Journal of Chromatography B (v.947-948, #C)

Rapid and sensitive LC-ESI-MS of gangliosides by Aldo D. Garcia; Jorge L. Chavez; Yehia Mechref (1-7).
Gangliosides are a class of sphingolipids characterized by a ceramide lipid chain attached to an anionic oligosaccharide moiety that varies in complexity based on the level of sialylation. Heterogeneity in the oligosaccharide chain of gangliosides is a direct result of the monosaccharide structure, content, sequence, and connections. Gangliosides are highly concentrated in the central nervous system, and are cell type-specific as well as development-dependent and their quantities and species can undergo drastic changes during cell differentiation. Specific localization of gangliosides also allows for interaction with a variety of bioeffectors, including glycoproteins, antibodies, peptide hormones, and growth factors. There are currently no rapid analytical assays capable of identifying and quantifying gangliosides. The aim of this study is to establish a reliable chromatographic mass spectrometry based assay capable of profiling ganglioside levels in complex biological samples at high sensitivity. We describe here a chromatographic method using an amino column on which the separation is based on hydrophilic interaction with the sugar moiety of gangliosides. Several gangliosides, including GM1–3, GD1a,b, GD2–3, and GT1a,b, were efficiently separated in less than 10 min at a limit of detection ranging between 10–50 pg on column with a concentration dynamic range extending over 4 orders of magnitude. The developed method allowed the sensitive quantitation of gangliosides derived from the blood serum of patients with different esophagus diseases, including, adenocarcinoma, high-grade dysplasia, and Barrett's.
Keywords: Glycolipids; Gangliosides; LC-ESI-MS; Hydrophilic interaction chromatography;

Rapid and direct analysis of statins in human plasma by column-switching liquid chromatography with restricted-access material by Vinicius Freire Fagundes; Camila Prado Leite; Gerson Antonio Pianetti; Christian Fernandes (8-16).
This study presents the development of a column-switching liquid chromatographic method with direct injection of human plasma for simultaneous determination of four statins (lovastatin, pravastatin, rosuvastatin and simvastatin), the main class of drugs used in the treatment of hyperlipidemia. By using a C18 (30 mm × 4.6 mm, 15 μm) a lab made bovine serum albumin restricted access material (RAM) column was prepared and compared with a commercial alquil-diol silica RAM column (C18, 25 mm × 4.0 mm, 25 μm) in terms of their protein exclusion capacity and micromolecules retention. Foreflush and backflush modes were compared for both RAM columns to the number of theoretical plates, asymmetry, resolution and chromatographic run time. The developed method was validated in the range from 125 to 876 ng mL−1 for lovastatin, rosuvastatin and simvastatin, and from 500 to 2000 ng mL−1 for pravastatin, presenting selectivity, precision and accuracy intra and inter-run. Total analysis time (sample preparation and chromatographic separation) was only 16 min when the backflush mode was employed in the column-switching system.
Keywords: Statins; Column-switching; Restricted access material; Online sample preparation;

Because of the plethora of exposure sources and routes through which humans are exposed to trihalomethanes (THM), the limitation of their short half-lives could be overcome, if a highly sensitive method was available to quantify urinary THM concentrations at sub-ppb levels. The objective of this study was to develop a fast and reliable method for the determination of the four THM analytes in human urine. A sensitive methodology was developed for THM in urine samples using gas chromatography coupled with triple quadrupole mass spectrometry (GC–QqQ-MS/MS) promoting its use in epidemiological and biomonitoring studies. The proposed methodology enjoys limits of detection similar to those reported in the literature (11–80 ng L−1) and the advantages of small initial urine volumes (15 mL) and fast analysis per sample (12 min) when compared with other methods. This is the first report using GC–QqQ-MS/MS for the determination of THM in urine samples. Because of its simplicity and less time-consuming nature, the proposed method could be incorporated into detailed (hundreds of participants’ urine samples) exposure assessment protocols providing valuable insight into the dose–response relationship of THM and cancer or pregnancy anomalies.
Keywords: Chlorine; Disinfection by products; Exposure; Mass spectrometry; Trihalomethanes; Urine;

In vitro drug release and ex vivo percutaneous absorption of resveratrol cream using HPLC with zirconized silica stationary phase by Hudson Caetano Polonini; Carina de Almeida Bastos; Marcone Augusto Leal de Oliveira; Carla Grazieli Azevedo da Silva; Carol Hollingworth Collins; Marcos Antônio Fernandes Brandão; Nádia Rezende Barbosa Raposo (23-31).
Since the designs of optimal formulations for resveratrol permeation via the skin are lacking, the aim of this study was to establish the profile of resveratrol permeability into and across human skin. For that, a laboratory-made chromatographic column was used (Zr-PMODS), with its performance being compared to a traditional C18 column. In vitro drug release was conducted with polysulfone membranes, and the flux (J S) was 30.49 μg cm−2  h−1), with a lag time (L T) of 0.04 h, following a pseudo-first-order kinetics. For ex vivo percutaneous absorption using excised female human skin, the kinetic profile was the same, but J S was 0.87 μg cm−2  h−1 and L T was 0.97 h. From the initials 49.30 μg applied to the skin, 9.50 μg were quantified in the receptor medium, 20.48 μg was retained at the stratum corneum (do not account as permeated) and 21.41 μg was retained at the viable epidermis + dermis (account as permeated), totalizing 30.90 μg of resveratrol permeated after 24 h of application (62.6%). From these results, one can conclude that a person using the 1-g emulsion dose released by the pump containing 20 mg of resveratrol will have, theoretically, 12.53 mg of it liberated into his bloodstream, gradually and continuously for 24 h.
Keywords: In vitro drug release; Percutaneous absorption; Transdermal dosage form; Resveratrol; Factorial design; Validation studies;

A novel strategy that combines the chemometrics method with high performance liquid chromatography with fluorescence detector (HPLC–FLD) was developed for the simultaneous determination of seven phenolic antioxidants in six kinds of oil samples. After a simple dilution step, oil samples can be directly injected into the detecting system and the data were measured in a short time with a chromatographic system operating in the gradient elution mode. Since the chromatographic and spectral peaks among interesting analytes and interferences were heavily overlapped, second-order calibration method based on alternating trilinear decomposition (ATLD) algorithm which fully exploiting the second-order advantage was adopted. Successful resolution was obtained in the presence of different matrix interferences in different oil samples, and the developed approach allows the quantification of the antioxidants at levels found in edible vegetable oils, without the necessity of applying either preconcentration or extraction steps, moreover, a column washing is also not required. Meanwhile, the effectiveness and reproducibility of the proposed method were also validated by some statistical parameters like root mean squared error of prediction (RMSEP), limits of detection (LOD) and relative standard deviation (RSD). Then the proposed method was compared with several commonly selected methods in sample preparation, elution time and LOD.
Keywords: HPLC–FLD; Antioxidants; Oil samples; Second-order advantage; Alternating trilinear decomposition;

In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil production, was achieved using combined column chromatography over macroporous and polyamide resins. To optimize the RA enrichment process, the performance and separation characteristics of nine selected macroporous resins with different chemical and physical properties were investigated. SP825 resin was the most effective: the content of RA increased from 0.27% in the original extract to 16.58% in the 50% ethanol fraction (a 61.4-fold increase). During further purification treatment on polyamide resin, 90.23% pure RA could be obtained in the 70% ethanol fraction. RA with a higher purity (>95%) could also be easily obtained using one crystallization operation. The proposed method is simple, easily operated, cost-effective, and environmentally friendly and is suitable for both large-scale RA production and waste management.
Keywords: Combination chromatography; Macroporous resin; Perilla seed meal; Polyamide resin; Rosmarinic acid;

Application of C18-functional magnetic nanoparticles for extraction of aromatic amines from human urine by Chunzhu Jiang; Ying Sun; Xi Yu; Yan Gao; Lei Zhang; Yuanpeng Wang; Hanqi Zhang; Daqian Song (49-56).
In this paper, a novel method using C18-functional ultrafine magnetic silica nanoparticles (C18-UMS NPs) as adsorbents was developed for rapid extraction and enrichment of aromatic amines from urine. C18-UMS NPs were prepared by chemical coprecipitation, silanization and alkylation. The aromatic amines can be adsorbed on C18-UMS NPs and isolated easily from the matrix with an external magnetic field. After desorption with acetonitrile, the aromatic amines were determined by ultra fast liquid chromatography. The experimental parameters, such as pH value of sample solution, amount of C18-UMS NPs, extraction time, type and volume of desorption solvent, and desorption time were optimized. The analytical performances of the present method were also evaluated. The limits of detection for 1-aminonaphthalene, 4-aminobiphenyl, 4,4′-diaminodiphenylmethane and 4-aminophenylthioether were 1.3, 0.88, 1.1 and 1.1 ng mL−1, respectively. The results showed that the present method was simple, highly efficient and rapid for the extraction and enrichment of aromatic amines from urine.
Keywords: Human urine; Aromatic amines; C18-functional ultrafine magnetic silica nanoparticles; Magnetic solid-phase extraction; Ultra fast liquid chromatography;

A sensitive, specific and rapid ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method has been established to study pharmacokinetic properties of xanthatin. Xanthatin, a compound which belongs to sesquiterpene lactone group, was determined in rat plasma with psoralen as internal standard. Chromatographic separation was performed on an Agilent Zorbax Eclipse plus C18 column (50 mm × 2.1 mm, 3.5 μm) with gradient elution system at a flow rate of 0.3 mL/min. The mobile phase was composed of methanol and 0.1% formic acid water solution. Analysis was performed under a triple-quadruple tandem mass-spectrometer with an electrospray ionization (ESI) source via the multiple reaction monitoring (MRM) mode to determine xanthatin at [M+H]+ m/z 247.3 →  m/z 205.2 and that of IS at [M+H]+ m/z 187.1 →  m/z 143.0 within 5 min. The assay method exhibited good separation of xanthatin from the interference of endogenous substances. The lower limit of quantification (LLOQ) was 1 ng/mL, with a good linearity within the concentration range of 1–5000 ng/mL (r  = 0.9990). Intra-day and inter-day precision RSD was less than 9.27%; intra-day and inter-day accuracy was 88.48% and 102.25% respectively. The extraction recoveries of xanthatin range from 82.12% to 89.55%, and the extraction RSD was less than 9.01%. The established LC–ESI-MS/MS method is rapid and sensitive, which has been successfully applied to quantify xanthatin in rat plasma for the first time.
Keywords: Xanthatin; Pharmacokinetics; Ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–ESI-MS/MS);

Preparative separation of polyphenols from the flowers of Paeonia lactiflora Pall. by high-speed counter-current chromatography by Xikai Shu; Wenjuan Duan; Feng Liu; Xingang Shi; Yanling Geng; Xiao Wang; Bingtian Yang (62-67).
High-speed counter-current chromatography was used to separate and purify polyphenols for the first time from the flowers of Paeonia lactiflora Pall. with two solvent systems as follows: petroleum ether–ethyl acetate–H2O (1:9:10, v/v/v/v) and petroleum ether–ethyl acetate–butanol–H2O (1:9:0.5:10, v/v/v/v). Eight compounds were separated successfully in single run which were identified as quercetin-3-O-(6″-O-galloyl)-glucoside (I, 41.5 mg), 1,2,3,4,6-trigalloyl-β-d-glucose (II, 106.2 mg), quercetin-3-O-β-d-glucoside (III, 42.3 mg), kaempferol-3-O-(6″-O-galloyl)-glucoside (IV, 23.5 mg), isohamnetin-3-O-β-d-glucoside (V, 34.1 mg), kaempferol (VI, 14.8 mg), kaempferol-3-O-β-d-glucoside (VII, 32.6 mg), kaempferol-7-O-β-d-glucoside (VIII, 23.8 mg) by electrospray ionization–mass spectrometry (ESI–MS) and nuclear magnetic resonance (NMR). The purities of compounds I–VIII were all over 97.0% as determined by HPLC.
Keywords: EtOAc; HPLC; H2O; MeOH; Pet; HSCCC; PTFE; Paeonia lactiflora Pall.; High-speed counter-current chromatography; Separation;

Bioanalysis of propylparaben and p-hydroxybenzoic acid, and their sulfate conjugates in rat plasma by liquid chromatography–tandem mass spectrometry by Yue Zhao; Guowen Liu; Hongwu Shen; Jim X. Shen; Anne-Françoise Aubry; Lakshmi Sivaraman; Mark E. Arnold (68-74).
Two rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods for the determination of propylparaben, its major metabolite, p-hydroxybenzoic acid (pHBA), and their sulfate conjugates have been developed and validated in citric acid-treated rat plasma. To prevent propylparaben being hydrolyzed to pHBA ex vivo, rat plasma was first treated with citric acid; then collected and processed at a reduced temperature (ice bath). Stable isotope labeled internal standards, d4-propylparaben, 13C6-pHBA, and the d4-labeled internal standards of their sulfate conjugates were used in the methods. The analytes were extracted from the matrix using protein precipitation, followed by chromatographic separation on a Waters ACQUITY UPLC HSS T3 column. Quantification using negative ion electrospray was performed on a Sciex API 4000 mass spectrometer. The analytical ranges were established from 2.00 to 200 ng/mL for propylparaben, 50.0–5000 ng/mL for pHBA, 50.0–10,000 ng/mL for the sulfate conjugate of propylparaben (SPP) and 200–40,000 ng/mL for the sulfate conjugate of pHBA (SHBA). Inter- and intra-run precision for the quality control samples were less than 5.3% and 4.4% for all analytes; and the overall accuracy was within ±5.7% of the nominal values. The validated bioanalytical methods demonstrated excellent sensitivity, specificity, accuracy and precision and were successfully applied to a rat toxicology study under the regulations of Good Laboratory Practices (GLP). Strategies have been developed and applied toward overcoming the challenges related to analyte stability, and environmental and endogenous background.
Keywords: LC–MS/MS; Propylparaben; Validation; Regulated bioanalysis;

Optimization of solvent bar microextraction combined with gas chromatography for preconcentration and determination of methadone in human urine and plasma samples by Homeira Ebrahimzadeh; Fatemeh Mirbabaei; Ali Akbar Asgharinezhad; Nafiseh Shekari; Narges Mollazadeh (75-82).
In this study, solvent bar microextraction combined with gas chromatography–flame ionization detector (GC–FID) was used for preconcentration and determination of methadone in human body fluids. The target drug was extracted from an aqueous sample with pH 11.5 (source phase) into an organic extracting solvent (1-Undecanol) located inside the pores and lumen of a polypropylene hollow fiber as a receiving phase. To obtain high extraction efficiency, the effect of different variables on the extraction efficiency was studied using an experimental design. The variables of interest were the organic phase type, source phase pH, ionic strength, stirring rate, extraction time, concentration of Triton X-100, and extraction temperature, which were first investigated by Plackett–Burman design and subsequently by central composite design (CCD). So that the optimum experimental condition was obtained when the sodium chloride concentration was 5% (w/v); stirring rate, 700 rpm; extraction temperature, 20 °C; extraction time, 45 min and pH of the aqueous sample, 11.5. Under the optimized conditions, the preconcentration factors were between 275 and 300. The calibration curves were linear in the concentration range of 10–1500 μg L−1. The limits of detection (LODs) were 2.7–7 and relative standard deviations (RSDs) of the proposed method were 5.9–7.3%. Ultimately, the applicability of the current method was evaluated by the extraction and determination of methadone in different biological samples.
Keywords: Solvent bar microextraction; Methadone; Preconcentration; Experimental design; Biological sample;

Screening and quantitative determination of drugs of abuse in diluted urine by UPLC–MS/MS by Solfrid Hegstad; Sigurd Hermansson; Ingvar Betnér; Olav Spigset; Berit Margrethe Hasle Falch (83-95).
The purpose of this work was to develop and evaluate a fast, robust and specific UPLC–MS/MS screening platform for the determination and quantification of a variety of commonly used drugs of abuse in urine, i.e. a high-throughput quantitative analysis. Substances in the drug classes opioids, central nervous system stimulants and benzodiazepines and related agents were included in addition to cannabis and pregabalin, a total of 35 different analytes. Based on the concentrations and the physico-chemical properties of the substances, three UPLC–MS/MS methods were developed in parallel. Prior to analysis, sample preparation consisted of two different simple dilutions with 60 and 100 μL urine, respectively, using a Tecan Freedom Evo pipetting robot platform. A Waters Xevo TQ-S tandem quadrupole mass spectrometer coupled to a Waters I-class UPLC was used for quantitative analysis of one quantitative and one qualifying MRM transition for each analyte, except for tramadol for which the metabolite O-desmethyl-tramadol was included in the MRM method to confirm tramadol identity. Deuterated analogs were included as internal standards. The between-assay relative standard deviations varied from 2% to 11% and the limits of quantification were in the range 1–200 ng/mL for the various analytes. After development and initial testing, the method has been successfully implemented and routinely used at our hospital for quantitative screening of drugs of abuse in more than 35,000 urinary samples.
Keywords: UPLC–MS/MS; Opioids; Benzodiazepines; Central nervous system stimulants; Drugs of abuse; Screening;

A rapid, sensitive and selective high performance liquid chromatography–electrospray ionization–tandem mass spectrometry method (HPLC–ESI–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of lomerizine in human plasma. Protein precipitation process was used to extract lomerizine from human plasma. Plasma samples were separated by HPLC on an Agela Venusil XBP Phenyl column (100 mm × 2.1 mm, 5 μm) using a mobile phase consisting of methanol-2 mM ammonium acetate-formic acid (70:30:0.1, v/v/v) and the flow rate was set at 0.35 mL/min. The total run time was 4.0 min and the elution of lomerizine was at 1.9 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective [M + H]+ ions m/z 469.2 → 181.0 for lomerizine and m/z 405.2 → 202.9 for the I.S. The calibration curve was linear over the range of 0.1–25 ng/mL (r 2  > 0.99) with a limit of quantitation (LOQ) of 0.1 ng/mL. The intra- and inter-day precision (relative standard deviation, RSD) values were below 9.65% and the mean accuracy was from 99.00 to 103.00% at four quality control levels. Lomerizine was stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of lomerizine after single oral doses of 10 mg lomerizine to 18 healthy volunteers.
Keywords: Lomerizine; LC–MS/MS; Pharmacokinetics;

A simple, rapid, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated for the determination of cefadroxil, a first-generation cephalosporin, in rat plasma and urine. Rat samples were deproteinized with methanol, and then injected into the LC–MS/MS system (electro-spray ionization, positive mode) for quantification. Drugs were separated on a Synergi™ 4 μm Polar-RP 80A column (150 mm × 2.0 mm, 4 μm) with a mixture of 0.1% formic acid and methanol (62:38, v/v) as the mobile phase at 0.2 mL/min. Detection was performed using multiple reaction-monitoring modes at m/z 364.1 → 208.1 (for cefadroxil) and m/z 368.1 → 174.2 (for cefaclor, the internal standard). Method was specific and linear over the concentration range of 10–10,000 ng/mL. Validation parameters for cefadroxil, including accuracy, precision, absolute matrix effect, and stability in rat plasma and urine, were acceptable according to the biological method validation guidelines of the FDA (2001) [16]. Cefadroxil levels in plasma up to 1440 min or 480 min and urine up to 96 h were quantifiable following oral and intravenous cefadroxil administrations to rats at a dose of 2 mg/kg, each, suggesting that the method is appropriate for routine pharmacokinetic studies including urinary recovery in rats.
Keywords: Cefadroxil; LC–MS/MS; Rat; Pharmacokinetics; Plasma; Urine;

A validated GC/MS method for the determination of amisulpride in whole blood by Ioannis Papoutsis; Anna Rizopoulou; Panagiota Nikolaou; Constantinos Pistos; Chara Spiliopoulou; Sotiris Athanaselis (111-116).
A sensitive GC/MS method for the determination of amisulpride in whole blood was developed, optimized and validated. Sample preparation included solid-phase extraction using HF Bond Elut C18 cartridges and further derivatization with heptafluorobutyric anhydride (HFBA). The limits of detection and quantification were 3.00 and 10.0 μg/L, respectively. The calibration curves were linear up to 1000 μg/L (R 2  ≥ 0.991). Absolute recovery ranged from 94.2 to 101%. Accuracy was found to be between −8.7 and 1.9% and imprecision was less than 10.0%. The developed method covers the generally accepted therapeutic range but it can also cover levels above them. This makes our method suitable for the determination of amisulpride not only for clinical purposes on psychiatric patients, but also during the investigation of forensic cases where amisulpride is involved.
Keywords: Amisulpride; GC/MS; Blood;

Kakkalide and irisolidone, the main isoflavones of Flos Puerariae, exhibit a wide spectrum of bioactivities. Intestinal bacteria biotransformation plays an important role in the metabolic pathways of flavones, and is directly related to the bioactivities of the prodrugs after oral administration. To the best of our knowledge, the metabolic pathways of kakkalide and irisolidone in vitro have not been comprehensively studied yet. This paper describes the strategy using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) for the rapid analysis of the metabolic profiles of kakkalide and irisolidone after incubated with human and rat intestinal bacteria. Bacteria incubated samples were prepared and analyzed after incubated under anaerobic conditions for 48 h. A total of 17 metabolites, including parent compounds, were detected in human and rat intestinal bacteria incubated samples. The results obtained indicate that hydrolysis, dehydroxylation, demethoxylation, demethylation, hydroxylation, decarbonylation, and reduction were the detected metabolic pathways of kakkalide and irisolidone in vitro. The conversion rate of irisolidone in human and rat bacteria was 8.57% and 6.51%, respectively. Biochanin A was the relatively main metabolite of irisolidone, and the content of biochanin A in human and rat bacteria was 3.68% and 4.25%, respectively. The conversion rate of kakkalide in human and rat bacteria was 99.92% and 98.58%, respectively. Irisolidone was the main metabolite of kakkalide, and the content of irisolidone in human and rat bacteria was 89.58% and 89.38%, respectively. This work not only provides the evidence of kakkalide and irisolidone metabolites in vivo, but also demonstrates a simple, fast, sensitive, and inexpensive method for identification of metabolites of other compounds transformed by intestinal bacteria.
Keywords: Kakkalide; Irisolidone; Metabolic profile; UHPLC/Q-TOF MS; Biotransformation; Intestinal bacteria;

A novel chlorotoxin-like toxin derived from Buthus martensii Karsch, namely BmKCT-13, is a potential candidate for glioma therapy and highly homologous to the chlorotoxin (CTX) derived from the venom of the scorpion Leiurus quinquestriatus. In this study, a simple, sensitive, and robust analytical method based on liquid chromatography–tandem mass spectrometry has been developed for the determination of BmKCT-13 in rat plasma using CTX as internal standard (IS). After sample preparation by protein precipitation with 0.1% formic acid in methanol, chromatography was performed on a Hanbon Dubhe C18 column (150 mm × 2.1 mm, 5 μm, and 100 Å) using a gradient elution with 0.1% formic acid in water and methanol. Mass spectrometry involved positive electrospray ionization and multiple reaction monitoring of the transitions at m/z 780.2→69.9 for BmKCT-13 and m/z 800.2→69.7 for CTX. The method was linear over the concentration range 10–1000 ng/mL with a lower limit of quantification of 10 ng/mL. Intra- and inter-day precision (expressed as relative standard deviation, RSD) were ≤8.1 and ≤7.9%, respectively, with intra-and inter-day accuracy of 94.5–99.0%. Recoveries of BmKCT-13 and IS were more than 65% and matrix effects were not significant. Stability studies showed that BmKCT-13 was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving intravenous administration of BmKCT-13 to rats.
Keywords: BmKCT-13; Chlorotoxin; Glioma; LC–MS/MS; Pharmacokinetics;

Determination of larotaxel and its metabolites in rat plasma by liquid chromatography–tandem mass spectrometry: Application for a pharmacokinetic study by Zhenzhen Liu; Lunhui Zhang; Ping Ju; Pengyi Hou; Yuanyuan Zhang; Xing Tang; Kaishun Bi; Xiaohui Chen (132-138).
A sensitive and reliable high-performance liquid chromatography–mass spectrometry (LC–MS/MS) method was developed and validated for determination of larotaxel (LTX) and its active metabolites (M1, M2 and M3) in rat plasma. The analytes were extracted by one-step protein precipitation and separated on a Capcell pak C18 column (2.0 mm × 100 mm; 2 μm; Shiseido) using methanol–water as mobile phase at a flow rate of 0.2 mL min−1 in gradient mode. The method was validated over the concentration range of 2.5–1250 ng mL−1 for LTX and 1.0–500 ng mL−1 for M1, respectively, while M2 and M3 were monitored semi-quantitatively and quantified as M1 equivalents. Intra- and inter-day accuracy and precision were within the acceptable limits of less than 15% at all concentrations. Coefficients of correlation (r) for the calibration curves were more than 0.99 for all analytes. The quantitation method was successfully applied for simultaneous estimation of LTX and its metabolites in a pharmacokinetic study after oral administration at different doses of 10, 20, and 40 mg/kg and intravenous administration at the dose 10 mg/kg to Wistar rats, respectively. The results indicated that larotaxel has linear pharmacokinetic characteristics in rats after oral administration and its absolute bioavailability in rats was 12.24%.
Keywords: LC–MS/MS; Larotaxel; Metabolites; Pharmacokinetics; Bioavailability;

Bradykinin analysis revived – A validated method for determination of its stable metabolite in whole blood by LC–MS/MS by Knut F. Seip; Kari C. Bjerknes; Harald T. Johansen; Erik W. Nielsen; Linn Landrø; Léon Reubsaet (139-144).
Investigation of bradykinin involvement in diseases like hereditary angioedema has been greatly hindered by its rapid metabolism and generation, induced by sampling. Because of this, reliable measurements of bradykinin have yet to be introduced in clinical practice. Prevention of bradykinin generation during sampling, and determination of the in vivo generated stable metabolite BK1–5, should allow a reliable indirect measure of bradykinin involvement. An LC–MS/MS method has been developed to determine BK1–5 in human whole blood. The method inactivates metabolizing enzymes with ethanol, followed by solid phase extraction (C18), separation (C8) and detection (linear ion trap) through the transitions 287.25 → 320.20 (y3, quantifier), 408.20 (b4, qualifier) for BK1–5, and 292.17 → 330.20 (y3, quantifier), 408.20 (b4, qualifier) for the heavy labelled internal standard. The method showed acceptable linearity (n  = 3, r 2  = 0.994), intra-run precision (CV < 15%), inter-run precision (CV < 15%) and accuracy (CV < 14%), without matrix effects. LLOQ was 265.5 pmol L−1 and LOD was 35.4 pmol L−1. The method was used on blood samples from patients with hereditary angioedema, where BK1–5 was measured during attacks and in remision. The samples showed elevated concentrations (up to 1.7 nmol L−1 during attacks and 265.5 pmol L−1 in remission) compared to healthy volunteers (<35.4 pmol L−1). This is the first time BK1–5 in hereditary angioedema patients has been measured.
Keywords: Bradykinin; Hereditary angioedema; LC–MS/MS; BK1–5; Validation;

Simvastatin attenuates airway inflammation and hyperreactivity, hallmarks of asthma, in allergen-challenged mice. As such, it is under consideration as a novel therapeutic, thus it is important to quantify the levels of simvastatin and its pharmacologically active and interconvertible metabolite, simvastatin hydroxy acid, that can be attained in the body. Methods exist to measure the concentrations of these compounds in biological media; however they do not maintain a physiological pH, and as a result do not accurately measure the ratio of these two compounds that exists in vivo. We developed a new method to measure simvastatin and simvastatin hydroxy acid more accurately in serum from mice by ultra high performance liquid chromatography–tandem mass spectrometry. We minimized the time that the compounds were in aqueous solution, and buffered samples to a physiological pH value of 7.4. Limits of quantification (LOQ) were 0.16 ng mL−1 extract (1.3 ng mL−1 serum) for simvastatin, and 8.3 ng mL−1 extract (66 ng mL−1 serum) for simvastatin hydroxy acid, respectively. No interconversion was observed, based on spike-and-recovery experiments of solutions containing both compounds. The method was applied using biological samples from mice challenged with house dust mite extract and simultaneously treated with subcutaneous simvastatin injection. Simvastatin hydroxy acid concentrations became significantly increased after a 2 week pre-treatment regime, whereas simvastatin concentrations were below the LOQ for all serum samples.
Keywords: Simvastatin; Simvastatin hydroxy acid; UHPLC/MS/MS; Interconversion;

A validated LC–MS/MS method to determine the content of dipfluzine (Dip) and its three metabolites (M1, M2, and M5) simultaneously within rat plasma samples was developed. After a single liquid–liquid extraction, the assay was performed by using a C18 column and positive electrospray ionisation mode (ESI) in the multiple reaction monitoring (MRM) mode with transitions of m/z 417.3→167.3, 251.2→165.2, 199.1→121.3, and 183.2→105.1 for Dip, M1, M2, and M5, respectively. Sulfamethoxazole (SMZ) was used as internal standard (IS). The method was linear ranged from 0.5–518, 0.5–524, 1.0–1036, and 0.5–514 ng/ml for Dip, M1, M2, and M5, respectively and all correlation coefficients were greater than 0.9919. The intra- and inter-day precision values obtained were less than 11.5% and the accuracy was between −3.2 and 9.7% for each analyte. The extraction recoveries of their three concentrations for Dip and its three metabolites were all higher than 71.9%. The technique was successfully applied to a pharmacokinetic study of Dip and its metabolites after a single oral administration of Dip (20 mg/kg) to rats. The results indicated that the metabolite formation was rapid and generated M5 as the predominant metabolite, followed by M1 and M2. The maximum plasma concentrations (C max) were 59 ± 7, 37 ± 4, 3 ± 0.2, and 55 ± 5 ng/ml; the time to maximum plasma concentration (T max) were 65 ± 12, 95 ± 12, 190 ± 25, and 90 ± 0 min and the areas under the concentration–time curves (AUC0→∞) were 17573 ± 704, 8328 ± 355, 5602 ± 753, and 16101 ± 429 ng min/ml for Dip, M1, M2, and M5, respectively. These results suggested that Dip was extensively metabolized and rapidly absorbed. The half-life (t 1/2) of Dip, M1, M2, and M5 were 329 ± 15, 767 ± 75, 2364 ± 434, and 378 ± 36 min, respectively, which indicated that Dip and M5 were eliminated quickly. M2 reached its T max later and exhibited a longer t 1/2 than the other metabolites, which indicated that there might be some type of flip-flop mechanism at work in the pharmacokinetics of M2.
Keywords: Dipfluzine; Pharmacokinetics; Metabolites; LC–MS/MS;

A liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of erythromycylamine, which is the predominant active metabolite of dirithromycin in human plasma. After solid-phase extraction, the analyte and internal standard (IS) were separated by using an isocratic mobile phase consisting of 20 mM ammonium acetate (pH 3.9, adjusted with formic acid)-acetonitrile (75:25, v/v) on a Phenyl–Hexyl column (150 × 2.1 mm, 3 μm) and then analyzed in positive ion mode under electrospray ionization. Azithromycin was selected as the IS because it has the most similar mass spectrometric and chromatographic behaviors to the analyte. The respective multiple reaction monitoring (MRM) transitions, m/z 368.5 > 83.2 for erythromycylamine and m/z 375.4 > 115.2 for IS were chosen to achieve high sensitivity and selectivity in determination. A more acidic mobile phase (pH 3.9) than those of previous reports and a special needle wash (ethylene glycol-acetonitrile-water, 50:30:20, v/v/v, adjusted to pH 3.9 using formic acid) were used to eliminate the carryover effects of the two macrolides. The method exhibited a linear dynamic range of 0.5–440.0 ng/mL for erythromycylamine in human plasma (r  = 0.9999). The lower limit of quantification (LLOQ) and limit of detection (LOD) were 0.5 and 0.05 ng/mL, respectively. The mean extraction recoveries were higher than 94.0% for the analyte and IS. The intra- and inter-day precisions ranged from 1.4 to 5.4% and from 1.6 to 4.0%, respectively. The accuracy varied between 91.2 and 101.2%. The established method was successfully applied to analyze the human plasma samples from 24 healthy subjects in a bioequivalence study of two dirithromycin enteric-coated formulations.
Keywords: Dirithromycin; Erythromycylamine; LC–MS/MS; Fragmentation pattern; Carryover effect; Bioequivalence;

Targeted peptide methods generally use HPLC–MS/MRM approaches. Although dependent on the instrumental resolution, interferences may occur while performing analysis of complex biological matrices. HPLC–MS/MRM3 is a technique, which provides a significantly better selectivity, compared with HPLC–MS/MRM assay. HPLC–MS/MRM3 allows the detection and quantitation by enriching standard MRM with secondary product ions that are generated within the linear ion trap. Substance P (SP) and neurokinin A (NKA) are tachykinin peptides playing a central role in pain transmission. The objective of this study was to verify whether HPLC–MS/MRM3 could provide significant advantages over a more traditional HPLC–MS/MRM assay for the quantification of SP and NKA in rat spinal cord. The results suggest that reconstructed MRM3 chromatograms display significant improvements with the nearly complete elimination of interfering peaks but the sensitivity (i.e. signal-to-noise ratio) was severely reduced. The precision (%CV) observed was between 3.5% and 24.1% using HPLC–MS/MRM and in the range of 4.3–13.1% with HPLC–MS/MRM3, for SP and NKA. The observed accuracy was within 10% of the theoretical concentrations tested. HPLC–MS/MRM3 may improve the assay sensitivity to detect difference between samples by reducing significantly the potential of interferences and therefore reduce instrumental errors.
Keywords: Tachykinins; Neuropeptides; Mass spectrometry; Multiple reaction monitoring; Biomarkers;

LC-ESI-MS/MS determination of simotinib, a novel epidermal growth factor receptor tyrosine kinase inhibitor: Application to a pharmacokinetic study by Lihua He; Sanwang Li; Feifan Xie; Zeneng Cheng; Liling Ran; Xingling Liu; Peng Yu (168-172).
Simotinib is a novel epidermal growth factor receptor tyrosine kinase inhibitor. This study presented a sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry method using erlotinib as internal standard for the determination of simotinib in human plasma. The method involved a simple liquid–liquid extraction using diethyl ether. The analytes were separated with isocratic gradient elution on an Agilent TC-C18 column (4.6 × 150 mm, 5 μm). Mass spectrometric detector equipped with electrospray ionization source was carried out in the mode of multiple reaction monitoring (MRM). The monitored transitions were m/z 501.2 → 182.1 for simotinib and m/z 394.4 → 278.1 for erlotinib. The calibration curve of simotinib was established over the range of 2.058–3000 μg L−1 (r 2  = 0.9924). The intra- and inter-day precisions were all less than 10%, and all the biases were not more than 7%. This validated method was then successfully applied to a pharmacokinetic study involving twelve healthy Chinese volunteers. The mean C max and T max for simotinib were 254.79 ± 98.30 μg L−1 and 1.71 ± 0.48 h, respectively. Plasma concentrations declined with a t 1/2 of 5.37 ± 2.32 h. AUC0–t and AUC0→∞ values obtained were 1262.59 ± 501.41 μg L−1  h and 1329.95 ± 517.42 μg L−1  h, respectively.
Keywords: High performance liquid chromatography-electrospray ionization-mass spectrometry; Simotinib; Human plasma; Pharmacokinetics;

A rapid, simple and sensitive method was developed for the determination of 8-iso-PGF in urine using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS); 8-iso-PGF-d4 was used as the internal standard (IS). Chromatographic separation was performed using an Acquity BEH C18 column with a mobile phase composition of A: 0.1% acetic acid in methanol:acetonitrile (1:1, v:v) and B: 0.1% acetic acid in water (A:B, 32.5:67.5, v:v). Detection was performed on a triple–quadrupole tandem mass spectrometer using atmospheric pressure chemical ionization (APCI) in negative mode and using multiple reaction monitoring (MRM). The MS/MS ion transitions monitored were m/z 353 → 193 and 357 → 197 for 8-iso-PGF and IS, respectively. The calibration curve was prepared in PBS buffer because of the presence of endogenous concentrations of analyte in the control matrix; the internal standard successfully correcting for matrix effects. Good linearity was observed over the concentration range of 0.025–20 ng/mL; the method proving to be accurate and reliable was successfully used in support of a pharmacodynamic study in humans.
Keywords: 8-Iso-PGF; UPLC–MS/MS; Pharmacodynamics; Analytical validation; Urine; Biomarker;

Quantitative determination of 11-nor-9-carboxy-tetrahydrocannabinol in hair by column switching LC–ESI-MS3 by Meejung Park; Jihyun Kim; Yuran Park; Sanghwan In; Eunmi Kim; Yonghoon Park (179-185).
Hair analysis has been regarded as an alternative method to urine analysis in forensic and criminal cases. Cannabis (marijuana) is one of the most widely used drugs in the world and it has been controlled in South Korea since 1976. Identification of 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair can be an important proof of cannabis use because it can exclude the possibility of passive cannabis smoke exposure. In this study, we described a quantitative method of THCCOOH in hair using simple liquid–liquid extraction (LLE), selective column switching liquid chromatography with electrospray ionization (ESI)-MS3. For the column switching system three columns (precolumn, trap column and analytical column) were used. Valve switch from the precolumn to the trap column was set from 3.0 to 4.0 min because THCCOOH appeared around 3.5 min with this precolumn. After 4.0 min the valve was switched to the original position and the analytes in the trap column were eluted onto the analytical column. Resolution occurred in this column and eluted into the ESI-MS3 system. The internal standard was THCCOOH-d3. We used ESI-negative-MS3 transition of ions at m/z 343 to 299 to 245 (343/299/245) and m/z 346 to 302 to 248 (346/302/248) for quantification of THCCOOH and THCCOOH-d3, respectively. The validation results of selectivity, matrix effect, recovery, linearity, precision and accuracy, and processed sample stability were satisfactory. The limit of detection (LOD) was 0.05 pg/mg and the limit of quantification (LOQ) was 0.10 pg/mg. The range of concentration of THCCOOH from 98 authentic human hair was 0.13–15.75 pg/mg. This method was successfully applied in the analysis of authentic human hair samples.
Keywords: Hair analysis; Cannabis; LC–MS3; Column switching HPLC;

A liquid chromatography-mass spectrometry assay for quantification of Exendin[9-39] in human plasma by Maria Lasaosa; Puja Patel; Stephanie Givler; Diva D. De León; Steven H. Seeholzer (186-191).
Exendin[9-39] is a glucagon-like peptide-1 receptor (GLP-R) antagonist and a potential therapeutic drug for treatment of congenital hyperinsulism by lowering insulin concentration in plasma. A specific and sensitive LC–MS/MS method was validated for quantification of Exendin[9-39] in human plasma. Exendin[9-39] and the stable isopically labeled internal standard eluted at 9.2 min and were analyzed by single reaction monitoring (SRM) of the transitions m/z 842.9 → 991.8 and 848.2 → 998.8, respectively. The calibration curve was linear in the range 15–1260 ng/mL with a limit of detection of 1.3 ng/mL. The CVs of the standards were 2.7–13.1% within-run and 3.1–13.2% between-run. The matrix effect was >100% and the SPE recovery was 98.4 ± 12.9%. In absence of protease inhibitors, short-term stability at room temperature was only one hour. Accordingly, samples were kept on ice and sample processing was kept below 1 h. Human plasma samples from a clinical pilot study in which Exendin[9-39] was administered intravenously were analyzed and concentrations up to 600 ng/mL were reported Plasma samples from the study were stored at −80 °C with internal standard and successfully reanalyzed after 12 months.
Keywords: Exendin[9-39]; Hyperinsulinism; Triple quadrupole mass spectrometry; LC–MS/MS; Peptide quantification; GLP-1;

A novel and efficient determination method for multi-class compounds including β-agonists, sedatives, nitro-imidazoles and aflatoxins in porcine formula feed based on a fast “one-pot” extraction/multifunction impurity adsorption (MFIA) clean-up procedure has been developed. 23 target analytes belonging to four different class compounds could be determined simultaneously in a single run. Conditions for “one-pot” extraction were studied in detail. Under the optimized conditions, the multi-class compounds in porcine formula feed samples were extracted and purified with methanol contained ammonia and absorbents by one step. The compounds in extracts were purified by using multi types of absorbent based on MFIA in one pot. The multi-walled carbon nanotubes were employed to improved clean-up efficiency. Shield BEH C18 column was used to separate 23 target analytes, followed by tandem mass spectrometry (MS/MS) detection using an electro-spray ionization source in positive mode. Recovery studies were done at three fortification levels. Overall average recoveries of target compounds in porcine formula feed at each levels were >51.6% based on matrix fortified calibration with coefficients of variation from 2.7% to 13.2% (n  = 6). The limit of determination (LOD) of these compounds in porcine formula feed sample matrix was <5.0 μg/kg. This method was successfully applied in screening and confirmation of target drugs in >30 porcine formula feed samples. It was demonstrated that the integration of the MFIA protocol with the MS/MS instrument could serve as a valuable strategy for rapid screening and reliable confirmatory analysis of multi-class compounds in real samples.
Keywords: Multi-class compounds; Multi-function impurity adsorption; Ultra performance liquid chromatography–tandem mass spectrometry; Porcine formula feed;

An integrated expanded bed adsorption process was developed in this study to purify lactoferrin and immunoglobulin G (IgG) from crude sweet whey. The process used two sequential expanded beds packed with a cationic exchanger (Fastline SP) and a mixed-mode resin (Streamline Direct CST-1), respectively. Lactoferrin was isolated in the first expanded bed packed with Fastline SP, and the flow through was loaded into the second expanded bed packed with Streamline Direct CST-1 to separate IgG. Three integration strategies were compared to improve the separation efficiency, especially for the purification of IgG in the second expanded bed. The purities of IgG obtained from these three strategies were 91.9%, 83.8% and 92.4%, and the recoveries were 14.3%, 63.7% and 29.7%, respectively. By efficient integration of the two expanded beds, lactoferrin and IgG were successfully recovered from crude sweet whey with high purities and reasonable recoveries. Moreover, the stream flowed out of the integrated process was collected and separated by ultrafiltration to produce whey protein concentrate. Therefore, sweet whey resource could be fully utilized. The results demonstrated that it is possible to purify multiple proteins from untreated crude resource with a rationally designed expanded bed adsorption process.
Keywords: Process integration; Expanded bed adsorption; Sweet whey; Lactoferrin; IgG; Whey protein concentrate;

Response to: Naming a novel hemoglobin variant by Emmanuel Bissé; Heinrich Wieland (208).

Naming a novel hemoglobin variant by Nejat Akar (208).