Journal of Chromatography B (v.913-914, #C)

► First report on simultaneous determination of FESO and 5-HMT in human plasma. ► Thorough investigation of ex vivo stability of fesoterodine in plasma samples. ► Mitigation of FESO degradation by pre-treatment of blood with sodium metabisulphite. ► The method is practically free of endogenous/exogenous matrix interference. ► Bioequivalence study in healthy Indian volunteers and incurred sample reanalysis.Fesoterodine is a non-selective muscarinic-receptor antagonist, used in the treatment of overactive bladder syndrome. A highly sensitive, selective and rapid method has been developed for the simultaneous determination of fesoterodine and its active metabolite, 5-hydroxymethyl tolterodine (5-HMT) in human plasma by liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS). Due to rapid conversion of parent drug to 5-HMT, ex vivo stability of fesoterodine in human plasma was extensively studied to optimize the extraction protocol. The analytes and their deuterated analogs were quantitatively extracted from 100 μL human plasma by liquid–liquid extraction in methyl tert-butyl ether: n-hexane. The chromatographic separation of analytes was achieved on a Kromasil C18 (100 mm × 4.6 mm, 5 μm) column under isocratic conditions. The method was validated over a dynamic concentration range of 0.01–10 ng/mL for both the analytes. Ion-suppression effects were investigated by post-column infusion of analytes. The precision (% CV) values for the calculated slopes of calibration curves, which would reflect the relative matrix effect, were less than 1.5% for both the analytes. The intra-batch and inter-batch precision (% CV) across quality control levels varied from 1.82 to 3.73% and the mean extraction recovery was >96% for both the analytes. The method was successfully applied to a bioequivalence study of 8 mg fesoterodine tablet formulation (test and reference) in 12 healthy Indian subjects under fasted and fed condition. The assay reproducibility estimated by reanalysis of incurred samples showed a change of ±12.0%.
Keywords: Fesoterodine; 5-Hydroxymethyl tolterodine; Ex vivo stability; Chromatographic separation; LC–MS/MS; Human plasma;

► This report describes the validation of an high precision and accuracy HPLC-fluorescence method for the simultaneous determination of letrozole, citralopram and their metabolites in human urine. ► This method was applied to the analysis of human urine samples from cancer patients. ► The method was performed using a urine sample with only a dilution step 1:2.This work reports the validation of a high precision and accuracy method for the simultaneous determination of letrozole, citalopram and their metabolites in urine by high performance liquid chromatography with fluorescence detection. Dilution (urine:mobile phase, 1:2, v/v) was the only sample preparation step. The separation was carried out in a Kromasil C18 (150 mm × 4.6 mm) column, and the mobile phase was phosphate buffer 80 mM (pH 3.0) and acetonitrile (65:35, v/v) at a flow rate of 1.0 mL/min. The analytes were detected at 295 nm after excitation at 230 nm. Linearity was observed in the range of 1.0–1000 ng/mL for letrozole and its metabolite and 2.5–1000 ng/mL for citalopram and their metabolites, with limits of detection and quantification between 0.09–1.0 and 0.27–1.65 ng/mL, respectively. The precisions were satisfactory with RSDs between 0.17 and 5.71%. The accuracy was studied by spiking three urines from healthy female volunteers, and the recoveries were from 85 to 103%. The method was applied to urine samples from women under treatment for breast cancer and depression diseases.
Keywords: Letrozole; Citalopram; HPLC; Fluorescence detection; Breast cancer;

Quantitation of aldosterone in human plasma by ultra high performance liquid chromatography tandem mass spectrometry by Edward Hinchliffe; Stephanie Carter; Laura J. Owen; Brian G. Keevil (19-23).
► We report a novel UPLC–MS/MS method for the quantitation of aldosterone from human plasma. ► Sample extraction utilises a rapid solid phase extraction technique. ► Chromatography uses highly selective pentaflurophenyl functional groups. ► Short chromatographic run times enable high throughput analysis of clinical samples. ► The LC–MS/MS method demonstrates improved specificity compared to immunoassay.Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC–MS/MS. Standards, quality controls and samples (250 μL) were extracted using Oasis® HLB 96-well plates. Extract (30 μL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5 μm guard column, coupled to a Kinetex PFP, 100 mm × 2.1 mm, 2.6 μm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters® Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0 > 189.1 for aldosterone and 366.0 > 194.1 for d7-aldosterone respectively, which co-eluted at 2.65 min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26 pmol/L and 30 pmol/L respectively. The assay was linear up to 3200 pmol/L (r 2  = 0.9999). Mean intra- and inter-assay imprecision and bias were all <10%. Comparison of the UPLC–MS/MS method with an immunoassay in routine clinical use in the UK yielded the equation UPLC–MS/MS = 0.789(RIA)–41.7, linear regression r 2  = 0.88, n  = 54. We have developed a sensitive and specific method for the extraction and measurement of aldosterone from human plasma. The method features a simple 96-well plate solid phase extraction procedure, highly selective column chemistry and short chromatographic run times.
Keywords: Aldosterone; Primary hyperaldosteronism; Liquid chromatography tandem mass spectrometry;

► A simple, rapid, and sensitive LC–ESI-MS/MS method of MEFWAY and MPPF. ► Validation for the determination of MEFWAY and MPPF in rat plasma and brain. ► The sample preparation followed by one-step protein precipitation using acetonitrile and methanol. ► The assay variability limits set forth in the FDA guidelines. ► The present method can be applied to plasma-brain pharmacokinetic studies to investigate brain penetration in rats.A simple, selective, and sensitive liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was validated for the determination of 4-fluoromethyl-N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexane-1-carboxamide (MEFWAY) and 4-fluoro-N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)benzamide (MPPF) in rat plasma and brain samples, respectively. Plasma and brain samples were extracted with a mixture of acetonitrile and methanol (1:1, v/v) and then separated on a C18 column (Gemini 3 μm 110 Å, 50 × 2.00 mm ID, Phenomenex, USA). Quantitation was performed using LC–ESI-MS/MS in multiple-reaction monitoring (MRM) mode with positive ion electrospray ionization (ESI). The limit of quantification (LOQ) of 5 ng/mL and 1 ng/mL were obtained in 50 μL brain homogenate and plasma, respectively. The analytical linear ranges of this method were 1–4000 ng/mL in plasma and 5–4000 ng/mL in brain homogenate with a correlation coefficients (R 2) greater than 0.9993. The intra- and inter-day precision and accuracy values were within the assay validation guideline (lower than 13.0%). The analytes in plasma and brain samples were stable after three freeze–thaw cycles, long-term storage (one month at −80 °C), and short-term (4 h) storage at room temperature. The present method was successfully applied to plasma-brain pharmacokinetic studies to investigate brain penetration of a single dose of MEFWAY and MPPF in rats.
Keywords: MEFWAY; MPPF; LC–MS/MS; Brain; Plasma;

► Simultaneous quantification of capecitabine and all its six metabolites in plasma. ► Assay fully validated according to current FDA guidelines. ► Rapid, robust, selective and sensitive method using HPLC–tandem mass spectrometry.Capecitabine is the oral prodrug of the anticancer drug 5-fluorouracil (5-FU). The purpose of this study was to quantify capecitabine and its metabolites including 5′-deoxy-5-fluorocytidine (5′-dFCR), 5′-deoxy-5-fluorouridine (5′-dFUR), 5-FU, dihydro-5-fluorouracil (FUH2), α-fluoro-ureidopropionic acid (FUPA) and fluoro-β-alanine (FBAL) in human plasma using liquid chromatography coupled to electrospray tandem mass spectrometry. To this end two individual assays were developed: one for the simultaneous quantification of capecitabine, 5′-dFCR and 5′-dFUR using reversed phase chromatography and gradient elution, and one assay for 5-FU, FUH2, FUPA and FBAL using hydrophilic interaction chromatography and isocratic elution. Both assays were fully validated according to current FDA guidelines. Total run time for the capecitabine assay was 9.0 min, and of the 5-FU assay 5.0 min. Analyte extraction was performed by protein precipitation. Stable labeled isotopes for each of the analytes were used as internal standards. The linear ranges of the analytes were 50–6000 ng/mL for the capecitabine assay and 50–5000 ng/mL for the 5-FU assay. Validation results demonstrate that capecitabine and its metabolites can be rapidly, accurately, precisely and robustly quantified in human plasma with the presented methods. Both assays are currently in extensive use in support of pharmacokinetic studies in patients treated with capecitabine or 5-FU.
Keywords: Capecitabine; HPLC–MS/MS; Bioanalysis; 5-Fluorouracil; Chromatography; Cancer;

A sensitive and selective quantification of catecholamine neurotransmitters in rat microdialysates by pre-column dansyl chloride derivatization using liquid chromatography–tandem mass spectrometry by Ramakrishna Nirogi; Prashanth Komarneni; Vishwottam Kandikere; Rajeshkumar Boggavarapu; Gopinadh Bhyrapuneni; Vijay Benade; Srinivasarao Gorentla (41-47).
► Dansyl chloride derivatization of dopamine and norepinephrine in rat microdialysates. ► Sensitive and selective method to determine the basal levels in rat prefrontal cortex. ► Good peak resolution and separation from the interferences using superficial porous micro particulate column. ► Selective method in the neurochemical monitoring for discovery of new chemical entities targeted for the treatment of ADHD. ► The method demonstrated can be applicable in other regions such as medial PFC, striatum and hippocampus.A rapid and sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of catecholamine neurotransmitters in microdialysates was developed. The catecholamine neurotransmitters dopamine (DA) and norepinephrine (NE) were pre-column derivatized with dansyl chloride and analyzed. A gradient elution method was used to separate the analytes from the interferences on an Agilent Poroshell 120 EC-C18 outer porous micro particulate column. The method was robust and sensitive to determine with the lower limit of quantification value of 0.068 pmol/mL and 0.059 pmol/mL for DA and NE, respectively. It has acceptable precision and accuracy for concentrations over the standard curve range. The method was successfully applied for simultaneous quantitation of DA and NE in the prefrontal cortex (PFC) dialysates of rats obtained from a microdialysis study dosed with vehicle and atomoxetine through intra peritoneal (i.p.) route at a dose of 3 mg/kg to monitor the change in extracellular concentrations. Thus, accomplishment of this method would facilitate the neurochemical monitoring for discovery of new chemical entities targeted for the treatment of attention deficit hyperactivity disorder (ADHD).
Keywords: Dopamine; Norepinephrine; Dansyl chloride derivatization; LC–MS/MS; Microdialysates; Rat prefrontal cortex;

► 17 keto acids as to ethoxime/tert-butyldimethylsilyl derivatives were analyzed by GC–MS. ► The optimal method showed good precision, accuracy and sensitivity. ► The method was suitable for simultaneous analysis of 17 keto acids in biological samples.Organic acids, including keto acids, are key intermediates of central pathways in cellular metabolism. In this study, a comprehensive and reliable method was developed and optimized for the simultaneous measurement of 17 keto acids in various biological samples. The keto acids were converted to solvent extractable forms by ethoximation followed by tert-butyldimethylsilylation for direct analysis by gas chromatography–mass spectrometry in selected ion monitoring mode. The proposed method was precise (0.05–8.3, % RSD) and accurate (−10.5 to 5.3, % RE) with low limit of detection (0.01–0.5 ng/mL) and good linearity (r  > 0.995) in the range of 0.01–5.0 μg/mL. This was suitable for profiling analysis of targeted keto acids in human plasma, urine and rat brain tissue.
Keywords: Keto acid; Ethoxime/tert-butyldimethylsilyl derivatives; Gas chromatography–mass spectrometry; Plasma; Urine; Brain tissue;

► A HILIC–ESI-MS was developed for the detection of Fuziline in rat plasma. ► The pharmacokinetic of fuziline in rats was studied for the first time. ► The absolute bioavailability of Fuziline after i.g. in rats (4 mg/kg) was 21.1 ± 7.0%.A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometric (HILIC–MS) method for the quantification of Fuziline (15α-Hydroxyneoline) in rat plasma was developed and validated. After liquid–liquid extraction with ethyl acetate, Fuziline and Guanfu base A (internal standard) were separated with HILIC Chrom Matrix HP amide column (5 μm, 10 cm × 3.0 mm I.D.) with isocratic elution at a flow-rate of 0.2 mL/min. The analytes were detected by using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration ranging from 1 to 1000 ng/mL (R 2  = 0.999) with the lower limit of quantification (LLOQ) at 1 ng/mL and limit of detection (LOD) at 0.5 ng/mL. The average recoveries of Fuziline in plasma at the concentrations of 2, 50, 1000 ng/mL ranged from 68.2 to 69.9%. Intra- and inter-batch relative standard deviations ranged from 1.5 to 3.3% and 2.6 to 8.3%, respectively. Fuziline was stable under different sample storage and processing conditions except three-cycle freeze–thaw treatment at 2 ng/mL. This method was successfully applied to the pharmacokinetic studies in Sprague-Dawley rats. The absolute bioavailability of Fuziline after oral administration 4 mg/kg Fuziline in rats was 21.1 ± 7.0%, with clearance rate at 1745.6 ± 818.1 mL/kg/h, and half-life at about 6.3 ± 2.6 h.
Keywords: Aconitum carmichaeli; Fuziline; Hydrophilic interaction liquid chromatography; LC/MS; Pharmacokinetics; Rat;

► Poly(ionic liquid)s with different functional anions were developed. ► The anion-functionalized poly(ionic liquid)s were achieved anion metathesis. ► The anion-functionalized polymers were upgraded with molecularly imprinting. ► Multi-phase dispersive extraction was developed for the extraction and clean-up.Molecularly imprinted anion-functionalized poly(ionic liquid)s (MAPILs) were prepared by radical polymerization for the multi-phase dispersive extraction (MPDE) of flavonoids from plants. Poly(ionic liquid)s were functionalized with different anions via anion metathesis to enhance their separation efficiency, called anion-functionalized poly(ionic liquid)s (APILs). A molecularly imprinting technique was introduced to produce specific recognition sites by forming complexes between the template molecules and anion-functionalized ionic liquid monomers to reduce the interactions with the interference substances and increase the selectivity. Multi-phase dispersive extraction (MPDE) was applied for separation instead of the traditional solid phase extraction method. The target compounds were first extracted by three-phase (sample–solvent–sorbent) dispersive extraction and cleaned up after removing the sample matrix. This method significantly decrease in the interference and analysis cost. A suitable sorbent for MPDE could be identified based on the adsorption behaviors of flavonoids on different MAPILs. The mean recovery yields of quercitrin, myricetin, and amentoflavone from Chamaecyparis obtusa under the optimized conditions were 88.07, 93.59, and 95.13%. This is a promising method for the extraction, separation and determination of flavonoids or other polyphenolic compounds from natural and other sources.
Keywords: Ionic liquid; Anion-functionalization; Molecularly imprinted polymer; Multi-phase dispersive extraction; Flavonoid;

► The “enzymatic hydrolysis-mild thermal oxidation” method has been developed. ► The new method makes beeflike flavours more similar to natural beef flavour. ► The precursors of oxidized tallow are identified using PLSR.The “enzymatic hydrolysis-mild thermal oxidation” method was employed to obtain oxidized tallow. Nine beeflike flavours (BFs) were prepared through Maillard reaction with oxidized tallow and other ingredients. Volatile compounds of oxidized tallow and beeflike flavours were analysed by SPME/GC–MS. Six sensory attributes (meaty, beefy, tallowy, simulate, burnt and off-flavour) were selected to assess BFs. Thirty four odour-active compounds were identified to represent beef odour through GC–O analysis based on detection frequency method. GC–MS profiles of oxidized tallow were correlated with GC–O responses and sensory attributes of BFs using partial least squares regression modelling (PLSR). Twenty nine compounds were considered as the potential precursors of oxidized tallow. Among them, tetradecanoic acid, d-limonene, 1,7-heptandiol, 2-butyltetrahydrofuran, (Z)-4-undecenal, (Z)-4-decenal, (E)-4-nonenal and 5-pentyl-2(3H)-furanone were unique products generated from enzymatic hydrolysis-mild thermal oxidation of tallow, while hexanal, heptanal, octanal, nonanal, decanal, pentanal, acetic acid, butanoic acid, hexanoic acid, 1-heptanol, 1-octanol, 3-methylbutanal, 2-pentylfuran, γ-nonalactone, 2-undecenal, (E,E)-2,4-decadienal, (E,E)-2,4-nonadienal, (E)-2-nonenal, (E)-2-octenal, (E)-2-decenal and (Z)-2-heptenal were common products generated from thermal oxidation of tallow.
Keywords: Enzymatic hydrolysis-mild thermal oxidation tallow; Characteristic flavour precursors; Odour-active components; Sensory attributes; PLSR;

► We develop an analytical method of metabolites of fluorine-containing pyrethroids. ► They are used widely recently as mosquito repellents or moth-repellents in houses. ► The metabolites from hydrolyzed urine are analyzed by GC/MS(EI) after derivatization. ► They can be measured accurately and precisely. ► The collected urine samples can be stored for up to 1 month at −20 °C in a freezer.An analytical method was developed for measurement of the major urinary metabolites in rats administered fluorine-containing pyrethroids (metofluthrin, profluthrin and transfluthrin) which are widely used recently as mosquito repellents or mothproof repellents. Eight metabolites, 2,3,5,6-tetrafluorobenzoic acid, 4-methyl-2,3,5,6-tetrafluorobenzoic acid, 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (carboxylic metabolites), 2,3,5,6-tetrafluorobenzyl alcohol, 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol, 4-methoxymethyl-2,3,5,6-tetrafluorobenzyl alcohol and 4-hydroxymethyl-2,3,5,6-tetrafluorobenzyl alcohol (alcoholic metabolites), were extracted from enzymatic hydrolyzed urine using toluene and then concentrated. After transformation to their tert-butyldimethylsilyl derivatives for carboxylic metabolites or their trimethylsilyl derivatives for alcoholic metabolites, analysis was conducted by gas chromatography/mass spectrometry in the electron impact ionization mode. The calibration curves for each metabolite were linear over the concentration range of 0–20 μg/ml in urine, and the quantification limits were between 0.009 and 0.03 μg/ml. The relative errors and the relative standard deviations on replicate assays were less than 6% and 5%, respectively, for all concentrations studied. The measurements were accurate and precise. The collected urine samples could be stored for up to 1 month at −20 °C in a freezer. The proposed method was applied to the analysis of several urine samples collected from rats treated with these pyrethroids.
Keywords: Gas chromatography/mass spectrometry; Rat; Urinary metabolite; Indoor air pollution; Mosquito repellent; Mothproof repellent;

Development and validation of an LC–MS/MS method for the determination of tolvaptan in human plasma and its application to a pharmacokinetic study by Qi Pei; Bikui zhang; Hongyi Tan; Lihua Liu; Xiangdong Peng; Zuojun Li; Panhao Huang; Mi Luo; Xiaocong Zuo; Chengxian Guo; Guoping Yang (84-89).
► A novel LC–MS/MS method for determination of tolvaptan in human plasma was developed. ► Simple protein precipitation was used for the sample preparation. ► The LLOQ of the method was 0.457 ng/mL, which was the lowest reported so far. ► The chromatographic run time was only 3.5 min. ► The method has been applied to a pharmacokinetic study of tovaptan in volunteers.Tolvaptan is a selective vasopressin V2-receptor antagonist mainly used for the treatment of hyponatremia. This study described the development and validation of an LC–MS/MS method for the determination of tolvaptan in human plasma. Sample preparation involved protein precipitation with acetonitrile containing 2-demethyl tolvaptan (internal standard, IS). Chromatographic separation was performed on a Zorbax XDB C18 column with an isocratic mobile phase consisting of water (containing 0.1% formic acid) and methanol (25:75, v/v). Determination of the analytes was achieved by tandem-mass spectrometry with positive electrospray ionization. The multiple reaction monitoring (MRM) transitions were performed at m/z 449.2 → 252.1 for tolvaptan and m/z 435.2 → 238.1 for IS. The assay was linear over the concentration range of 0.457–1000 ng/mL, with a lower limit of quantification of 0.457 ng/mL. The intra- and inter-day precisions at three concentration levels (0.914, 111 and 800 ng/mL) were less than 15% and their accuracies were within the range of 97.7–107.8%. The mean recovery ranged from 99.2 to 104.6% and the matrix effect from 89.3 to 99.5%. Tolvaptan was stable under all tested conditions. This validated method was successfully applied to a pharmacokinetic study in healthy volunteers after oral administration of single-dose tolvaptan tablets.
Keywords: Tolvaptan; LC–MS/MS; Human plasma; Pharmacokinetics;

► First MEPS/HPLC method for analysis of amiodarone and desethylamiodarone in plasma. ► Enables rapid and simultaneous analysis of both analytes. ► Fully validated in the range of 0.1–10 μg/mL for both analytes. ► The method was applied to real plasma samples of patients under amiodarone therapy.For the first time a simple and fast high-performance liquid chromatography (HPLC) method using a novel sample preparation procedure based on microextraction by packed sorbent (MEPS) was developed and validated for the determination of amiodarone (AM) and its main metabolite desethylamiodarone (DEA) in human plasma. Chromatographic separation of the analytes (AM and DEA) and tamoxifen, used as internal standard (IS), was achieved within less than 5 min on a LiChroCART Purospher® Star C18 column (55 mm × 4 mm, 3 μm). The mobile phase consisting of 50 mM phosphate buffer with 0.1% formic acid (pH 3.1)/methanol/acetonitrile (45:5:50, v/v/v) was pumped isocratically at a flow rate of 1.2 mL/min. The detection was carried out at 254 nm. Calibration curves were linear (r 2  ≥ 0.9976) in the ranges of 0.1–10 μg/mL for AM and DEA. The limits of quantification were established at 0.1 μg/mL for AM and DEA. The overall imprecision did not exceed 6.67% and inaccuracy was within ±9.84%. The overall mean recovery of AM and DEA ranged from 58.6% to 68.2%. Neither endogenous nor tested exogenous compounds were found to interfere at retention times of the analytes (AM and DEA) and IS. This new MEPS/HPLC method was also applied to real samples obtained from polymedicated patients receiving AM therapy. Thus, this bioanalytical method seems to be a useful tool for therapeutic drug monitoring of patients under AM treatment and also to support other clinical pharmacokinetic-based studies involving this drug, such as bioavailability/bioequivalence studies.
Keywords: Amiodarone and desethylamiodarone; Microextraction by packed sorbent; HPLC; Bioanalytical method validation; Human plasma; Therapeutic drug monitoring;

Gas chromatographic–mass spectrometry method for the detection of busulphan and its metabolites in plasma and urine by Ibrahim El-Serafi; Ylva Terelius; Brigitte Twelkmeyer; Ann-Louise Hagbjörk; Zuzana Hassan; Moustapha Hassan (98-105).
► We designed a new method for the detection of busulphan and its four major metabolites. ► Different extraction forms were utilized according to the compound chemical properties. ► One single GC–MS was used to detect busulphan and its four metabolites using fused silica non-polar phase column. ► The method showed acceptable results in validation conduction and quantitative analysis. ► Clinical application was carried out using patient plasma and urine samples.Busulphan is an alkylating agent used as conditioning regimen prior to stem cell transplantation. Busulphan is metabolized in the liver and four major metabolites have been identified. The first metabolite is tetrahydrothiophene which is oxidized to tetrahydrothiophene 1-oxide, then sulfolane and finally 3-hydroxy sulfolane. Despite the low molecular weight and wide polarity range of busulphan and its four metabolites, the use of a fused silica non-polar column significantly enhanced the automated gas chromatography–mass spectrometry of their detection in one simple method. The limit of quantification was 0.5 μM for busulphan and all its metabolites except 3-OH sulfolane, which was 1.25 μM. This method was validated for all the compounds in both human plasma and urine. Lower limits of quantifications (LLOQs) were run in pentaplicate per compound and all results were within 20% of the nominal values. The recovery was determined by comparing the peak area of two quality control (QC) samples, before and after extraction in plasma and urine, in triplicate. Acceptable precision and accuracy have been obtained; at least 3 standard curves have been run for each compound using three different QCs covering the calibration curve in triplicate. The QC values were within 15% (SD) of the nominal values. Selectivity and sensitivity of all compounds have been measured. Compounds were stable up to 50 days after extraction in −20 °C and 48 h at RT. Moreover, the compounds were stable for three cycles of freezing and thawing. The method was applied in a clinical case where the patient received high dose busulphan; all the compounds have been detected, identified and quantified both in plasma and urine.
Keywords: Busulphan; Tetrahydrothiophene; Tetrahydrothiophene 1-oxide; Sulfolane; 3-Hydroxysulfolane; Metabolism; Stem cell transplantation;

► Activation of eugenol and isoeugenol into protein reactive haptens was achieved. ► Electrochemistry coupled to liquid chromatography and mass spectrometry was used. ► Protein modification as a key step in the hapten concept was performed. ► EC/LC/MS serves as tool for the assessment of pre- and pro-haptens.Skin sensitization is initiated by the modification of proteins located in the skin. After oxidative activation, eugenol and isoeugenol have the potential to modify skin proteins and therefore cause sensitization processes. Despite their known skin sensitizing properties, they are of common use in cosmetic products. According to the European Commission regulation No. 1223/2009, animal tests have to be banned for substances intended for cosmetic use. Therefore, alternative methods of investigation need to be developed for the approval of future substances. For this reason, eugenol and isoeugenol were selected as model substances to be investigated in a purely instrumental approach comprising electrochemistry, liquid chromatography and mass spectrometry. In the present work, reactive oxidation products of eugenol and isoeugenol were electrochemically generated. Reactive quinones and quinone methides were formed. Surprisingly, eugenol and isoeugenol differ significantly in their oxidation behaviour. Isoeugenol exhibits the formation of quinones and quinone methides of an alkylated and dealkylated species, respectively, whereas eugenol shows the formation of quinoid species only after dealkylation. Reactive quinoid species could be trapped with glutathione and the protein β-lactoglobulin A. The results are comparable to the ones with conventional animal studies in literature, which attribute the adverse effects of eugenol and isoeugenol to the formation of reactive quinones or quinone methides, which are reactive intermediates, able to react with proteins. Such species were successfully generated and investigated by the use of electrochemistry coupled to mass spectrometry. Above all, the investigation of adduct formation by the additional use of liquid chromatography allowed the assessment of the mechanism of oxidation, as it might happen in the skin. Both substances were proven to be trapped by the protein β-lactoglobulin A after electrochemical oxidation. However, isoeugenol formed the larger variety of adducts compared to eugenol.
Keywords: Electrochemistry; Eugenol; Isoeugenol; Metabolism; Protein modification;

► The relative mobility of DNA fragments was determined by capillary electrophoresis. ► The electrophoretic mobility was dependent on the 3′-terminal residue. ► 3′-Phosphoglycolate termini have anomalous mobility in capillary electrophoresis. ► Important for the elucidation of the sequence specificity of DNA damaging agents.An automated capillary DNA sequencer with laser-induced fluorescence detection can be utilised for DNA fragment analysis. The precise mobilities of DNA fragments with different chemical termini are especially important in the determination of the sequence specificity of DNA damaging agents. The aim of this study was to examine the electrophoretic mobility profile of DNA fragments with different 3′-termini. The nature of the 3′-teminal residue was found to have a major effect on the electrophoretic mobility of the DNA fragment, especially for 3′-phosphoglycolate termini that migrated anomalously by 3–6 nucleotides. Using the automated capillary sequencer, the electrophoretic mobilities of DNA fragments with different 3′-termini including 3′-hydrogen, 3′-hydroxyl, 3′-phosphate, and 3′-phosphoglycolate were extensively quantified and compared relative to each other. The 3′-hydrogen termini were generated by dideoxy sequencing; 3′-hydroxyl ends by minus sequencing; 3′-phosphate by Maxam–Gilbert chemical sequencing; and 3′-phosphoglycolate by bleomycin cleavage. The mobilities of these DNA fragments with different 3′-termini were found to be: (slowest) 3′-hydroxyl < 3′-hydrogen < 3′-phosphate < 3′-phosphoglycolate (fastest); with average relative mobilities of 0.00 < 0.12 < 0.63 < 4.42 nucleotides, respectively. The possible causes of the unusual electrophoretic mobility of the 3′-phosphoglycolate termini were discussed.
Keywords: Bleomycin; Capillary gel electrophoresis; DNA damage; Electrophoretic mobility; 3′-Phosphoglycolate;

Determination of malachite green, crystal violet and their leuco-metabolites in fish by HPLC–VIS detection after immunoaffinity column clean-up by Jie Xie; Tao Peng; Dong-Dong Chen; Qing-Jie Zhang; Guo-Min Wang; Xiong Wang; Qi Guo; Fan Jiang; Dan Chen; Jian Deng (123-128).
► We developed successfully an immunoaffinity column of MG–CV for the first time. ► We optimized the condition of the MG–CV column. ► An IAC–HPLC–VIS method was successfully developed for the first time for determination of MG, CV, and their leuco-metabolites in fish samples.A high performance liquid chromatography method with visible detection (HPLC–VIS) for the determination of malachite green (MG), crystal violet (CV), leucomalachite green (LMG), and leucocrystal violet (LCV) in fish has been developed after clean-up through an immunoaffinity column (IAC). Residues were simultaneously extracted from fish muscle with acetonitrile and ammonium acetate buffer. The leuco-forms, LMG and LCV, were oxidized quantitatively to the chromic CV and MG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Extracts were then purified on an IAC which prepared by immobilizing the anti-MG–CV antibodies by the sol–gel method. Finally, the eluents were analyzed by HPLC–VIS. The limits of detection were 0.15, 0.1, 0.18 and 0.14 ng/g for MG, CV, LMG and LCV, respectively. The average recoveries in samples fortified with MG, CV, LMG and LCV over the range 0.5–10 ng/g were from 71.6% to 96.8% with RSDs of 5.1–12.3% (n  = 6). This novel method was confirmed by liquid chromatography–tandem mass spectrometry with electrospray interface in positive mode using multiple reaction monitoring.
Keywords: Malachite green; Crystal violet; Leuco metabolites; Immunoaffinity column; High performance liquid chromatography;

► Fluorescence properties allow accurate Ko143 quantification down to 2 ng/mL. ► Selective sample pretreatment using liquid–liquid extraction with diethyl ether. ► Fumitremorgin C is used as the internal standard. ► The assay is validated for human plasma, mouse plasma and mouse tumor samples. ► Esterase mediated degradation of Ko143 in mouse plasma is inhibited by NaF.The fumitremorgin C analogue Ko143 is a potent and selective inhibitor of the ATP-binding cassette transporter ABCG2. To support in vivo ABCG2 resistance studies, we developed a sensitive and selective method for Ko143 quantification in plasma and tumor samples, using the parent compound fumitremorgin C as internal standard. Sample pretreatment by liquid–liquid extraction in diethyl ether yielded a recovery of 50% from human and mouse plasma. Pretreated samples were separated by reversed-phase high-performance liquid chromatography with fluorescence detection at 295 nm excitation and 350 nm emission wavelengths. Sharp chromatographic peaks were obtained with a 5 μm GraceSmart C18 column. The mobile phase consisted of methanol:10 mM ammonium acetate pH 5.0 (62:38, v/v), delivered at a flow rate of 0.2 mL/min. Acceptable accuracy and precision of ±15% were achieved within the linear dynamic range of the calibration curve (2–500 ng/mL) for human and mouse plasma samples. Mouse tumor tissue samples required the use of a calibration curve prepared in the same matrix due to the lower recovery of 40% from this matrix. Then, accuracy and precision were within the generally accepted range of ±15% for bioanalytical assays. Ko143 was stable in human plasma for up to 3 repeated freeze–thaw cycles and when stored at room temperature for up to 72 h. However, when kept at room temperature in mouse plasma, Ko143 was rapidly degraded by esterase activity, which could be prevented by collection of blood into sodium fluoride-containing tubes and maintaining samples on ice during pretreatment. A preliminary pharmacokinetics study in mice demonstrated the applicability of this assay for ABCG2 resistance studies in vivo.
Keywords: Ko143; Fumitremorgin C; Roquefortine C; Topotecan; ABCG2; Drug resistance;

Citrulline and metabolomics in acute kidney injury by Carlo Chiarla; Ivo Giovannini (137).

► Combination of electromembrane and dispersive liquid–liquid extraction was introduced. ► The combination was applied for extraction of antidepressants from human plasma. ► GC–FID was applied for determination of the drugs. ► Preconcentration factors of TCAs in water, urine, and plasma were from 383 to 1065. ► The system is a promising combination for analysis of TCAs in biological matrices.For the first time, combination of electromembrane extraction (EME) and dispersive liquid–liquid microextraction (DLLME) followed by gas chromatography–flame ionization detection (GC/FID) was developed for determination of tricyclic antidepressants (TCAs) in untreated human plasma and urine samples. Response surface methodology (RSM) was used for optimization of experimental parameters, so that extraction time of 14 min, voltage of 240 V, donor phase of 64 mM HCl and acceptor phase of 100 mM HCl were obtained as optimal extraction conditions. Matrix effect and carry-over were investigated in this work. The results indicated matrix effect for urine and plasma samples in comparison with neat solutions, so match matrix method was used for drawing working calibration curves. However, no carry-over was appeared at the retention time of investigated TCAs (S/N < 3). With application of optimized values, good linearity in the range of 2–500 μg L−1 was obtained for TCAs with the correlation of determination values (r 2) above 0.9968. The limits of detection (S/N = 3) for TCAs were found 0.25, 3.0, and 15 μg L−1 in water, urine, and plasma, respectively. The preconcentration factors of TCAs in water, urine, and plasma were from 383 to 1065. The intra- and inter-assay precisions (%) were in the ranges 6.4–11.8% and 6.2–10.8%, respectively, and the intra- and inter-assay accuracies were >86.5%. The results showed that EME–DLLME–GC/FID is a promising combination for analysis of TCAs present at low concentrations in biological matrices.
Keywords: Antidepressant drugs; Dispersive liquid–liquid microextraction; Electromembrane; Gas chromatography; Plasma; Urine;

Simultaneous quantification of vinblastine and desacetylvinblastine concentrations in canine plasma and urine samples using LC–APCI–MS/MS by Satyanarayana Achanta; Minh Ngo; Allison Veitenheimer; Lara K. Maxwell; Jarrad R. Wagner (147-154).
► HLC/MS/MS method to quantitate vinblastine/desacetylvinblastine in canine matrices. ► Linear over wide range of concentrations in plasma and urine, LLOQ 0.125 ng/mL. ► Intra and interday accuracy >89% and imprecision <8.6% in all analytes/matrices. ► The method was applied to in vivo vinblastine pharmacokinetic studies in dogs.A highly sensitive and specific liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC/APCI–MS/MS) method has been developed and validated for simultaneous quantification of vinblastine and its metabolite, desacetylvinblastine, in canine plasma and urine samples. Plasma and urine samples were processed by a solid phase extraction procedure. The optimal chromatographic behavior of these analytes was achieved on pentafluorophenyl (PFP) propyl analytical column (5 μm, 50 × 2.1 mm) under isocratic elution of 0.75 mL/min with a mobile phase of 5 mM ammonium acetate and methanol. The samples were analyzed in positive ion, multiple reaction monitoring mode. The calibration curves were linear over 0.125–2 ng/mL (lower calibration curve); 2–100 ng/mL (higher calibration curve) and 0.125–5 ng/mL for vinblastine and desacetylvinblastine in plasma, and over 1–2000 ng/mL and 0.5–100 ng/mL for vinblastine and desacetylvinblastine in urine samples, respectively. The limits of quantitation of vinblastine and desacetylvinblastine were 0.125 ng/mL in both matrices. The intra and interday accuracy was above 89% and precision below 8.6% for both analytes in both matrices. The developed method was successfully applied to ongoing in vivo vinblastine pharmacokinetic studies in dogs.
Keywords: Vinblastine; Desacetylvinblastine; Vinorelbine; Dog; LC/MS/MS; Analysis;

► We develop a method for gaseous alkanes generation analysis. ► This generation is based on the reaction of water and Alkyl-Grignard reagents. ► We apply this method to perform butane precise quantification. ► All gaseous alkanes can be investigated following this method. ► The method is especially designed for forensic applications.The aim of our study was to provide an innovative HS-GC/MS method applicable to the routine determination of butane concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature concerning butane measurement was the absence of a specific butane internal standard necessary to perform quantification. Because no stable isotope of butane is commercially available, it is essential to develop a new approach by an in situ generation of standards. To avoid the manipulation of a stable isotope-labelled gas, we have chosen to generate in situ an internal labelled standard gas (C4H9D) following the basis of the stoichiometric formation of butane by the reaction of deuterated water (D2O) with Grignard reagent butylmagnesium chloride (C4H9MgCl). This method allows a precise measurement of butane concentration and therefore, a full validation by accuracy profile was presented.
Keywords: In situ stable labelled isotope generation; Butane; Deuterated butane; Grignard reagent; Headspace gas extraction;

Simultaneous quantification of F2-isoprostanes and prostaglandins in human urine by liquid chromatography tandem-mass spectrometry by Jeevan K. Prasain; Alireza Arabshahi; Pam R. Taub; Scott Sweeney; Ray Moore; J. Daniel Sharer; Stephen Barnes (161-168).
► Measurement of F2-isoprostanes and prostaglandins in human urine by LC–MS/MS. ► Base line separation of the isomers measured. ► Quantification of analytes over a linear dynamic range (0.05–50 ng/mL). ► Excellent recoveries (79–100%), no major matrix effects and absence of carry over. ► Urinary F2-isoprostanes and PGs analysis in patients undergoing cardiac surgery.A specific and sensitive LC–MS/MS method for analysis of F2-isoprostanes (F2-IsoPs) and prostaglandins (PGs) in urine was developed and validated to examine the levels of F2-IsoPs and prostaglandin F (PGF), in human urine in patients undergoing cardiac surgery. The rapid extraction for F2-IsoPs and PGs from urine was achieved using a polymeric weak anion solid phase extraction cartridge. The base-line separation of 8-iso-PGF, 8-iso-15(R)-PGF, PGF, and 15(R)-PGF was carried out on a Hydro-RP column (250 mm × 2.0 mm i.d., Phenomenex, CA) using a linear gradient of methanol:acetonitrile (1:1, v/v) in 0.1% formic acid at a flow rate of 0.2 mL/min. The method was proved to be accurate and precise for simultaneous quantification of each analyte over a linear dynamic range of 0.05–50 ng/mL with correlation coefficients greater than 0.99. The intra-day and inter-day assay precision at the lowest quality control (0.07 ng/mL) level were less than 17%. The mean extraction recoveries of F2-IsoPs and PGs were in a range of 79–100%. In applications of this method to patients undergoing cardiac surgery, post-surgery urinary concentrations of 8-iso-PGF increased significantly in patients (n  = 14) who did not develop acute kidney (AKI) (pre-surgery 0.344 ± 0.039 vs. post-surgery 0.682 ± 0.094 ng/mg creatinine, p  < 0.01), whereas there was no significant change in this isoprostane in the patients (n  = 4) who developed AKI (pre-surgery 0.298 ± 0.062 vs. post-surgery 0.383 ± 0.117 ng/mg creatinine, NS). Therefore, the method is suitable for the analysis of individual F2-IsoPs and PGF’s in both clinical and research studies.
Keywords: F2-isoprostanes; Prostaglandins; LC–MS/MS; Analysis;

► Validation of a HPLC-fluorescence method for the determination of VRC in human BAL samples. ► Freeze-drying step to concentrate BAL samples, attaining high sensitivity with LLOQ of 2.5 ng/mL. ► Successful application in a clinical study in immunocompromised lung transplant recipients, who are at risk of low VRC concentrations.The quantification of voriconazole concentration in lung epithelial lining fluid to facilitate the management of pulmonary fungal colonisation or aspergillosis is of increasing interest. An accurate and reproducible high-performance liquid chromatography method to quantify voriconazole in human bronchoalveolar lavage (BAL) fluid was developed and validated. BAL samples were concentrated by freeze-drying and reconstituted with water prior to deproteinisation. Separation was achieved with a C18 column employing fluorescence detection (excitation: 260 nm, emission: 370 nm). The calibration curves were linear from 2.5 to 500 ng/mL. The intra- and inter-day precisions were within 7%. Accuracies ranged from 102% to 107%. The clinical applicability was established by successful measurement of voriconazole concentrations in lung transplant recipients. The assay provides an alternative approach for those with negligible access to liquid chromatography–tandem mass spectrometry instrumentation.
Keywords: Voriconazole; HPLC; Fluorescence; Bronchoalveolar lavage fluid;