1:10:50)} were prepared thermally through bulk polymerization by using QC as the template molecule, 4-vinylpyridine (4-VP), methacrylic acid (MAA), acrylamide (AA) as the functional monomers, ethylene glycol dimethacrylate (EDMA) as the cross-linker and 2,2′-azobisisobutyronitrile (AIBN) as initiator in the porogens of acetone and tetrahydrofuran. Their recognition and selectivity properties were investigated in solutions containing QC and other similar-structure phenolics by equilibrium binding experiments using different proportions of acetonitrile (ACN)-dimethylsulfoxide (DMSO) mixtures and methanol (MeOH) as solvents. The MIP with 1:4:20 molar ratio of QC:4-VP:EDMA was established as the most suitable for recognition of QC. Sorption parameters of the MIP and the NIP (non-imprinted polymer) were calculated by using Freundlich and Langmuir isotherms with QC solutions in ACN:DMSO (98:2, v/v). The mentioned MIP was found to be highly selective for quercetin over other phenolic compounds (rutin, catechin, etc.). Thus, molecularly imprinted solid-phase extraction (MISPE) procedures were applied for selective pre-concentration and purification of QC from synthetic mixtures of phenolic compounds and nettle extract, known as a source of official and folk medicine. The results demonstrated the possibility of direct extraction of certain pharmacophoric constituents such as QC and QC derivatives from nettle by MIP separation.
Keywords: Quercetin recovery; Molecularly imprinted polymer; Quercetin imprinted solid phase extraction; Nettle;

A stereoselective analytical method was developed and validated for the quantification of bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion and threohydrobupropion in human plasma. Separation of individual enantiomers (R)-bupropion, (S)-bupropion, (R,R)-hydroxybupropion, (S,S-hydroxybupropion), (1S,2S)-threohydrobupropion, (1R,2R)-threohydrobupropion, (1R,2S)-erythrohydrobupropion, and (1S,2R)-erythrohydrobupropion was achieved utilizing an α1-acid glycoprotein column within a 12-min run time. Chromatograph separation was significantly influenced by mobile phase pH and variability between columns. Analytes were quantified by positive ion electrospray tandem mass spectrometry following plasma protein precipitation with 20% trichloroacetic acid. Identification of erythrohydrobupropion enantiomer peaks and threohydrobupropion enantiomer peaks was achieved by sodium borohydride reduction of enantiopure (R)- and (S)-bupropion. Initial assay validation and sensitivity determination was on AB Sciex 3200, 4000 QTRAP, and 6500 mass spectrometers. Accuracy and precision were within 15% for each analyte. The assay was fully validated over analyte-specific concentrations using an AB Sciex 3200 mass spectrometer. Intra- and inter-assay precision and accuracy were within 12% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), and erythrohydrobupropion (1R,2S and 1S,2R) were 0.5, 2, 1, and 1 ng/mL, respectively. All analytes were stable following freeze thaw cycles at −80 °C and while stored at 4 °C in the instrument autosampler. This method was applicable to clinical pharmacokinetic investigations of bupropion in patients. This is the first chromatographic method to resolve erythrohydrobupropion and threohydrobupropion enantiomers, and the first stereoselective LC–MS/MS assay to quantify bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma.
Keywords: Bupropion; Hydroxybupropion; Erythrohydrobupropion; Threohydrobupropion; LC–MS/MS; Stereoselectivity;

A simple and validated ultra high pressure liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of the dopaminergic agents pramipexole and ropinirole in plasma of patients with Parkinson’s disease. Following liquid–liquid extraction with tert-butyl methyl ether from 250 μL plasma, the separation of the analytes was achieved on a Gemini NX3 column using 10 mM pH 6.0 ammonium formate and 10 mM ammonium formate in methanol as binary gradient mobile phase at a flow rate of 0.3 mL/min. The MS/MS ion transitions were 212.10 → 153.03 for pramipexole, 261.2 → 114.2 for ropinirole and 256.1 → 211 for the internal standard (lamotrigine). The lower limit of quantitation (LLOQ) for both analytes was 80 pg/mL and the linearity was determined from 80 to 4000 pg/mL for pramipexole and from 200 to 10000 pg/mL for ropinirole. Mean recoveries were 94% for PRA and 73% for ROP. Both intra- and inter-assay precision and accuracy were ≤20% at LLOQ concentration and ≤15% at other concentrations. The proposed validated method was successfully applied to measure plasma concentrations of pramipexole and ropinirole in a series of patients with Parkinson’s disease on chronic treatment. By grouping the two dopaminergic agents in the same assay, the method allows a large series of patient samples to be processed in a single analytical session.
Keywords: Pramipexole; Ropinirole; Ultra high performance liquid chromatography–tandem mass spectrometry; Dopamine receptor agonist; Parkinson’s disease;

Simultaneous determination of moxifloxacin and ofloxacin in physiological fluids using high performance liquid chromatography with ultraviolet detection by Fahim Ullah Khan; Fazli Nasir; Zafar Iqbal; Ismail Khan; Naila Shahbaz; Muhammad Hassan; Farhad Ullah (120-128).
A novel, sensitive and validated RP-HPLC–UV method was developed for simultaneous determination of moxifloxacin and ofloxacin using timolol maleate as internal standard in physiological fluids. Different experimental parameters were optimized and validated according to international guidelines. Complete separation of the analytes was achieved with Kromasil 100-5C18 analytical column (250 mm × 4.6 mm × 5 μm), methanol and 0.05% trifloroacetic acid (TFA) (38:62 v/v) were used as mobile phase, pumped at flow rate of 1.1 ml/min in isocratic phase, column oven temperature maintained at 45 °C and detection wavelength of 290 nm. Protein precipitation method was applied to extract the drugs from human plasma and bovine aqueous humor samples using methanol as precipitating solvent. This method is linear in concentration range of 0.018–100 μg/ml for moxifloxacin and 0.014–20 μg/ml for ofloxacin. The recoveries of the method were 97.52 and 97.39% in human plasma for MX and OFN respectively, while in aqueous humor 94.48% for MX. The LOD values in plasma were found to be 10.0 and 8.00 ng/ml for MX and OFN respectively, while their respective LOQ values were 18.0 and 14 ng/ml. In aqueous humor the LOD and LOQ for MX were 16.0 and 24 ng/ml respectively. In future, this method will be used to study the pharmacokinetic profile of moxifloxacin and ofloxacin in biological fluids and pharmaceutical products.
Keywords: HPLC–UV; Moxifloxacin; Ofloxacin;

Functional hydrophilic polystyrene beads with uniformly size and high cross-linking degree facilitated rapid separation of exenatide by Qiang Li; Lan Zhao; Rongyue Zhang; Yongdong Huang; Yan Zhang; Kun Zhang; Xuexing Wu; Zhigang Zhang; Fangling Gong; Zhiguo Su; Guanghui Ma (129-135).
A high cross-linking polystyrene(PSt)-based anion-exchange material with uniformly size, high ion exchange capacity, and high hydrophilicity was synthesized by a novel surface functionalization approach in this study. Uniformly sized PSt microspheres were prepared by the membrane emulsion polymerization strategy, and then modified by (1) conversing resid ual surface vinyl groups to epoxy groups followed by quaternization, and (2) decorating aromatic ring matrix including nitration, reduction and attachment of glycidyltrimethylammonium chloride. The 3-D morphology and porous features of microspheres were observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The surface of the modified PSt became roughness but the particle size remained same. Meanwhile, FT-IR spectra and laser scanning confocal microscope (LCSM) indicated that the modification groups had been successfully covalently coated onto the PSt microspheres. Modified PSt microspheres showed greatly improved hydrophilicity and biocompatibility with 0.387 mmol/mL ion exchange capacity (IEC). In the application evaluation procedure, exenatide can be purified from 42.9% (peptide crudes) to 88.6% by modified PSt column with 97.1% recovery yield. This modified PSt microspheres had a large potential in application for efficient separation of peptides.
Keywords: Polystyrene microspheres; Hydrophilicity; Anion-exchange; Peptide separation;

Development and validation of a liquid chromatography-tandem mass spectrometric assay for quantitative analyses of triptans in hair by Daniele Vandelli; Federica Palazzoli; Patrizia Verri; Cecilia Rustichelli; Filippo Marchesi; Anna Ferrari; Carlo Baraldi; Enrico Giuliani; Manuela Licata; Enrico Silingardi (136-144).
Triptans are specific drugs widely used for acute treatment of migraine, being selective 5HT1B/1D receptor agonists. A proper assumption of triptans is very important for an effective treatment; nevertheless patients often underuse, misuse, overuse or use triptans inconsistently, i.e., not following the prescribed therapy. Drug analysis in hair can represent a powerful tool for monitoring the compliance of the patient to the therapy, since it can greatly increase the time-window of detection compared to analyses in biological fluids, such as plasma or urine. In the present study, a liquid chromatography-tandem mass spectrometric (LC–MS/MS) method has been developed and validated for the quantitative analysis in human hair of five triptans commonly prescribed in Italy: almotriptan (AL), eletriptan (EP), rizatriptan (RIZ), sumatriptan (SUM) and zolmitriptan (ZP). Hair samples were decontaminated and incubated overnight in diluted hydrochloric acid; the extracts were purified by mixed-mode SPE cartridges and analyzed by LC–MS/MS under gradient elution in positive multiple reaction monitoring (MRM) mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection (LOD) and lower limit of quantitation (LLOQ), accuracy, precision, carry-over, recovery, matrix effect and dilution integrity. The method was linear in the range 10–1000 pg/mg hair, with R2 values of at least 0.990; the validated LLOQ values were in the range 5–7 pg/mg hair. The method offered satisfactory precision (RSD <10%), accuracy (90–110%) and recovery (>85%) values. The validated procedure was applied on 147 authentic hair samples from subjects being treated in the Headache Centre of Modena University Hospital in order to verify the possibility of monitoring the corresponding hair levels for the taken triptans.
Keywords: Triptans; HPLC–MS/MS; Hair samples; Migraine; Overuse;

A novel method for simultaneous quantification of alpha-aminoadipic semialdehyde/piperideine-6-carboxylate and pipecolic acid in plasma and urine by Tatiana Yuzyuk; Aiping Liu; Amanda Thomas; JoDell E. Wilson; Irene De Biase; Nicola Longo; Marzia Pasquali (145-152).
Elevated levels of pipecolic acid (PA), α-aminoadipic semialdehyde (AASA) and its cyclic form Δ1-piperideine-6-carboxylate (P6C) are characteristic of pyridoxine dependent epilepsy (PDE), a rare disorder of inborn error of metabolism. Recent studies showed the effectiveness of dietary therapy in PDE patients and emphasized the importance of the assessment of these metabolites for monitoring treatment efficacy. The objective of this study was to develop a robust and sensitive method for simultaneous quantification of AASA-P6C and PA in plasma and urine.Plasma and urine samples were derivatized with 3 N HCl in n-butanol (v/v) and injected onto ACQUITY BEH-C18 column. A gradient of water/methanol containing 0.1% formic acid was used for the chromatographic separation of AASA, P6C and PA. The analytes’ concentrations were calculated using their calibration curves and the sum of AASA and P6C (AASA-P6C) was calculated. To evaluate the clinical utility of this test, samples from unaffected controls and patients with confirmed PDE were analyzed.The performance characteristics of the assay as well as sample stability and interferences were determined. The intra- and inter- assay CVs were ≤2.9% and ≤10.9% for AASA-P6C, and ≤3.3% and ≤12.6% for PA, respectively. Reference ranges for AASA-P6C and PA in plasma and urine were established. Comparison of values obtained from unaffected controls and PDE patients showed high clinical sensitivity and specificity of the assay.This novel method for the simultaneous quantification of AASA-P6C and PA in plasma and urine can be used in a clinical laboratory setting for the diagnosis and monitoring of patients with PDE.
Keywords: 1-piperideine-6-carboxylate; Alpha-aminoadipic semialdehyde; Pipecolic acid; Pyridoxine dependent epilepsy; UPLC–MS/MS;

Tomato style is the pathway for pollen germination and pollen tubes growth from the stigma to the ovules where fertilization occurs. It is essential to supplying the nutrients for pollen tube growth and guidance for the pollen tubes. To our knowledge, style also regulates gametophytic self-incompatibility (SI) in tomato species. This study identified the metabolites and monitored the metabolic changes of self-incompatible and self-compatible tomato with self-pollinated or unpollinated styles by gas chromatography–mass spectrometry (GC–MS). A total of 9 classes of compounds were identified in SI and self-compatibility (SC) self-pollinated and unpollinated styles which included amino acids, sugars, fatty acids/lipids, amines, organic acids, alcohols, nitriles, inorganic acids and other compounds. The contents of d-Mannose-6-phosphate, Cellobiose, Myristic acid, 2,4-Diaminobutyric acid, Inositol and Urea were significantly decreased and the rest did not significantly change in SI styles. But change of metabolites content significantly happened in SC styles. In addition, among the total 9 classes of compounds, the different metabolites accounted for a different proportion in amino acids, sugars, amines, organic acids and alcohols compared SC and SI. The result indicated that the physiological changes of styles existed differences in SC and SI after self pollination.
Keywords: GC–MS; Metabolomics; Style; Self-incompatibility; Self-compatibility; Tomato;

Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography by Cecília Alves Mourão; Gabriela Pannunzio Carmignotto; Sonia Maria Alves Bueno (163-173).
This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9 mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7 mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution.
Keywords: Human Fab fragments; IMAC; CM-Asp (carboxymethylaspartate); Adsorption; Negative chromatography;

A novel type of aptamer-functionalized immunoadsorbent was prepared and characterized to remove HCV particles, a promising option of extracorporeal immunoadsorption (ECI) therapy against HCV. Herein, we fabricated a HCV-specific immunoadsorbent where single-stranded DNA aptamers reported and studied previously, modified with amino group at the 5′ terminus, was immobilized covalently onto surfaces of carboxylated-derivative sepharose 4FF beads through N-hydroxysuccinimide (NHS) linkage. Then the adsorbents was evaluated and characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Subsequently, we also confirmed that proposed immunoadsorbents exhibited a favorable biocompatibility as well as specificity. In addition, time-dependent effects of the eradication capacity of aptamer functionalized sepharose beads against HCV was investigated. With the optimized time point, the decontamination performance of HCV particles was assessed by real-time quantitative PCR (qPCR) followed by nucleic acid-based hybridization (NAH), which shows sorbents with an aptamer density of 2 nmol ligand/ml resin could remove approximately 80% (i.e. 8.9 × 106 HCV particles/ml resin) of the HCV genotype 2a cultivated in vitro and 75% (vary with the intial concentration of HCV from about 7.5 × 104–4.4 × 105 HCV particles/ml resin) of the HCV samples from human plasma samples. All these results indicated that the novel aptamer-based adsorbents could effectively remove HCV particles and likely serve as a novel therapy option or at least supplementary for the treatment regimen of HCV.
Keywords: Immunoadsorbent; Aptamer; HCV; NHS linkage; Biocompatibility; qPCR; NAH;

Purification of porcine reproductive and respiratory syndrome virus using ultrafiltration and liquid chromatography by Liu Bohua; Sun Ming; Yang Lu; Deng Xiaoyu; Li Baochun; Shentu Fenqin; Zhang Li; Chen Xizhao (182-186).
Porcine reproductive and respiratory syndrome (PRRS) virus causes severe and persistent disease in pigs worldwide. Its heterogeneity poses a major challenge for the effective prevention and control of PRRS. Purified viruses are essential for serological studies. Traditional methods for purifying PRRS virus are time- and labor-intensive and difficult to scale-up and requires long processing time. Here, we describe a rapid, simple, scalable process for PRRS virus purification. Highly pure viral particles were obtained after ultrafiltration and liquid chromatography, as confirmed by SDS-PAGE and electron microscopy. The overall process achieved a recovery of 50% of raw virus, with a purity close to that obtained by CsCl coupled with sucrose density gradients. The purification process described here should be useful in large-scale production of highly pure PRRS virus.
Keywords: Porcine reproductive and respiratory syndrome virus; Purification; Chromatography;

A new method using solid phase extraction (SPE) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of six polyether antibiotics, including lasalocid, salinomycin, monensin, narasin, madubamycin and nigericin residues, in foods of animal origin. The samples were extracted with acetonitrile and purified by ENVI-Carb SPE columns after comparing the impurity effect and maneuverability of several SPE cartridges. Subsequently, the analytes were separated on a Hypersil Gold column (2.1 × 150 mm, 5 μm) and analyzed by MS/MS detection. The limit of quantization (LOQ) for milk and chicken was 0.4 μg/kg, and for chicken livers and eggs, it was 1 μg/kg. The linearity was satisfactory with a correlation coefficient of >0.9995 at concentrations ranging from 2 to 100 μg/L. The average recoveries of the analytes fortified at three levels ranged from 68.2 to 114.3%, and the relative standard deviations ranged from 4.5 to 12.1%. The method was suitable for quantitative analysis and confirmation of polyether antibiotic residues in foods of animal origin.
Keywords: SPE; LC–MS/MS; Polyether antibiotics; Foods of animal origin;

Comparative analysis of bioactive N-alkylamides produced by tissue culture raised versus field plantlets of Spilanthes ciliata using LC-Q-TOF (HRMS) by Zubair Shanib Bhat; Neeharika Jaladi; Ravi Kant Khajuria; Zeeshan Hamid Shah; Neelakantan Arumugam (195-203).
Spilanthes ciliata (S. ciliata) is a perennial herb of global importance owing to its luscious source of bioactive fatty acid derived amides known as N-alkylamides. It finds application in skin creams, mouth gels and toothpastes. Despite multifaceted applications, a major limitation associated for its commercial application is the scarcity of contamination free plant source, fluctuations in active metabolites due to variation in extraction procedures, and lack of rapid qualitative method for alkylamide profiling. In the current work, attempts were made to 1) optimize conditions for mass propagation of contamination free plants of S. ciliata by tissue culture using leaf discs as explants, 2) establish an optimum extraction ratio of plant/solvent (w/v) for maximum elution of alkylamides and 3) develop a rapid method for qualitative estimation of alkylamide from in vitro raised plants in comparison with that of the field grown counterpart by using LC-Q-TOF (HRMS). To the best of our knowledge, this is the first qualitative report on alkylamide profile of micropropagated whole plant of Spilanthes. The correlation pattern reported in this study may form the basis for using tissue culture raised plantlets of S. ciliata as potential source of bioactive alkylamides on industrial scale.
Keywords: Tissue culture; Spilanthes ciliata; Alkylamides; Ethanol extract; LC–MS;

UFLC–MS/MS analysis of four tanshinone components in Salvia miltiorrhizae after ultrasound-assisted extraction by Shanshan Zhang; Qiutao Liu; Hongli Luo; Ping Chen; Xiaoru Wu; Meihua Yang; Weijun Kong; Weiying Guo (204-210).
A sensitive and rapid ultra-fast liquid chromatography with tandem mass spectrometry (UFLC–MS/MS) method was developed for simultaneous qualitative and quantitative of four characteristic tanshione components including tanshinone IIA, cryptotanshinone, tanshinone I and dihydrotanshinone I in Salvia miltiorrhizae after ultrasound-assisted extraction. By using a C18 column, the four analytes were separated by gradient elution with acetonitrile and water both containing 0.1% formic acid at the flow rate of 0.3 mL/min. Multiple-reaction monitoring (MRM) was used for quantification, and an information-dependent acquisition (IDA) method was used to trigger enhanced product ion scans (EPI) for supplementary characteristic identification for qualitative research. Calibration curves showed good linearities with correlation coefficients (r) higher than 0.9990. The method showed high sensitivity with limits of detection (LODs) and quantification (LOQs) less than 0.0002 ng/mL and 0.0008 ng/mL, respectively, as well as good precision and reproducibility. Mean recoveries for four analytes ranged from 92.5% to 106.2% with relative standard deviations (RSDs) lower than 14.59%. Real application of the developed method in 32 batches of S. miltiorrhizae samples demonstrated that the total contents of four analytes in all samples were in the range of 2.258–52.342 mg/g. Ultrasound-assisted extraction technique took a small amount of sample and low time but giving high extraction efficiency. Combining with UFLC–MS/MS method in MRM-IDA-EPI mode, more components in other complicated matrices can be simultaneously analyzed for qualitation and quantitation in one run.
Keywords: Salvia miltiorrhizae; Tanshinone; Ultrasound-assisted extraction; UFLC–MS/MS; MRM-IDA-EPI;

Multi-residue analysis of 26 organochlorine pesticides in Alpinia oxyphylla by GC-ECD after solid phase extraction and acid cleanup by Xiangsheng Zhao; Yakui Zhou; Weijun Kong; Bao Gong; Deli Chen; Jianhe Wei; Meihua Yang (211-220).
A simple and effective multi-residue method was developed and validated for the analysis of 26 organochlorine pesticide residues in Alpinia oxyphylla by a gas chromatography with an electron capture detector (GC-ECD). The target pesticides were extracted by sonication and cleaned up with florisil solid phase extraction and sulphuric acid. Some crucial parameters, including extraction solvent and time, sorbent type, elute solvent and concentration of sulphuric acid were optimized to improve the performance of sample preparation procedure. The optimized method gave high sensitivity with detection limit ranging from 0.1 to 2.0 μg/kg. Matrix-matched calibration was employed for the quantification, and a wide linear range (from 1.0 to 1000 μg/kg) with r2 values ranging from 0.9971 to 0.9998 was obtained. For the majority of the tested pesticides, the average recoveries were in acceptable range (between 70% and 110%) with relative standard deviation values below 15.0%. Matrix effect was evaluated for target compounds through the study of ratio of peak area obtained in the solvent and blank matrix. The proposed method was applied to simultaneously analyze 26 pesticides in 55 batches of Alpinia oxyphylla samples. 3 samples were found to be positive with four pesticides (α-BHC, quintozene, trans-chlordane and op'-DDD), which were confirmed by gas chromatography-mass spectrometry (GC–MS) in selective ion monitoring (SIM) mode.
Keywords: Alpinia oxyphylla; Organochlorine pesticides; Solid-phased extraction; Gas chromatography-electron capture detection;

Separation of catechins and O-methylated (−)-epigallocatechin gallate using polyamide thin-layer chromatography by Kunbo Wang; Qincao Chen; Yong Lin; Shuangshang Yu; Haiyan Lin; Jianan Huang; Zhonghua Liu (221-225).
Thin-layer chromatography (TLC) method for the separation and quantitative determination of seven related compounds: (+)-catechin (C), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3″Me) and (−)-epigallocatechin- 3-O-(4-O-methyl) gallate (EGCG4″Me) has been developed. The above-mentioned seven compounds have been resolved using polyamide TLC plates using a double-development with methanol followed by acetone/acetic acid (2:1, v/v). In addition, separation of the phenolic acids namely gallic acid, chlorogenic acid, and caffeic acid was achieved using the same solvent system. The applicability of the method was checked by screening of extracts of green, black, oolong, white tea and tea cultivars leaves.
Keywords: Catechins; O-methylated (−)-epigallocatechin gallate; Thin-layer chromatography; Double-development; Tea;

Nimodipine is a dihydropyridine calcium-channel blocker that has been recently shown to be effective on the function of central nervous system. It has been reported that treatment against deficits of learning and memory in animals and human by maintain the calcium homeostasis in hippocampus with nimodipine may be promising therapeutic strategies. A rapid and sensitive liquid chromatography-tandem mass spectrometric method was developed to determination the nimodipine in hippocampus using microdialysis technique. The separation was accomplished on an Agilent Zorbax SB-C18 column (100 mm × 2.1 mm ID, 3.5 μm) with the mobile phase composed of methanol-water (80:20, v/v) containing 0.2% formic acid at a flow rate of 0.2 ml/min. Multiple reaction monitoring of the precursor-product ion transitions 419 → 343 for nimodipine and 361 → 315 nitrendipine (IS) was used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.2 ng/ml for nimodipine, with good linearity in the range of 0.2–20 ng/ml. All the validation data, such as accuracy, precision, intra- and inter-day repeatability and stability were within the required limits. The method was successfully applied to p harmacokinetic study of the nimodipine in the guinea pig hippocampus.
Keywords: Microdialysis; LC–MS/MS; Nimodipine; Hippocampus; Pharmacokinetics;

In this study, two simple pretreatment methods were comprehensively evaluated for the determination of 135 pesticide residues in roots and rhizomes of Chinese herbal medicines (CHMs). The studied methodologies are (a) solid-phase extraction (SPE) and (b) Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS). For SPE, extraction solvents, SPE cartridges and types and volume of eluent were accessed and optimized. For QuEChERS, different versions, acetic acid concentration and dispersive solid-phase extraction (dSPE) sorbent materials were tested. SPE and QuEChERS were estimated in recovery range, the number of pesticides that were recovered ranging from 90% to 110% and expenses in Corydalis Rhizoma, Chuanxiong Rhizoma and Angelicae Sinensis Radix. QuEChERS method showed better performance than SPE. The method showed good linearity over the range assayed 0.9986–0.9999 (1–80 ng/mL for 124 pesticides, 1–50 ng/mL for 10 pesticides, 1–20 ng/mL for satisfar). The matrix effect was compensated by matrix-based calibration curves with internal standard. The average recoveries of all pesticides were ranging from 70% to 120% at three levels of 10, 50 and 100 ng/g with relative standard deviations less than 20%. The limits of quantification of the 135 pesticides in three matrices were 1–5 ng/g, which were below the maximum residue levels (MRLs) established by the European Union. The verified QuEChERS method was successfully applied to the analysis of 65 actual samples from eight different types of roots and rhizomes of CHMs. Angelicae Sinensis Radix was the most susceptible to pesticides among these samples, and the most frequently detected pesticide was carbendazim with levels below MRLs. Metalaxyl, phorate, atrazine, diniconazole, coumaphos and paclobutrazol were also detected in some samples.
Keywords: Pesticides; Solid-phase extraction; QuEChERS; Chinese herbal medicines; UHPLC–MS/MS;

Rapid, easy and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analysis method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography-mass spectrometry was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Optimization of the SPE cleanup yielded recovery rates of minimum 95% for all amino acids (except arginine). Variations in accuracy and precision did not exceed 12.5%, except for cysteine, histidine and tryptophane, which were excluded from analysis. Quantification of overlapping peaks for isoleucine/threonine and proline/asparagine was possible by selection of two specific fragment ions for each amino acid. Of the 16 selected amino acids, 14 were quantified successfully in at least 75% of the samples, while methionine and tyrosine were only quantifiable in 6% and 42%, respectively. A case study on the aspartate super pathway confirmed the applicability of the optimized method on wild type and genetically modified plants: external supplementation of methionine or lysine yielded a 146-fold or 27-fold increase in the respective absolute amino acid levels compared with the control treatment. Induced expression of dhdps-r1 (a mutated lysine biosynthesis gene encoding a feedback insensitive enzyme) caused an 83-fold increase in absolute lysine levels.
Keywords: GC–MS; Amino acid profiling; Aspartate super pathway; Methyl chloroformate; Solid phase extraction; Arabidopsis thaliana;