Journal of Chromatography B (v.997, #C)

In some developing countries, p-phenylenediamine (PPD) is used in combination with Henna as hair dye or skin decoration. A sensitive LC–MS/MS method was developed and validated for the simultaneous determination of p-phenylenediamine (PPD) and its metabolites N-acetyl-p-phenylenediamine (MAPPD) and N,N-diacetyl-p-phenylenediamine (DAPPD) in human blood. Acetanilide was used as an internal standard (IS). The LC–MS/MS was operated under multiple reaction-monitoring mode using the electrospray positive ionization technique. The transition ions m/z 109 → 92, m/z 151 → 92, m/z 193 → 92, and m/z 136 → 77 were selected for the quantification of PPD, MAPPD, DAPPD, and IS, respectively. The linear range was 10–2000 ng/mL for all the compounds. The absolute recoveries were 51.94, 56.20 and 54.88% for PPD, MAPPD and DAPPD, respectively. Intra- and inter-assay imprecision were lower than 14% (RSD), and the bias of the assay was lower than 15% for all the compounds. The stability studies demonstrated that critical degradation for PPD in blood samples and autosampler occurred after 6 h, while MAPPD and DAPPD were stable in blood samples and the autosampler up to 48 h and 24 h, respectively. This newly developed method allows for the detection of PPD and its metabolites in blood samples in the clinical and forensic setting.
Keywords: p-Phenylenediamine; Metabolites; Blood; LC–MS/MS; Validation; Stability;

Ormeloxifene (ORM) is a non-steroidal, selective estrogen receptor modulator used as a once a week oral contraceptive and is intended for long-term clinical use by females. Therefore, a simple, sensitive and rapid LC–MS/MS method was developed and validated for simultaneous quantification of ORM and its active metabolite (7-desmethyl ormeloxifene, 7-DMO) in rat plasma. Also, the method was partially validated in human plasma. Chromatographic separation was achieved on Discovery HS C-18 column (5 μm, 50 × 4.6 mm) with mobile phase [acetonitrile: aqueous ammonium acetate (0.01 M) buffer (85: 15, %v/v)] at a flow rate of 0.8 mL/min. The calibration curve was linear (r  ≥ 0.99) for a concentration range of 0.78–100 ng/mL for both the analytes. The precision (%RSD; ORM, 1.3 to 13.4; 7-DMO, 3.1 to 15.0) and accuracy (%bias; ORM, −13.8 to 12.5; 7-DMO, −10.6 to 6.8) in both rat and human plasma were within the acceptable limits. The recovery for ORM and 7-DMO was always >79.1% and >72.9%, respectively. Both the analytes were found stable in rat plasma even after 30 days of storage at −80 °C and on being subjected to three freeze–thaw cycles. The method has not only short run time (3.5 min) but requires a low plasma sample volume (20 μL) and is the first reported LC–MS/MS method for simultaneous quantification of the marketed drug known as centchroman (INN: ormeloxifene) and the metabolite 7-DMO in plasma. The method was applied to evaluate drug–drug interaction of ORM with the commonly prescribed antidepressant drug sertraline using serial sampling in rats.
Keywords: Centchroman; Drug interaction; LC–MS/MS; Sertraline;

An efficient hydrophilic interaction liquid chromatographic method for the simultaneous determination of metformin and pioglitazone using high-purity silica column by Abdel-Maaboud Ismail Mohamed; Fardous Abdel-Fattah Mohamed; Sameh Ahmed; Yahya Abduh Salim Mohamed (16-22).
Hydrophilic interaction liquid chromatography (HILIC) provides a feasible approach to effectively separate polar compounds in complex matrices. Herein, a simple, reproducible and efficient HILIC method was developed for the simultaneous determination of pioglitazone. HCl (PIO) and metformin HCl (MET) in rabbit plasma. High-purity silica column was used for rapid and efficient separation of these co-administered drugs. The chromatographic parameters were optimized for best separation. The proposed HILIC system provides high separation efficiency with good peak shape compared to reversed phase (RP) chromatography. Additionally, a simple isocratic elution mode with a mobile phase composed of a mixture of methanol and 10 mM phosphate buffer (pH 3.0) (94:6, v/v) was used and the effluent was monitored at 230 nm. The method was validated in accordance with the requirements of US-FDA guidelines and was found to behave efficiently for the intended purpose. The correlation coefficient of 0.9992 was obtained in the concentration ranges of 0.5–100 μg mL−1. The limits of detection (S/N = 3) and quantification (S/N = 10) were 0.16 and 0.5 ng mL−1, respectively. The retention times were 3.4 and 5.0 min for PIO and MET, respectively. Plasma levels were successfully determined in rabbit with satisfactory precision and accuracy. In addition, the stability tests in rabbit plasma proved reliable stability under the experimental conditions. The developed HILIC method was applied successfully to study the pharmacokinetic behaviors of the studied analytes in rabbit plasma after a single oral dose containing PIO and MET.
Keywords: Pioglitazone HCl; Metformin HCl; Oral hypoglycemic; Hydrophilic interaction liquid chromatography; Binary mixture; Pharmacokinetics;

Due to increasing regulatory awareness of their hepatotoxic, genotoxic and possibly carcinogenic potential, pyrrolizidine alkaloid (PA) content has to be thoroughly monitored in herbal medicinal preparations. Recently, new very low PA regulatory threshold concentrations have been requested by the authorities. Therefore, a highly sensitive and reproducible UPLC TOF MS method for the quantification of the PAs senkirkine, senecionine, seneciphylline, senecionine-N-oxide and seneciphylline-N-oxide in a CO2-extract of Petasites hybridus leaves (Ze 339) has been developed.The limit of quantification (LOQ) was 2 ppb for all PAs. Recovery at the LOQ was between 88.9 and 141.9%, the repeatability precision between 3.5 and 13.6%. Linearity of the five PAs showed correlation coefficients between 0.9995 and 0.9998 and coefficients of variation between 7.44 and 8.56%. A working range between 2 ppb and 200 ppb could be fixed. In the tested batches of the P. hybridus extract Ze 339, the absence of PAs could be demonstrated. In conclusion, this assay allows to determine trace PA concentrations in P. hybridus extract Ze 339, making it suitable for analytical PA monitoring in accordance with regulatory requirements.
Keywords: Pyrrolizidine alkaloids; UPLC; Mass spectroscopy;

Characterization of cysteine related variants in an IgG2 antibody by LC–MS with an automated data analysis approach by Yuling Zhang; Robert Bailey; Nancy Nightlinger; Alison Gillespie; Alain Balland; Richard Rogers (30-37).
In this communication, a high-throughput method for automated data analysis of cysteine-related product quality attributes (PQAs) in IgG2 antibodies is reported. This method leverages recent advances in the relative quantification of PQAs to facilitate the characterization of disulfide variants and free sulfhydryls (SHs) in IgG2 antibodies. The method uses samples labeled with a mass tag (N-ethyl maleimide [NEM]) followed by enzymatic digestion under non-reducing conditions to maintain the cysteine connectivity. The digested IgG2 samples are separated and detected by mass spectrometry (MS) and the resulting peptide map is analyzed in an automated fashion using Pinpoint software (Thermo Scientific). Previous knowledge of IgG2 disulfide structures can be fed into the Pinpoint software to create workbooks for various disulfide linkages and hinge disulfide variants. In addition, the NEM mass tag can be added to the workbooks for targeted analysis of labeled cysteine-containing peptides. The established Pinpoint workbooks are a high-throughput approach to quantify relative abundances of unpaired cysteines and disulfide linkages, including complicated hinge disulfide variants. This approach is especially efficient for comparing large sets of similar samples such as those created in comparability and stability studies or chromatographic fractions. Here, the high throughput method is applied to quantify the relative abundance of hinge disulfide variants and unpaired cysteines in the IgG2 fractions from non-reduced reversed-phase high-performance liquid chromatography (nrRP-HPLC). The LC–MS data analyzed by the Pinpoint workbook suggests that the nrRP-HPLC separated peaks contain hinge disulfide isoforms and free cysteine pairs for each major disulfide isoform structure.
Keywords: IgG2; Monoclonal antibody; Disulfide; Free sulfhydryls and cysteine; LC–MS; nrRP-HPLC; Pinpoint software;

A sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed for simultaneous chiral analysis of an antiasthma drug bambuterol, its key intermediate monocarbamate bambuterol and its active drug terbutaline in human plasma. All samples were extracted with ethyl acetate and separated on an Astec Chirobiotic T column under isocratic elution with a mobile phase consisting of methanol and water with the addition of 20 mm ammonium acetate and 0.005% (v/v) formic acid at 0.6 mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method has high sensitivity with the lower limit of quantifications of 25.00 pg/mL for bambuterol enantiomers, and 50.00 pg/mL for monocarbamate bambuterol and terbutaline enantiomers, respectively. The calibration curves for bambuterol enantiomers were linear in the range of 25.00–2500 pg/mL, and for monocarbamate bambuterol and terbutaline enantiomers were linear in the range of 50.00–5000 pg/mL. The intra- and inter-day precisions were <12.4%. All the analytes were separated in 18.0 min. For the first time, the validated method was successfully applied to an enantioselective pharmacokinetic study of rac-bambuterol in 8 healthy volunteers. According to the results, this chiral LC-MS/MS assay provides a suitable and robust method for the enantioselectivity and interaction study of the prodrug bambuterol, the key intermediate monocarbamate bambuterol and its active drug terbutaline in human.
Keywords: Chiral analysis; Bambuterol; Intermediate; LC–MS/MS; Clinical pharmacokinetics;

Most lipids are best characterized by their fatty acids which may differ in (a) chain length, (b) degree of unsaturation, (c) configuration and position of the double bonds, and (d) the presence of other functionalities. Thus, a fast, simple, and quantitative analytical technique to determine naturally occurring free fatty acids (FFA) in different samples is very important. Just as for saponified acylglycerols, the determination of FFA’s has generally been carried out by high resolution gas chromatography (HRGC). The use of an open tubular capillary column coupled with a flame ionization or mass spectrometric detector provides for both high resolution and quantification of FFA’s but only after conversion of all free fatty acids to fatty acid methyl esters (FAME) or pentafluorobenzyl esters. Unfortunately, volatilization of labile ester derivatives of mono- and poly-unsaturated FFA’s can cause both thermal degradation and isomerization of the fatty acid during HRGC. The employment of a second generation instrument (here referred to as UltraHigh Performance Supercritical Fluid Chromatograph, UHPSFC) with high precision for modified flow and repeated back pressure adjustment in conjunction with sub-2 μm various bonded silica particles (coupled with evaporative light scattering, ELSD, and mass spectrometric, MS, detection) for separation and detection of the following mixtures is described: (a) 31 free fatty acids, (b) isomeric FFA’s, and (c) lipophilic materials in two real world fish oil samples. Limits of detection for FFA’s via UHPSFC/MS and UHPSFC/ELSD versus detection of FAME’s via HRGC/MS are quantitatively compared.
Keywords: Free fatty acids; Supercritical fluid chromatography; Mass spectrometry; Evaporative light; scattering; High resolution gas chromatography; Fish oil; Detection limit; Fatty acid ester derivatization;

Regulated bioanalysis of conformers – A case study with ASP2151 in dog plasma and urine by Yoshiaki Ohtsu; Shohei Otsuka; Takeshi Nakamura; Kiyoshi Noguchi (56-63).
We developed and validated bioanalytical methods for a potent helicase–primase inhibitor ASP2151 that has two conformers. These conformers elute as unseparated broad peaks under ordinary high-performance liquid chromatographic conditions, indicating discernable differences in hydrophobicity. We observed that column temperature and mobile phase pH have no effect on these peaks and that conformers form a single symmetrical peak when tetrahydrofuran is added to the mobile phase. In addition, we needed to develop semi-automated methods where inter-conversion of the conformers is unlikely to cause sample-to-sample extraction variability. Briefly, following the addition of deuterium-labeled ASP2151 as an internal standard (IS), dog plasma samples or acetonitrile-added urine samples were filtrated. The filtrates were then injected into a column-switching liquid chromatography–tandem mass spectrometry (LC–MS/MS) system and trapped onto an extraction column. Extracts were back-flushed onto an analytical C18 column (4.6 × 50 mm, 3 μm) with a mobile phase consisting of methanol, tetrahydrofuran, and 20 mmol/L ammonium acetate (45:5:50, v/v/v). The eluent was monitored in the negative atmospheric pressure chemical ionization mode. The calibration curve was linear over a range of 5–1000 ng/mL for plasma and 0.5–100 μg/mL for urine. Validation data met the acceptance criteria in accordance with regulatory guidance and demonstrated that these methods were selective, accurate, and reproducible. In addition, the present methods were successfully applied to a pharmacokinetic study in dogs.
Keywords: ASP2151; Plasma; Regulated bioanalysis; Column-switching; LC–MS/MS; Conformer;

A rapid, selective and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to simultaneously determine trantinterol and its major metabolites in human urine. Waters Oasis HLB C18 solid phase extraction cartridges were used in the urine sample preparation. The separation was carried out on an ACQUITY UPLC™ BEH C18 column with methanol-0.2% formic acid (30:70, v/v) as the mobile phase at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The linear calibration curves for trantinterol, arylhydroxylamine trantinterol (N-OH-trantinterol), the tert-butyl hydroxylated trantinterol (tert-OH-trantinterol) and the 1-carbonyl trantinterol (trantinterol-COOH) were obtained in the concentration range of 0.414–207, 0.578–385, 0.168–84.0, and 0.954–477 ng/mL, respectively. The linear correlation coefficients were greater than 0.990. The intra and inter-day precision (relative standard deviation, RSD) values were less than 12% and the accuracy (relative error, RE) was 6.7–11%. The method herein described was superior to previous methods in sample throughput and sensitivity and successfully applied to the human excretion study.
Keywords: Trantinterol; Metabolites; UPLC–MS/MS; Human urine; Excretion;

An UPLC–MS/MS method for the quantitation of vortioxetine in rat plasma: Application to a pharmacokinetic study by Er-min Gu; Chengke Huang; Bingqing Liang; Lingjing Yuan; Tian Lan; Guoxin Hu; Hongyu Zhou (70-74).
In this work, a simple, sensitive and fast ultra performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the quantitative determination of vortioxetine in rat plasma. Plasma samples were processed with a protein precipitation. The separation was achieved by an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) column with a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM). The validated method had an excellent linearity in the range of 0.05–20 ng/mL (R 2  > 0.997) with a lower limit of quantification (0.05 ng/mL). The extraction recovery was in the range of 78.3–88.4% for vortioxetine and 80.3% for carbamazepine (internal standard, IS). The intra- and inter-day precision was below 8.5% and accuracy was from −11.2% to 9.5%. No notable matrix effect and astaticism was observed for vortioxetine. The method has been successfully applied to a pharmacokinetic study of vortioxetine in rats for the first time, which provides the basis for the further development and application of vortioxetine.
Keywords: Vortioxetine; Rat; UPLC–MS/MS; Plasma; Pharmacokinetics;

Quantification of hypoglycin A in serum using aTRAQ® assay by François Boemer; Michelle Deberg; Roland Schoos; Etienne Baise; Hélène Amory; Gilbert Gault; Jeremy Carlier; Yvan Gaillard; Christel Marcillaud-Pitel; Dominique Votion (75-80).
Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine’s biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication.Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition.Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50 μmol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals.The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy.
Keywords: Hypoglycin A; Atypical myopathy; Amino acids; Mass spectrometry; aTRAQ;

Quantitation of the anaesthetic xylazine in ovine plasma by LC–MS/MS by Gregory S. Doran; Leah A. Bradbury (81-84).
Removal of the wool-bearing skin around a young lamb’s rump (mulesing) provides long term health benefits for the animal, and the use of a sedative and analgesic agent such as xylazine may assist with pain relief to reduce discomfort and stress. Sensitive analytical methods are essential for monitoring pharmaceuticals and their metabolites in animals destined for human consumption. The following work reports a method that is 200 times more sensitive for xylazine detection than previously published methods, with lower limits of quantitation for xylazine and its primary metabolite in animals of 0.5 pg and 2 pg on-column, respectively. The use of a square wave solvent gradient immediately prior to analyte elution resulted in larger MS/MS peaks and a reduction in baseline noise, allowing reliable detection of lower analyte concentrations. The method uses as little as 1 mL of plasma which allows replication within a sample if required, and requires simple sample preparation, minimising the introduction of matrix components into the MS/MS.
Keywords: Xylazine; 2,6-dimethylaniline; Sheep; Ovine; LC–MS/MS; Chromatography;

Quantification of alpha-amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection by Juliana Garcia; Vera M. Costa; Paula Baptista; Maria de Lourdes Bastos; Félix Carvalho (85-95).
α-Amanitin is a natural bicyclic octapeptide, from the family of amatoxins, present in the deadly mushroom species Amanita phalloides. The toxicological and clinical interests raised by this toxin, require highly sensitive, accurate and reproducible quantification methods for pharmacokinetic studies. In the present work, a high-performance liquid chromatographic (HPLC) method with in-line connected diode-array (DAD) and electrochemical (EC) detection was developed and validated to quantify α-amanitin in biological samples (namely liver and kidney). Sample pre-treatment consisted of a simple and unique deproteinization step with 5% perchloric acid followed by centrifugation at 16,000  × g, 4 °C, for 20 min. The high recovery found for α-amanitin (≥96.8%) makes this procedure suitable for extracting α-amanitin from liver and kidney homogenates. The resulting supernatant was collected and injected into the HPLC. Mobile phase was composed by 20% methanol in 50 mM citric acid, and 0.46 mM octanessulfonic acid, adjusted to pH 5.5. The chromatographic runs took less than 22 min and no significant endogenous interferences were observed at the α-amanitin retention time. Calibration curves were linear with regression coefficients higher than 0.994. The overall inter- and intra-assay precision did not exceed 15.3%.The present method has low interferences with simple and fast processing steps, being a suitable procedure to support in vivo toxicokinetic studies involving α-amanitin. In fact, the validated method was successfully applied to quantify α-amanitin in biological samples following intraperitoneal α-amanitin administration to rats. Moreover, human plasma was also used as matrix and the purposed method was adequate for detection of α-amanitin in that matrix. The results clearly indicate that the proposed method is suitable to investigate the pharmacokinetic and tissue distribution of α-amanitin. Additionally, the method will be very useful in the development of novel and potent antidotes against amatoxins poisoning and to improve the knowledge of α-amanitin toxicity.
Keywords: High-performance liquid chromatographic; Diode-array; Electrochemical; α-amanitin;

Application of GC/MS-based metabonomic profiling in studying the therapeutic effects of Huangbai–Zhimu herb-pair (HZ) extract on streptozotocin-induced type 2 diabetes in mice by Lili Song; Hongyue Liu; Yan Wang; Yuming Wang; Jinbiao Liu; Zhensheng Zhou; Huilun Chu; Pengwei Zhuang; Yanjun Zhang (96-104).
A protocol for metabolic profiling of mice urine was developed based on gas chromatograph–mass spectrometer (GC–MS) to explore metabolic state directly. The intra-day, inter-day, repeatability, and stability RSD for most endogenous compounds were less than 3%. Type 2 diabetic mellitus (T2DM) mice model was induced by high calorie diet combined with streptozocin. Urine from the control, T2DM and Huangbai–Zhimu herb-pair (HZ) treatment mice were enrolled in the subsequent study to show the usefulness of the method. OPLS-DA scores plots demonstrate that the cluster of T2DM mice is separated from that of control mice, while HZ-T2DM mice are located close to control mice, indicating that metabolic profiles of these HZ-T2DM mice are placed toward those of control group. The results illustrate that HZ treatment could lower the level of d-glucose, hexadecanoic acid, octadecanoic acid, propanoic acid, 3-hydroxybutyric acid, and 2,3-dihydroxybutanoic acid in urine of DM mice, meanwhile the results show that HZ treatment could ameliorate T2DM symptoms by intervening the fatty acid metabolism, starch and sucrose metabolism, and glyoxylate and dicarboxylate metabolism. This preliminary application indicated that the method is suitable and reliable for urine metabolic profiling. This study might explain the metabolic effects of T2DM and the mechanisms of action of HZ against T2DM.
Keywords: GC–MS; Type 2 diabetes; Huangbai–Zhimu herb-pair; Urine metabonomics;

Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41 nt ssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13 mm, 20 mm and 25 mm diameter columns having a bed height of 60 mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A.
Keywords: Concanavalin A; DNA aptamer; Aptamer-affinity chromatography; Canavalia ensiformis; NHS-activated agarose;

An ultra-high performance liquid chromatographic-tandem mass spectrometry (UHPLC–MS/MS) method for quantification of potato steroidal alkaloids, namely α-solanine, α-chaconine, solanidine and demissidine was developed and validated. Three different column chemistries, i.e. ethylene bridged hybrid (BEH) C18, hydrophilic lipophilic interaction and amide columns, were assessed. The BEH C18 column showed best separation and sensitivity for the alkaloids. Validation data (inter-day and intra-day combined) for accuracy and recovery ranged from 94.3 to 107.7% and 97.0 to 103.5%, respectively. The accuracy data were within the acceptable range of 15% as outlined in the United States Food and Drug Administration (USFDA) guidelines. The recovery data were consistent and reproducible with a coefficient of variation (CV) ranging from 6.2 to 9.7%. In addition, precision of the method also met the criteria of the USFDA with CV values lower than 15% even at lower limit of quantification (LLOQ), while the permissible variation is considered acceptable below 20%. The limit of detection and LLOQ of the four alkaloids were in the range of 0.001–0.004 μg/mL whereas the linearities of the standard curves were between 0.980 and 0.995.
Keywords: Potato; Steroidal alkaloids; Validation; Multiple reaction monitoring; UHPLC–MS/MS;

Determination of sex-based differences in serum γ-linoleic acid and dihomo-γ-linoleic acid using gas chromatography–mass spectrometry by Mayu Onozato; Minami Nishikiori; Hideaki Iizuka; Hideaki Ichiba; Kiyomi Sadamoto; Takeshi Fukushima (116-121).
Because serum unsaturated fatty acids can provide useful information on disease diagnosis, the simultaneous determination of several fatty acids in small volumes of human serum would be beneficial for clinical applications. In the present study, serum fatty acids were extracted with n-heptane/chloroform from 10 μL of serum collected from 26 healthy Japanese subjects (11 men, ages 23–37 years; 15 women, ages 18–37 years) after deproteinization with perchloric acid, derivatization to their methyl ester using p-toluenesulfonic acid as an acid catalyst, and subsequent separation and measurement by gas chromatography–mass spectrometry (GC–MS) in the selected ion monitoring mode. Nine types of fatty acids (palmitoleic acid [PLA], oleic acid [OA], lenoleic acid [LA], γ-linolenic acid [GLA], α-linolenic acid [ALA], dihomo-GLA [DGLA], arachidonic acid [AA], eicosapentaenoic acid [EPA], and docosahexaenoic acid [DHA]) were analyzed in the serum within 35 min by GC–MS. The concentrations of these fatty acids in serum ranged from 3.64 ± 0.38 μM (GLA) to 413 ± 26.3 μM (LA). Among these nine fatty acids, GLA and DGLA levels were significantly lower in women than in men (p  = 0.0034 and 0.0012, respectively), suggesting that there may be sex-based differences in the biosynthetic production or metabolic processes of GLA and DGLA in humans.
Keywords: Polyunsaturated fatty acid; γ-Linoleic acid; Dihomo-γ-linoleic acid; Human serum; Sex-based difference;

LC–MS/MS method for the simultaneous quantification of 11 compounds of Ginkgo biloba extract in lysates of mesangial cell cultured by high glucose by Jing-ying Qiu; Xu Chen; Zheng Li; Shi-rui Wang; Xiao-wen Wu; Yin-jie Li; Dong-zhi Yang; Yan-yan Yu; Xiao-xing Yin; Dao-quan Tang (122-128).
The mesangial cell (MC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN), but the compounds interacting with cell are still unknown, which may be potential bioactive components. Based on this, the determination of GBE in MC lysates was proposed by high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) in this study. The MC was cultured with normal or high glucose with GBE for 4, 8, 12, 16, 24 and 48 h. The harvested cell was extracted with 40% acetic acid in water and further analyzed by LC–MS/MS. All the validation data including linearity, intra-day and inter-day precision, limit of detection and quantification, matrix effect, and stability were within the required limits. The validated method was successfully applied to quantify 11 compounds of GBE in cell lysates. The results showed that high glucose prolonged the peak time of all observed 11 compounds and peak concentrations of bilobalide, ginkgolide C, ginkgolide B, quercetin, luteolin, kaempferol, isorhamnetin and genkwanin in cell lysates, which hinted that these compounds may be the potential bioactive components of GBE with preventive effect against DN.
Keywords: Ginkgo biloba extract; Mesangial cell; High performance liquid chromatography tandem mass spectrometry; Quantification; Diabetic nephropathy;

To evaluate the taste characteristics of Chinese rice wine, wine samples sourced from different vintage years were analyzed using liquid chromatographic analysis, sensory evaluation, and an electronic tongue. Six organic acids and seventeen amino acids were measured using high performance liquid chromatography (HPLC). Five monosaccharides were measured using anion-exchange chromatography. The global taste attributes were analyzed using an electronic tongue (E-tongue). The correlations between the 28 taste-active compounds and the sensory attributes, and the correlations between the E-tongue response and the sensory attributes were established via partial least square discriminant analysis (PLSDA). E-tongue response data combined with linear discriminant analysis (LDA) were used to discriminate the Chinese rice wine samples sourced from different vintage years. Sensory evaluation indicated significant differences in the Chinese rice wine samples sourced from 2003, 2005, 2008, and 2010 vintage years in the sensory attributes of harmony and mellow. The PLSDA model for the taste-active compounds and the sensory attributes showed that proline, fucose, arabinose, lactic acid, glutamic acid, arginine, isoleucine, valine, threonine, and lysine had an influence on the taste characteristic of Chinese rice wine. The Chinese rice wine samples were all correctly classified using the E-tongue and LDA. The electronic tongue was an effective tool for rapid discrimination of Chinese rice wine.
Keywords: Chinese rice wine; Vintage year; Sensory evaluation; Electronic tongue; Liquid chromatographic analysis;

Peroxiredoxins (Prxs) are a family of thiol peroxidases, which have been suggested to serve as biomarkers for diseases caused by oxidative stress. In this study, we established a high performance liquid chromatography (HPLC) method for quantifying the amount of Prx2 in red blood cells (RBCs). RBC proteins were separated using HPLC, and a single peak was detected that matched that produced by recombinant Prx2. Under improved conditions, the calibration curve for Prx2 (reduced form) was linear over the range of 0.5-20.0 μg with a correlation coefficient of 0.999. The minimum detectable level of the recombinant Prx2 was 0.2 μg, with a signal-to-noise ratio of 3 per 20 μl of injection volume. SDS-PAGE and mass spectrometric analysis showed that the proteins comprising the peak were almost exclusively Prx2. Further high-resolution analysis using nanoLC–MS/MS demonstrated that the oxidation sensitive, Cys-51 was carbamidomethylated by iodoacetamide-alkylation during in-gel digestion but was not modified with sulfinic acid (−SO2H) or sulfonic acid (−SO3H). These results indicated that the separated Prx2 was the reduced form and not the hyperoxidized form. These basic experiments allowed us to determine the relative amounts of native Prx2 in RBCs taken from healthy subjects. The average levels of Prx2 in male and female subjects were 7.28 ng/mg and 8.29 ng/mg, respectively, and no significant difference was observed between the sexes. Therefore, the HPLC method with UV detection described herein offers a convenient method to quantitatively determine the levels of reduced form of Prx2 and its oxidative decrease in human RBCs.
Keywords: Oxidative stress; Biomarker; High performance liquid chromatography; Peroxiredoxin 2; Red blood cell;

A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13 min with mobile phase pH 10.5 and a small gradient of 9–13% acetonitrile, and detected using UV at 260 nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD <2%. Limit of detection was 1.6 μg/mL for nicotine and below 1 μg/mL for all other alkaloids. The limit of quantification was 2.8 and 4.8 μg/mL for nornicotine and nicotine respectively, and below 2 μg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.
Keywords: Tobacco alkaloids; Reversed-phase HPLC; Quantitative method; Nicotiana benthamiana; Alkaline mobile phase;

Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography–tandem mass spectrometry by Bing Li; Jie Zhang; Xu-Zheng Zhou; Jian-Yong Li; Ya-Jun Yang; Xiao-Juan Wei; Jian-Rong Niu; Xi-Wang Liu; Jin-Shan Li; Ji-Yu Zhang (146-153).
A rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneous quantify artesunate and its metabolite in sheep plasma. The plasma samples were prepared by liquid–liquid extraction. Chromatographic separation was achieved on a C18 column (250 × 4.6 mm, 5 μm) using methanol: water (60:40, v/v) (the water included 1 mM ammonium acetate, 0.1% formic acid, and 0.02% acetic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 1 ng/mL to 400 ng/mL (r 2  = 0.9992 for artesunate, r 2  = 0.9993 for its metabolite). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations for both artesunate and its metabolite. The recoveries ranged from 92% to 98% at the three concentrations for both. In summary, the LC–MS/MS metho described herein was fully successfully applied to pharmacokinetic studies of artesunate nanoemulsion after intramuscular delivery to sheep.
Keywords: Antiparasitic; Artesunate nanoemulsion; LC/MS/MS; Pharmacokinetics; Sheep plasma;

A tandem mass spectrometry method combined with an ion-pair chromatographic separation after weak cation exchange solid phase sample extraction for epinephrine (E), norepinephrine (NE) and dopamine (DA) has been developed. Two surrogate matrixes for plasma and urine as well as stable isotope labeled internal standards were utilized for quantitation. The observed dynamic range of E, NE and DA was 0.025–100 ng/ml for plasma, and 0.25–1000 ng/ml for urine with a r 2 regression coefficient >0.99. Extraction recoveries were greater than 60% and the lower limit of quantitation was 25 pg/ml for all three analytes in plasma. This method provided excellent sensitivity and selectivity for use with small sample volumes (≤25 uL), enabling high-throughput pharmacodynamic animal model development and screening of adverse effects.
Keywords: Catecholamines; Epinephrine; Norepinephrine; Dopamine; High performance liquid; chromatography; Tandem mass spectrometry; Pharmacokinetics; Pharmacodynamics; Solid phase; extraction;

Comparison of orthogonal chromatographic and lectin-affinity microarray methods for glycan profiling of a therapeutic monoclonal antibody by Matthew C. Cook; Sherif J. Kaldas; Gauri Muradia; Michael Rosu-Myles; Jeremy P. Kunkel (162-178).
The N-linked glycosylation of four lots of a marketed human therapeutic monoclonal antibody (mAb) was assessed by three orthogonal chromatographic methods and a commercial lectin microarray. For chromatography, the N-glycans were removed enzymatically from the mAbs using PNGase F. Native glycans were determined by HPAEC-PAD using a panel of 21 N-glycan standards and a multi-stage linear gradient eluent profile for sequential analyses of typical neutral and sialylated glycans in one chromatographic run. The monosaccharide contents of these glycans following acid hydrolysis were confirmed by HPAEC-PAD with monosaccharide standards. Glycosylation analysis by HILIC-FD after stoichiometric labelling with two different fluorescent tags (2-AA and 2-AB) enabled direct quantitation. The 2-AA- and 2-AB-labelled versions of the same glycan standard panel yielded distinctive separation profiles suitable for orthogonal identification of mAb glycans. Glycan profiling with the lectin microarray required partial denaturation of the intact mAbs to expose the sequestered Fc N-glycans. Glycosylation fingerprints were obtained using a fluorescently labelled antibody directed against human IgG Fc. Fluorescence intensities from the fingerprints were deconvoluted with a proprietary algorithm to obtain semi-quantitative “glycan structural class” information. Glycosylation analyses of the four mAb lots by these four methods, which separate and detect oligosaccharides according to different principles, provided complementary and corroboratory qualitative and quantitative information. The predominant N-linked structures were core-fucosylated asialo diantennary structures with varying galactosylation. There were also trace amounts of afucosyl and bisected glycans, but no detectable sialylation by any of the four methods. The therapeutic mAb demonstrated a high degree of consistency in the types and amounts of N-linked glycans in the four lots (<6% CV), and between all four analysis methods (<6% CV). The described methods are co-supported by the excellent quantitative agreement of their results, which is particularly notable considering the orthogonality of their separation and detection mechanisms.
Keywords: Monoclonal antibody; Glycosylation; Glycan; Oligosaccharide; HPAEC-PAD; HILIC-FD; Lectin microarray; Glycoscope;

Online polar two phase countercurrent chromatography × high performance liquid chromatography for preparative isolation of polar polyphenols from tea extract in a single step by Wei-Bin Chen; Shu-Qi Li; Long-Jiang Chen; Mei-Juan Fang; Quan-Cheng Chen; Zhen Wu; Yun-Long Wu; Ying-Kun Qiu (179-186).
Herein, we report an on-line two-dimensional system constructed by counter-current chromatography (CCC) coupling with preparative high-performance liquid chromatography (prep-HPLC) for the separation and purification of polar natural products. The CCC was used as the first dimensional isolation column, where an environmental friendly polar two-phase solvent system of isopropanol and 16% sodium chloride aqueous solution (1:1.2, v/v) was introduced for low toxicity and favorable resolution. In addition, by applying the stop-and-go flow technique, effluents pre-fractionated by CCC was further purified by a preparative column packed with octadecyl silane (ODS) as the second dimension. The interface between the two dimensions was comprised of a 6-port switching valve and an electronically controlled 2-position 10-port switching valve connected with two equivalent holding columns. To be highlighted here, this rationally designed interface for the purpose of smooth desalination, absorption and desorption, successfully solved the solvent compatibility problem between the two dimensional separation systems. The present integrated system was successfully applied in a one-step preparative separation and identification of 10 pure compounds from the water extracts of Tieguanyin tea (Chinese oolong tea). In short, all the results demonstrated that the on-line 2D CCC × LC method is an efficient and green approach for harvesting polar targets in a single step, which showed great promise in drug discovery.
Keywords: Counter-current chromatography; Two-dimensional liquid chromatography; Tieguanyin tea; Polar two-phase solvent system; Tea polyphenols;

In order to clarify the mechanism of fucoidan transport, we developed the chromatographic determination method.A size-exclusion chromatography (SEC) method for the determination of Okinawa-fucoidan using Develosil 300 Diol-5 (60 × 8.0 mm I.D., 30 nm pore-diameter) with the eluent containing 1% non-ionic detergent is developed. Determination range (UV at 210 nm) is from 0 to 100 ng of fucoidan with the linear calibration line inserting to zero.A transport activity of fucoidan is demonstrated by using Caco-2 cells (model of gut transport system); i.e., the initial transport velocity 12 nmol/h/mg of protein (25-fold slower rate as compared to a bacterial l-alanine active-transport activity 300 nmol/h/mg of protein) is found to occur. Since this fucoidan transport is inhibited by 10 mM sodium azide (respiration inhibitor) and 0.05 mM FCCP (uncoupler), this transport by Caco-2 cells is found to be an active one requiring energy-source. On the other hand, colchicine (inhibitor of phagocytosis/pinocytosis) and mannitol (putative competitive-inhibitor of tight-junction transport) cannot inhibit the fucoidan transport at all.We firstly report that the active transport occurs for such a high molecular-weight sulphated–polyfucose of fucoidan in vitro using Caco-2 cells.
Keywords: Fucoidan; Caco-2; Active transport; SEC; Azide; Uncoupler;

Simultaneous determination of intracellular UDP-sugars in hyaluronic acid-producing Streptococcus zooepidemicus by Lukáš Franke; Dagmar Čožíková; Dzianis Smirnou; Martina Hermannová; Tereza Hanová; Andrea Růžičková; Vladimír Velebný (194-199).
Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R 2) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80–90% recoveries and low LODs (≤6.19 μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD ≤ 5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells.
Keywords: Hyaluronan; UDP-sugars; Ion exchange chromatography;

Pharmacokinetics and tissue distribution study of camellianin A and its major metabolite in rats by liquid chromatography with tandem mass spectrometry by Yunliang Zheng; Xingjiang Hu; You Zhai; Jian Liu; Guolan Wu; Lihua Wu; Jianzhong ShenTu (200-209).
Camellianin A is a major active constituent of Adinandra nitida. A LC–MS/MS method for the determination of camellianin A and its metabolite (camellianin B) in rat plasma and tissues was developed and applied to a pharmacokinetics and tissue distribution study. Samples were separated on a Waters HSS T3 column with a mobile phase consisted of methanol and water (containing 0.1% formic acid). MS/MS detection was carried out on a triple-quadruple mass spectrometer under negative ESI mode. Pharmacokinetics study showed that camellianin A was rapidly eliminated with a t 1/2 of 92.6 ± 41.4 h and CL of 3.19 ± 0.471 L/min/kg. Additionally, camellianin A showed a low oral bioavailability of 2.99% and a narrow tissue distribution; however, camellianin B was proved to have a wide tissue distribution with brain penetration. The data presented in this study provides useful information for the further applications of A. nitida and camellianin A.
Keywords: Camellianin A; Camellianin B; Pharmacokinetics; Tissue distribution; HPLC–MS/MS;

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed for the simultaneous determination of the four major active ingredients, danshensu, protocatechuic aldehyde, rosmarinic acid, and ligustrazine, in the traditional Chinese medicine Shenxiong glucose injection in rat plasma. Acidified and alkalized plasma samples were extracted using ethyl acetate, and separated on a Waters C18 column (2.1 mm × 50 mm, 1.7 μm) by using a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid and luteoloside as an internal standard. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to identify and quantitate the active components. All calibration curves showed good linearity (r  > 0.994) over the concentration range, with a lower limit of quantification (LLOQ) between 0.02 and 0.21 μg/mL. The precision of the in vivo study was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation was within 15%. Moreover, satisfactory extraction efficiency was obtained (between 83.94 and 117.81%) by liquid–liquid extraction. The validated method was successfully applied in a pharmacokinetic study in rats after intravenous administration of Shenxiong glucose injection. The results showed that the four bioactive ingredients in Shenxiong glucose injection have linear pharmacokinetic properties in rats after intravenous injection within the administered dose range and partially different ones compared to single ingredient.
Keywords: UPLC–MS/MS; Pharmacokinetic; Shenxiong glucose injection; Danshensu; Ligustrazine;

Analysis of metformin, sitagliptin and creatinine in human dried blood spots by Maike Scherf-Clavel; Petra Högger (218-228).
For analysis of the anti-diabetic drugs metformin and sitagliptin and the renal clearance marker creatinine in the same human dried blood spot extract liquid chromatography methods employing HPLC/UV and LC-ESI–MS/MS have been developed and fully validated.Display OmittedFor analysis of the anti-diabetic drugs metformin and sitagliptin and the renal clearance marker creatinine in the same human dried blood spot (DBS) extract two liquid chromatography methods employing HPLC/UV and LC-ESI–MS/MS have been developed and validated. An accurate volume of 40 μL blood was spotted on a sampling paper which was extracted using 90% acetonitrile with 10% formic acid. The new methods were shown to be selective, accurate and precise. The validated ranges were 0.2–5 μg/mL for metformin, 1.5–15 μg/mL for creatinine and 3–500 ng/mL for sitagliptin. Since drug analysis in DBS determines whole blood concentrations as opposed to the typically used plasma levels the partition ratios between human plasma and blood cells, c(P)/c(BC), were elucidated in vitro to gain insight into the significance of blood cells as compartment of distribution for both compounds. The c(P)/c(BC) was found to be 4.65 ± 0.73 for metformin and 5.58 ± 0.98 for sitagliptin. While an accumulation of metformin in erythrocytes was already known we now present the first evidence that sitagliptin distributes into human blood cells. The analytical methods were also successfully applied to authentic capillary blood samples from two diabetic patients regularly taking a combination of metformin and sitagliptin. Both samples revealed analyte trough concentrations well above the lower limit of quantification of the respective compounds. Therefore, the present study offers a methodological basis for the DBS analysis of metformin and sitagliptin in relation to the patients' creatinine concentration.
Keywords: Human; Capillary blood; Metformin; Sitagliptin; Creatinine; Distribution; Blood cells;

Improved method for rapid detection of phthalates in bottled water by gas chromatography–mass spectrometry by Paz Otero; Sushanta Kumar Saha; Siobhan Moane; John Barron; Gerard Clancy; Patrick Murray (229-235).
An improved gas chromatography–mass spectrometry (GC–MS) method for simple, rapid and precise quantification of phthalates in drinking water is presented. This method was validated for bis (2-n-butoxyethyl) phthalate (DBEP), bis (2-n-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP), di-butyl phthalate (DBP), diethyl phthalate (DEP), dihexyl phthalate (DHP), dimethyl phthalate (DMP), di-n-octyl phthalate (DNOP) and dinonyl phthalate (DINP). Linearity of 0.9984 >  r 2  > 0.9975 in the range of 0.075–4.8 μg/mL for the selected phthalates was obtained. Accuracy values were in the range of 93–114% and RSD% for the analysis of 1.2 μg/mL of each phthalate was below 2.3% (n  = 9). This new method design has significantly improved the detection in terms of rapidity, specificity, repeatability and accuracy compared to available methods. The procedure has been applied to the analyses of three different brands of commercially available bottled mineral water and the corresponding plastic bottles. Phthalates were extracted with dichloromethane and re-constituted in cyclohexane prior to GC–MS analysis. When the validated GC–MS method was applied to the quantification of the selected phthalates in the samples, only DBP (up to 0.0675 ± 0.0018 μg/mL) and DEHP (up to 1.6848 ± 0.1631 μg/mL) were found. Furthermore, we provide specific data about the concentration of DBP and DEHP in bottled water attributable to migration of phthalates from respective plastic bottles.
Keywords: Phthalate esters; Gas chromatography–mass spectrometry (GC–MS); Water samples;

Metabolites characterization of timosaponin AIII in vivo and in vitro by using liquid chromatography-mass spectrometry by Yu Sun; Liyin Liu; Ying Peng; Bingjie Liu; Dongju Lin; Lingzhi Li; Shaojiang Song (236-243).
Timosaponin AIII, a major saponin found in Anemarrhena asphodeloides Bge., exhibits a wide spectrum of bioactivities. It is believed that it may be further developed into a promising new drug. To better understand the pharmacological activities of the component, the investigation of its in vivo and in vitro metabolism was necessary. In this study, the metabolic profile of timosaponin AIII was investigated using liquid chromatography-mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers-aquadrupole time-of-flight (Q-TOF) mass spectrometer and hybrid quadrupole/linear ion trap (Q-TRAP) mass spectrometer were employed to acquire structural information on timosaponin AIII metabolites. Plasma, bile, urine and feces were collected from rats after a single oral dose of 400 mg/kg of water solution. A total of 19 metabolites were detected and tentatively identified based on the mass spectral fragmentation patterns, elution order or confirmed using available reference standard. Two metabolites were detected after incubating with rat liver microsomal. What’s more, we isolated sarsasapogenin from the collection of urine samples after timosaponin AIII (5.0 g) giving orally to 20 rats at a dose of 150.0 mg/kg in an interval of 7 days. The present study provided important information about the metabolism of timosaponin AIII which will be helpful for fully understanding the mechanism of this compound’s action.
Keywords: Timosaponin AIII; Metabolites characterization; HPLC-Q-TOF-MS; HPLC-Q-TRAP-MS;