Journal of Chromatography B (v.885-886, #C)

► Online LC/MS–MS sensitivity allows drugs quantification in small blood volume. ► Illicit drugs analysis in dried blood spots is a suitable alternative to whole blood. ► DBS is a precise, low-cost option in cases of driving under the influence of drugs.A novel approach has been developed for the illicit drugs quantitative determination using dried blood spots (DBS) on filter paper. The illicit drugs tested were opiates (morphine and its 3- and 6-glucuronide metabolites, codeine, 6-monoacetylmorphine), cocainics (ecgonine methylester, benzoylecgonine, cocaine, cocaethylene) and amphetamines (amphetamine, methamphetamine, MDA, MDMA, MDEA). The described method, requiring a small blood volume, is based on high performance liquid chromatography coupled to tandem mass spectrometry using on-line extraction. A Whatman card 903 was spotted with 30 μL of whole blood and left overnight to dry at room temperature. A 3-mm diameter disk was removed using a manual punch, suspended in 150 μL of water for 10 min with ultrasonication, and then 100 μL was injected in the on-line LC–MS/MS system. An Oasis HLB was used as an extraction column and a C18 Atlantis as an analytical column. The chromatographic cycle was performed with 20 mM ammonium formate buffer (pH 2.8) (solvent A) and acetonitrile/solvent A (90:10, v/v) gradient in 16 min. Detection was performed in positive electrospray ionization mode (ESI+) with a Quattro Micro (Waters). Recoveries of all analytes were up to 80%. DBS were stored in duplicate at 4 °C and −20 °C for up to 6 months. Illicit drugs seemed to be much more stabled at −20 °C. Furthermore, it was tested whether analysis of DBS may be as reliable as that of whole blood investigating authentic samples; significant correlations were obtained. This DBS assay has potential as rapid, sensitive and inexpensive option for the illicit drugs determination in small blood volumes, which seems of great interest in suspected cases of driving under the influence of drugs.
Keywords: Dried blood spot; Illicit drugs; On-line LC–MS/MS;

► An LC–MS/MS method for the analysis of 5-thio-d-glucose in plasma was developed. ► The validated quantitation range was from 10 to 3000 ng/mL. ► This is the first method that does not require radioisotope-labelled compounds. ► We succeeded in the removal of interferences by unique sample preparation procedure. ► The method was successfully applied to evaluate pharmacokinetic studies in rats.A highly selective, sensitive, and robust liquid chromatography–tandem mass spectrometric method for the determination of 5-thio-d-glucose concentrations in rat and human plasma was developed and validated. The sample preparation procedure involved protein precipitation and solid phase extraction, which efficiently removed sources of interference present in the plasma. Chromatographic separation was obtained using an NH2-column with distilled water and acetonitrile as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in negative ion mode. The selected reaction monitoring (SRM) transitions for 5-thio-d-glucose and an internal standard (5-thio-d-glucose-13C6) were m/z 195 →  m/z 105 and m/z 201 →  m/z 108, respectively. The correlation coefficients of the calibration curves ranged from 0.9997 to 0.9999 over a concentration range from 10 to 3000 ng/mL plasma. The validated method was successfully applied to a pharmacokinetic study in rats.
Keywords: 5-Thio-d-glucose; Monomeric sugar; LC–MS/MS; Plasma; Quantification; SPE;

► An LC/MS–MS assay was developed to measure odanacatib concentrations in human plasma. ► Sample preparation was based on automated liquid–liquid extraction. ► The linear calibration range was from 0.5 to 500 ng/mL. ► Assay precision was within 5.88%, and accuracy was between 95.6 and 106%. ► The methodology was fully validated and widely applicable to clinical bioanalysis.Odanacatib (ODN, MK-0822) is an investigational drug under development for the treatment of osteoporosis. A quantitative LC/MS–MS methodology was developed and validated to determine ODN concentrations in human plasma, with a linear calibration range from 0.500 to 500 ng/mL. Stable isotope 13C6-labeled ODN was employed as the internal standard (IS). Sample preparation was based on liquid–liquid extraction of basified plasma with methyl t-butyl ether in a 96-well plate format. The extracted samples were analyzed on a liquid chromatography–tandem mass spectrometry system equipped with a turbo ion spray source. Chromatographic separation of the analyte and IS was achieved on a Phenomenex Luna C18 (50 mm × 2.0 mm, 5 μm) column. Ion pairs m/z 526 → 313 for the analyte and m/z 532 → 319 for the IS were monitored in positive ionization mode for MS detection. This methodology has been fully validated and proved to be rugged and reproducible. Intra- and inter-run variability was within 5.88%, with accuracy between 95.6 and 106% of the nominal concentrations. Analyte stability was evaluated under various sample preparation, analysis and storage conditions. This assay has been utilized to analyze human plasma samples obtained from phase I to III clinical trials.
Keywords: Odanacatib; MK-0822; Cathepsin K inhibitor; Bioanalysis; Human plasma; Liquid chromatography–tandem mass spectrometry; Liquid–liquid extraction;

A validated high-performance liquid chromatographic method with diode-array detection for the estimation of xyloketal B in rat plasma by Wei Zhang; Yan Liu; Hongtu Yang; Zongyi Li; Yadong Huang; Zhongliang Xu; Yongcheng Lin; Qi Xiang; Jiyan Pang (24-29).
A sensitive and specific HPLC–UV method was developed and validated for the determination of xyloketal B in rat plasma. Following liquid–liquid extraction, the separation was performed using an isocratic mobile phase of methanol–acetonitrile–water (30/30/40, v/v/v) on a Phenomenex C18 column (4.6 mm × 250 mm, 5 μm). The eluent was monitored at 220 nm and at a flow rate of 0.8 ml min−1. A linear curve over the concentration range of 1–128 μg/ml (r  > 0.999) was established. The LLOQ of the method was 1 μg/ml. Good precision and accuracy at concentrations of 2.5, 25 and 100 μg/ml were obtained. The recovery of xyloketal B in plasma was >87.91%. The validated method was found to be specific, precise and accurate in the study. The analytic method was satisfactorily applied to perform preclinical pharmacokinetic study of xyloketal B in rat plasma.
Keywords: Xyloketal B; Pharmacokinetics; HPLC-DAD; Rat plasma;

► A sensitive and robust LC–MS/MS method was developed to quantify basal adenosine concentrations. ► A strong cation exchange cartridge was used to enrich analyte and reduce biological complexity. ► Patients undergoing on-pump coronary artery bypass grafting (CABG) surgery were studied. ► Elevation of adenosine basal level after on-pump CABG surgery was observed.A sensitive and robust LC–MS/MS method was developed to quantify basal adenosine concentrations in human plasma of patients undergoing on-pump coronary artery bypass grafting (CABG) surgery. A strong cation exchange (SCX) monolithic cartridge was used to enrich analyte, improve robustness, and reduce biological complexity. A simple modifier-free mobile phase was employed to improve sensitivity and reproducibility. This method exhibits consistent precision and accuracy, and the RSDs or REs of all the intraday and interday determinations were within 10%. The calibration curve was linear across the examined dynamic range from 1 nM to 500 nM (r 2  = 0.996). LOD and LOQ were determined to be 0.257 nM and 0.857 nM respectively, while LLOQ was below 10 nM. This method was used to monitor changes of adenosine levels in patient plasma drawn intraoperatively during on-pump CABG surgery. The analysis of 84 patients revealed that the mean concentration of adenosine in coronary sinus plasma after cardiopulmonary bypass (CPB) is higher than that in coronary sinus before CPB (p  = 0.0024; two-tailed t-test) and that in radial artery plasma after CPB (p  = 0.0409; two-tailed t-test). These findings suggest that the equilibrium between adenosine production and elimination has favored the elevation of adenosine basal level during on-pump CABG surgery and the change is specific to heart tissues. Evaluation of adenosine with a sensitive and robust analytical method has important implications on providing consistent results and meaningful insights into adenosine regulation, as well as its steady state and sustained action on the heart. Relating patient characteristics or clinical outcomes with basal adenosine concentration can be used to optimize the CABG–CPB maneuver by regulating adenosine level via pharmacological intervention, and differentiating adenosine's contribution to cardioprotection from other modulatory factors.
Keywords: Basal adenosine concentration; Liquid chromatography tandem mass spectrometry (LC–MS/MS); On-pump coronary artery bypass grafting (CABG); Cardiopulmonary bypass (CPB); Cardioprotection;

► UPLC–MS/MS method for the simultaneous determination of GHB, BHB, pregabalin, 1,4BD and GBL. ► Sample preparation with 96-well SPE format. ► Method successfully used for routine screening of whole blood and urine samples, including forensic autopsies.The demand of high throughput methods for the determination of gamma-hydroxybutyrate (GHB) and its precursors gamma-butyrolactone (GBL) and 1,4-butane-diol (1,4BD) as well as for pregabalin is increasing. Here we present two analytical methods using ultra-high pressure liquid chromatography (UPLC) and tandem mass spectrometric (MS/MS) detection for the determination of GHB, beta-hydroxybutyrate (BHB), pregabalin, 1,4BD and GBL in whole blood and urine. Using the 96-well formate, the whole blood method is a simple high-throughput method suitable for screening of large sample amounts. With an easy sample preparation for urine including only dilution and filtration of the sample, the method is suitable for fast screening of urine samples. Both methods showed acceptable linearity, acceptable limits of detection, and limits of quantification. The within-day and between-day precisions of all analytes were lower than 10% RSD. The analytes were extracted from matrices with recoveries near 100%, and no major matrix effects were observed. Both methods have been used as routine screening analyses of whole blood and urine samples since January 2010.
Keywords: GHB; BHB; 1,4BD; GBL; Pregabalin; UPLC–MS/MS; Whole blood; Urine;

Optimisation of an HPLC method for the simultaneous quantification of the major sugars and organic acids in grapevine berries by Hans A. Eyéghé-Bickong; Erik O. Alexandersson; Liezel M. Gouws; Philip R. Young; Melané A. Vivier (43-49).
► High performance liquid chromatographic method optimised and validated. ► Simultaneous quantification of major sugars and organic acids in grapevine berries. ► Samples directly injected after sample extraction. ► Extraction step downscaled to allow the use of milligrams of sample material. ► A set of equations proposed to calculate concentrations of malic acid and fructose.A high performance liquid chromatographic method was developed to profile major sugars and organic acids in grapevine berries. Sugars and organic acids in grapevine berries were extracted by chloroform/polyvinylpolypyrrolidone purification. The extracts were chromatographed on an Aminex HPX-87H ion-exchange HPLC column with 5 mM sulphuric acid as mobile phase. Chromatography was visualised via a diode array detector combined with a refractive index detector. The analysis was calibrated using external standard calibration and a novel equation was used to calculate the concentrations of malic acid and fructose from unresolved separation. For the method to be utilised for analysing a large numbers of berry samples, each sample was directly injected after sample extraction and the extraction step was downscaled to allow the use of small amounts of sample material. The concentrations of sugars and organic acids in grapevine berry samples were normalised to the internal standard concentrations obtained after extraction of an internal standard mixture. The analysis method exhibits a good precision and a high analyte recovery from samples spiked with the standard mixture and is suitable for the profiling of major sugars and organic acids in grapevine berry samples at different stages of berry development. This is the first report on the combined profiling of the major sugars and organic acids in grapevine berries using milligram amounts of plant material with direct injection after sample extraction.
Keywords: Sugars; Organic acids; Grapevine berries; High performance liquid chromatography;

Development and validation of a fully automated online human dried blood spot analysis of bosentan and its metabolites using the Sample Card And Prep DBS System by Norbert Ganz; Maharajah Singrasa; Laurent Nicolas; Marcelo Gutierrez; Jasper Dingemanse; Werner Döbelin; Mirko Glinski (50-60).
► Validation of the online determination of bosentan and its metabolites in human DBS. ► The SCAP DBS System allows the fully automated online bioanalysis of DBS samples. ► No manual sample processing is necessary prior to online DBS analysis. ► The sample throughput is competitive with conventional liquid-based bioanalysis.This paper describes the development and validation of a liquid chromatography (LC)–electrospray ionization tandem mass spectrometry assay for the fully automated simultaneous determination of bosentan, a dual endothelin receptor antagonist used in the treatment of pulmonary arterial hypertension, and its three primary metabolites hydroxy bosentan (Ro 48-5033), desmethyl bosentan (Ro 47-8634), and hydroxy desmethyl bosentan (Ro 64-1056) in human dried blood spots (DBS) by use of the Sample Card And Prep (SCAP) DBS System. The system enabled the online extraction of compounds from filter paper cards without the need for punching and sample pretreatment. This was realized by automatic introduction of DBS sample cards into the LC flow via a pneumatically controlled clamp module. Using a three-column setup comprised of two pre columns for successive online DBS sample cleanup and a Synergi™ POLAR-RP C18 analytical column for chromatographic separation under gradient conditions with a mobile phase A consisting of 1% acetic acid and a mobile phase B consisting of 1% acetic acid in methanol/2-propanol (80/20, v/v). MS/MS detection was performed in the positive multiple reaction monitoring mode using a Sciex API 4000 triple quadrupole LC–MS/MS system equipped with a TurboIonSpray™ source. The total run time was 9.0 min. The individual phases of online human DBS analysis were synchronized by automated valve switching. The analytical method was shown to be sensitive and selective with inter-day accuracy and precision of 91.6–108.0% and 3.4–14.6%, respectively, and it exhibited good linearity (r 2  ≥ 0.9951 for all analytes) over the concentration range of 2 ng/mL (5 ng/mL for Ro 47-8634)–1500 ng/mL. The analytes were stable in human DBS over 3.5 months at ambient temperature and accurate and precise results were obtained when using a blood spot volume between 20 and 30 μL. Furthermore, no apparent (−8.9 to 12.6%) impact of hematocrit values ranging from 0.35 to 0.65 was observed on the quantification of the analytes. The system allowed very good recoveries of all analytes, between 83.0% and 92.3% for bosentan, between 94.4% and 100% for Ro 48-5033, between 98.0% and 100% for Ro 47-8634, and between 94.3% and 100% for Ro 64-1056. The validation demonstrated that the SCAP DBS System provides a robust automated platform for DBS analysis.
Keywords: Bosentan; Fully automated; Dried blood spot; Online extraction; Pulmonary arterial hypertension; SCAP DBS;

Determination of caprolactam and 6-aminocaproic acid in human urine using hydrophilic interaction liquid chromatography-tandem mass spectrometry by Ya-Hsueh Wu; Ming-Ling Wu; Chun-Chi Lin; Wei-Lan Chu; Chen-Chang Yang; Robert Tate Lin; Jou-Fang Deng (61-65).
► We developed a method for determination of caprolactam and its metabolite in urine. ► The method fulfilled our analytical standard criteria, and it is simple and rapid. ► The method was successfully applied to an unusual case of caprolactam poisoning. ► The method is ready to be used in the analysis of clinical and toxicological samples.A simple and rapid assay based on hydrophilic interaction liquid chromatography with tandem mass spectrometry has been first developed and validated for simultaneous determination of caprolactam (CA) and 6-aminocaproic acid (6-ANCA) in human urine using 8-aminocaprylic acid as internal standard. A 20 μL aliquot of urine was injected directly into the liquid chromatography tandem mass spectrometry (LC–MS–MS) system. The analytes were separated on a Phenomenex Luna HILIC column with gradient elution. Detection was performed on Triple Quadrupole LC–MS in positive ions multiple reaction monitoring mode using electrospray ionization. The calibration curves were linear (r 2  ≥ 0.995) over the concentration range from 62.5 to 1250 ng/mL for CA and 31.25 to 1000 ng/mL for 6-ANCA. The detection limits of CA and 6-ANCA were 62.5 and 15.6 ng/mL, respectively. The intra-day and inter-day precisions were within 8.7% and 9.9%, respectively. The intra-day and inter-day accuracy were between 5.3% and 3.5%, and between 6.1% and 6.6%, respectively. The method proved to be simple and time efficient, and was successfully applied to evaluate the kinetics of caprolactam in one unusual case of caprolactam poisoning.
Keywords: Caprolactam (CA); 6-Aminocaproic acid (6-ANCA); Hydrophilic interaction liquid chromatography (HILIC); Liquid chromatography tandem mass spectrometry (LC–MS–MS);

Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography by Maria Bergström; Eva Åström; Peter Påhlsson; Sten Ohlson (66-72).
► Affinity columns with engineered Aleuria aurantia lectin (AAL) were constructed. ► Weak affinity chromatography (WAC) was used to determine AAL binding profile. ► K d values of non-labeled saccharides from 5 μM to 7 mM were accurately measured. ► All disaccharides had a K d about 10 μM while oligosaccharides varied in binding. ► Single site AAL differed slightly in binding compared to full-length AAL.Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coli as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked α1–3 to GlcNAc interacted with higher affinity compared to fucose linked α1–6 or α1–4 and the obtained dissociation constants (K d ) were in the range of 10 μM for all AAL forms. Tetra- and pentasaccharides with fucose in α1–2, α1–3 or α1–4 had K d values ranging from 0.1 to 7 mM while a large α1–6 fucosylated oligosaccharide had a K d of about 20 μM. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.
Keywords: Affinity; Aleuria aurantia lectin; Glycan interaction; Recombinant protein; Weak affinity chromatography;

► Propose a novel strategy to systematically identify the in vivo metabolites of SNS. ► Accurate structural elucidation using MS, UV data and n-octanol/water partition coefficient. ► Use neutral loss scan to screen glucuronide and sulfate conjugated metabolites. ► Profile metabolites in normal dosage bio-samples using multiple reaction monitoring.Si-Ni-San (SNS) is a widely used traditional Chinese medicine formula (TCMF) in treating various diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, a liquid chromatography coupled with diode array detection and triple–quadrupole spectrometry (LC-DAD–MS/MS) method was developed for detection and identification of SNS metabolites in rat plasma and urine at a normal clinical dosage. Accurate structural elucidation was performed using MS/MS, UV data and n-octanol/water partition coefficient. Based on the proposed strategy, 36 absorbed compounds and 29 metabolites in plasma and 33 metabolites in urine were detected by a highly sensitive MRM method. Our results indicated that phase II reactions (e.g., methylation, glucuronidation and sulfation) were the main metabolic pathways of gallic acid and flavanones, while phase I reactions (e.g., hydroxylation) were the major metabolic reaction for triterpenoid saponins. The metabolite profile analysis of SNS provided a comprehensive understanding of the in vivo metabolic fates of constituents in SNS. Moreover, the results in this work demonstrated the present strategy based on the combination of chromatographic, spectrophotometric, mass-spectrometric, and software prediction to detect and identify metabolites was effective and reliable. And such a strategy may also be extended to investigate the metabolism of other TCMF.
Keywords: Si-Ni-San; Metabolite; Liquid chromatography coupled with diode array detection; Triple–quadrupole spectrometry; n-Octanol/water partition coefficient;

► Isolation of indole alkaloids from the stem bark of Geissospermum vellosii using high performance counter-current chromatography. ► Identification of indole alkaloids by nuclear magnetic resonance and mass spectrometry. ► Structural characterization of indole alkaloids by electrospray ionization tandem mass spectrometry. ► Establishment of mass spectrometry fragmentation pattern of indole alkaloids.Alkaloids from the stem bark of Geissospermum vellosii possess a variety of therapeutic properties including antimalarial activities, activity as a sexual stimulant and inhibition of the proliferation of HIV and herpes viruses. Methods currently used to isolate the active components from G. vellosii are time-consuming, labor intensive, and result in low recovery. In addition, there is a lack of sensitive and accurate analytical methods for the structural characterization and identification of alkaloid components in minor quantities. A combination of high performance counter-current chromatography and ESI tandem mass spectrometry (MS n ) was established to isolate alkaloids from the stem bark of G. vellosii, and study their electrospray ionization mass spectrometry fragmentation behavior. Five indole alkaloids were successfully isolated and identified by nuclear magnetic resonance and mass spectrometry. The multi-stage tandem mass spectrometric data were used to study their fragmentation pattern and set a model for detailed structure characterization of related indole alkaloids. The presence of the even mass fragment ion suggestive of an odd number of nitrogen at m/z 144 corresponding to C10H9N was characteristic to indole alkaloids. The results of the experiments demonstrated that the combination of high performance counter current chromatography and ESI-MS n is a sensitive, selective and effective approach for rapid isolation and characterization of alkaloids from G. vellosii.
Keywords: Electrospray ionization tandem mass spectrometry; High performance counter-current chromatography; Indole alkaloids; Structural elucidation;

One-step extraction for gas chromatography with flame photometric detection of 18 organophosphorus pesticides in Chinese medicine health wines by Qianzhen Liu; Weijun Kong; Feng Qiu; Jianhe Wei; Shihai Yang; Yuguo Zheng; Meihua Yang (90-96).
► An easy, rapid and selective GC-FPD method was established for determining 18 OPPs in Chinese medicine health wines. ► The OPPs in health wines were extracted by a simple one-step extraction procedure. ► This extraction procedure only needed a little solvent without any further cleanup steps. ► These pesticides were successfully confirmed by GC–MS.An easy, rapid and selective gas chromatography with flame photometric detection (GC-FPD) method was established for simultaneously determining 18 organophosphorus pesticides (OPPs) in 80 Chinese medicine (CM) health wines. This method was based on a simple one-step extraction procedure using a little solvent without any further cleanup steps. The optimized extraction solvent for the pesticides is acetone:dichloromethane (1:1, V/V) with extraction recovery of 79.0–109.1% and relative standard deviation (RSD) of 0.36–12.68%, respectively. The limits of detection (LODs) of the established GC-FPD method for all investigated pesticides ranged from 1 to 15 ng mL−1 and limits of quantification (LOQs) from 4 to 50 ng mL−1. Out of all 80 CM health wines, 18 OPPs were found in 8 samples at low concentrations of 8.2–37.9 ng mL−1. These pesticides were successfully confirmed by GC–MS. This is the first report of determining OPPs in CM health wines, providing references for monitoring the quality of CM health wine in routine analysis.
Keywords: GC-FPD; GC–MS; One-step extraction; Organophosphorus pesticides; Chinese medicine health wine;

► A sensitive and reliable LC–MS/MS method for the simultaneous determination of l-FMAU and d-FMAU in human plasma has been developed and validated. ► LLOQ of 10 ng/mL was achieved using only 200 μL of plasma. ► It is the first method for enantioselective determination of FMAU in human plasma.A new, simple and enantioselective normal-phase liquid chromatography–mass spectrometry method was presented for the quantification of clevudine and its enantiomer in human plasma. A C18 cartridge was used in this method to extract the enantiomers in 200 μL plasma followed by a chiral separation on a cellulose-based LC column with mobile phase consisted of hexane, methanol and ethanol (62:28:10, V/V/V). The eluate was directed to a mass spectrometry through an electrospray ionization interface. A transition of m/z 261.0 to m/z 126.8 was used for monitoring of clevudine and its enantiomer. This method showed good linearity (R  > 0.997), precision (<9.6%) and accuracy (within 95.48–105.9%) within a range of 10–1000 ng/mL for the enantiomers and has been applied to the pharmacokinetics study of clevudine capsules in human plasma.
Keywords: Clevudine; d-FMAU; Chiral separation; Normal-phase HPLC; Pharmacokinetics;

► A sensitive LC–MS/MS assay method for two metabolites of atomoxetine was developed. ► LLOQ was 0.05 ng/mL for 4-hydroxyatomoxetine and 0.1 ng/mL for N-desmethylatomoxetine. ► The validated method was successfully applied to a pharmacokinetic study in humans.4-Hydroxyatomoxetine (4-HAT) and N-desmethylatomoxetine (N-DAT) are major metabolites of atomoxetine, a potent and selective inhibitor of the presynaptic norepinephrine transporter that is used for the treatment of attention deficit/hyperactivity disorder. The pharmacological activity of 4-HAT is similar to that of atomoxetine. We have developed and validated a simple, rapid and sensitive liquid chromatography analytical method with tandem mass spectrometry (LC–MS/MS) for the determination of 4-HAT and N-DAT in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of analytes was performed using a reversed-phase Luna C18 column (2.0 mm × 100 mm, 3 μm particles) with a mobile phase of 10 mM ammonium formate buffer (pH 3.5)–methanol (10:90, v/v) and quantified by MS/MS detection in ESI positive ion mode. The flow rate of the mobile phase was 250 μL/min and the retention times of 4-HAT, N-DAT and internal standard (IS, metoprolol) were 0.9, 1.0 and 1.0 min, respectively. The calibration curves were linear over the range of 0.05–20 ng/mL for 4-HAT and 0.1–20 ng/mL for N-DAT. The lower limits of quantification, using 200 μL human plasma, were 0.05 and 0.1 ng/mL for 4-HAT and N-DAT, respectively. The mean accuracy and precision for intra- and inter-day validation of 4-HAT and N-DAT were both within the acceptable limits. This LC–MS/MS method showed improved sensitivity for quantification of the two main metabolites of atomoxetine in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.
Keywords: 4-Hydroxyatomoxetine; N-Desmethylatomoxetine; LC–MS/MS; Human plasma; Pharmacokinetics;

► Volatile compounds of fresh royal jelly. ► Bactericidal and repelling activities. ► Markers of inappropriate storage. ► Extractable compounds of royal jelly. ► Possible source of free amino acids.Using headspace solid-phase microextraction (HS-SPME) followed by diethyl ether and methanol extraction, it was possible to isolate as many as 185 organic compounds out of 17 samples of crude royal jelly (RJ). Of the above compound number, 169 compounds were positively identified by means of gas chromatography–mass spectrometry. The volatile fraction of RJ consists of 25 different compounds where approximately 47% of the total ion current (TIC) of volatile compound chromatograms were composed of substances characterized by bactericidal (phenols) and repelling (octanoic acid and 2-heptanone) activities. Preliminary investigations have shown that RJ stored for 10 months at −18 °C and 4 °C keeps its composition of volatile compounds unchanged, however, at the same time at room temperature RJ phenol contents is decreased twice, whereas the fraction of aliphatic acids is increased 2.8 times due to the presence of both acetic and butyric acids. The chromatogram of RJ ether extracts showed 85 different compounds, however about 88% of TIC consisted exclusively of 8 compounds, i.e. 10-hydroxy-2-decenoic, 10-hydroxydecanoic, 3,10-dihydroxydecanoic, 8-hydroxyoctanoic, 2-decene-1,10-dioc and (Z)-9-hydroxy-2-decenoic acids. Nine aliphatic acids, which were detected for the first time, are the homologues of hydroxy- and oxo-acids identified earlier in RJ. In the RJ methanol extracts 82 compounds were identified, mainly carbohydrates and their derivatives. Approximately 87% of TIC consisted of fructose, glucose and sucrose. Special attention was paid to discrepancies between obtained and literature data concerning the presence of free amino acids in RJ. It was suggested that these inconsistencies can be explained by the differences in the methods of RJ collection and/or sample preparation.
Keywords: Gas chromatography–mass spectrometry; Royal jelly; Chemical composition; Volatile compounds; Hydroxy fatty acids; Carbohydrates; Free amino acids;

► A SPE-HPLC–MS method was developed for the quantification of SSRI drugs in plasma. ► Matrix effects were reduced thanks to SPE and adequate chromatographic separation. ► Stable isotope-labeled IS were used to compensate for the global method variability. ► Very good validation performances were obtained, which were verified during routine use. ► This method is suitable for both routine TDM and pharmacokinetic studies.A simple and sensitive liquid chromatography–electrospray ionization mass spectrometry method was developed for the simultaneous quantification in human plasma of all selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their main active metabolites (desmethyl-citalopram and norfluoxetine). A stable isotope-labeled internal standard was used for each analyte to compensate for the global method variability, including extraction and ionization variations. After sample (250 μl) pre-treatment with acetonitrile (500 μl) to precipitate proteins, a fast solid-phase extraction procedure was performed using mixed mode Oasis MCX 96-well plate. Chromatographic separation was achieved in less than 9.0 min on a XBridge C18 column (2.1 × 100 mm; 3.5 μm) using a gradient of ammonium acetate (pH 8.1; 50 mM) and acetonitrile as mobile phase at a flow rate of 0.3 ml/min. The method was fully validated according to Société Française des Sciences et Techniques Pharmaceutiques protocols and the latest Food and Drug Administration guidelines. Six point calibration curves were used to cover a large concentration range of 1–500 ng/ml for citalopram, desmethyl-citalopram, paroxetine and sertraline, 1–1000 ng/ml for fluoxetine and fluvoxamine, and 2–1000 ng/ml for norfluoxetine. Good quantitative performances were achieved in terms of trueness (84.2–109.6%), repeatability (0.9–14.6%) and intermediate precision (1.8–18.0%) in the entire assay range including the lower limit of quantification. Internal standard-normalized matrix effects were lower than 13%. The accuracy profiles (total error) were mainly included in the acceptance limits of ±30% for biological samples. The method was successfully applied for routine therapeutic drug monitoring of more than 1600 patient plasma samples over 9 months. The β-expectation tolerance intervals determined during the validation phase were coherent with the results of quality control samples analyzed during routine use. This method is therefore precise and suitable both for therapeutic drug monitoring and pharmacokinetic studies in most clinical laboratories.
Keywords: Antidepressants; Solid-phase extraction; HPLC–ESI-MS; Stable isotope-labeled internal standard; Method validation; Therapeutic drug monitoring;

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for monitoring drug exposure in hematopoietic stem cell transplant recipients by Isabelle Laverdière; Patrick Caron; Félix Couture; Éric Lévesque; Chantal Guillemette (131-137).
► We developed a LC–MS/MS method for the simultaneous monitoring of multiple immunosuppressive drugs in blood. ► The validated method demonstrates sensitivity, accuracy and reproducibility. ► Its clinical application has been validated in samples from transplant recipients. ► The method is convenient for therapeutic drug monitoring and large-scale studies.A liquid chromatography–tandem mass spectrometry method was developed for the quantification of circulating levels of multiple immunosuppressant drugs including cyclosporine (CsA), tacrolimus, methotrexate (Mtx), prednisone, prednisolone, methylprednisone, total and free mycophenolic acid (MPA), as well as MPA phenolic (MPAG) and acyl (AcMPAG) glucuronide metabolites. Linearity, precision and accuracy were validated within the typical therapeutic range of concentrations for each compound. The assay was linear over 0.125–25 ng/mL for tacrolimus, 1–500 ng/mL for prednisone/methylprednisone, 2–400 ng/mL for Mtx, 2–1000 ng/mL for prednisolone and from 7.5 to 1500 ng/mL for CsA with the lowest limit of quantification (LLOQ) being 0.125, 1.00, 2.00, 2.00 and 7.5 ng/mL, respectively. The calibration curve concentrations for MPA and MPAG ranged from 50 to 50,000 ng/mL (LLOQ: 50 ng/mL) and 10 to 10,000 ng/mL (LLOQ: 10 ng/mL) for AcMPAG. Mean recoveries in blood and plasma were 84% ± 5.7%. The method could measure individual drugs with high sensitivity, accuracy (bias ≤ 14%), and reproducibility (CV ≤ 12.8%). Its clinical application was validated by measuring levels of these drugs in samples obtained from hematopoietic stem cell transplant recipients treated with combined immunosuppressive drug therapy. Our results indicate that this approach is suitable for simultaneous determination of in vivo levels of immunosuppressive drugs commonly used in combined therapies.
Keywords: Chromatography; Mass spectrometry; Calcineurin inhibitor; Corticosteroid; Methotrexate; Mycophenolic acid;

Comparison of extraction procedures for assessment of matrix effect for selective and reliable determination of atazanavir in human plasma by LC–ESI-MS/MS by Manish Yadav; Vikas Trivedi; Vivek Upadhyay; Gaurang Shah; Girin A Baxi; Sailendra Goswami; Pranav S. Shrivastav (138-149).
► A reliable SPE-LC–ESI-MS/MS is proposed for determination of atazanavir in human plasma. ► A comparative study with PP, LLE and SPE is demonstrated to show the magnitude of ion suppression. ► Matrix effect assessment is done by post-column analyte infusion and post extraction spiking methods. ► The method is practically free from matrix interference based on relative matrix effect in different lots of plasma. ► The application is demonstrated by a bioequivalence study in healthy volunteers and incurred sample reanalysis.A comparative study with three conventional extraction techniques namely protein precipitation (PP), liquid–liquid extraction (LLE) and solid phase extraction (SPE) has been demonstrated to assess the magnitude of matrix interference by post-column analyte infusion and post extraction analyte spiking for the determination of atazanavir from human plasma. Severe ion suppression observed in PP and to a lesser extent in LLE was circumvented by SPE on LiChrosep Sequence extraction cartridge. Based on these observations a selective, rugged and high throughput SPE-LC–MS/MS method has been developed for reliable determination of atazanavir in human plasma. The chromatographic separation was achieved on a Hypersil Gold C18 (50 mm × 4.6 mm, 5 μm) analytical column using 5 mM ammonium formate in water:methanol (10:90, v/v) as the mobile phase under isocratic conditions. The method was validated over a wide dynamic concentration range of 10–6000 ng/mL. The mean relative recovery and absolute matrix effect across quality controls were 84.9 and 93.2%, respectively. The precision value for relative matrix effect between eight different lots of plasma, expressed as %CV of the slopes of the calibration lines was 2.41. The stability of atazanavir under different storage conditions varied from −8.4 to 5.4%. The method was successfully applied to a bioequivalence study of 300 mg atazanavir capsule formulation in 24 healthy Indian males under fasting condition.
Keywords: Atazanavir; Indinavir; Post-column infusion; Ion-suppression; Extraction procedures; LC–ESI-MS/MS; Human plasma;

Simultaneous determination of fluoroquinolones in foods of animal origin by a high performance liquid chromatography and a liquid chromatography tandem mass spectrometry with accelerated solvent extraction by Huan Yu; Yanfei Tao; Dongmei Chen; Yuanhu Pan; Zhenli Liu; Yulian Wang; Lingli Huang; Menghong Dai; Dapeng Peng; Xu Wang; Zonghui Yuan (150-159).
► A rapid method for quantitative determination of 15 fluoroquinolones. ► The method reduced the analysis time and improved the extraction efficiency. ► The method is more high throughput.A confirmatory and quantitative method based on a high performance liquid chromatography UV detector (HPLC-UV) and a liquid chromatography tandem mass spectrometry (LC–MS/MS) with an extraction procedure of accelerated solvent extraction (ASE) has been developed for simultaneous determination of 15 kinds of fluoroquinolones in various animal origin food samples. The sample preparation procedures consist of an extraction step with acetonitrile and a cleaning-up step with Oasis HLB cartridge. Parameters for extraction pressure and temperature, cycle of ASE, clean-up, and analysis procedure have been optimized systematically. The recoveries of FQNs spiked in the tissues as the muscle, liver, kidney of swine, bovine, chicken and fish at a concentration range of 10–800 μg/kg were found between 70.6% and 111.1% with relative standard deviations (RSD) less than 15% in HPLC. The LOD and LOQ of the HPLC for the 15 FQNs were 3 μg/kg and 10 μg/kg, respectively, and those of the LC–MS/MS were 0.3 and 1 μg/kg, respectively. These rapid and reliable methods can be used to efficiently separate, characterize and quantify the residues of 15 FQNs (Marbofloxacin, Enoxacin, Fleroxacin, Ofloxacin, Pefloxacin, Lomefloxacin, Danofloxacin, Enrofloxacin, Orbifloxacin, Cinoxacin, Gatifloxacin, Sarafloxacin, Difloxacin, Nalidixic Acid, Flumequine) in food of animal origin.
Keywords: Fluoroquinolones; Accelerated solvent extraction; High performance liquid chromatography; Liquid chromatography tandem mass spectrometry; Food of animal origin;

Development and validation of a rapid and sensitive liquid chromatography–tandem mass spectrometry method for benvitimod quantification in human plasma by Libo Zhao; Baoying Zhu; Xin Chen; Genghui Chen; Haibo Chen; Yuzhen Li; Shan Jing; Yi Fang (160-165).
► Validation of a HPLC–MS/MS method for the determination of benvitimod for the first time. ► Highly sensitive, with LLOQ of 0.1 ng/ml using only 0.2 ml of plasma. ► Applying to a pharmacokinetic study in patients with mild to moderate psoriasis. ► Confirming the low absorption of this drug.Benvitimod is a newly synthesized non-steroid small molecule being developed as a candidate drug for the treatment of inflammatory skin diseases. Here a rapid, sensitive and specific high performance liquid chromatography–tandem mass spectrometry (LC/ESI/MS/MS) method was developed for the determination of benvitimod in human plasma. The samples were alkalified with disodium tetraborate firstly, and then extracted by methyl tert-butyl ether. Fluorophenyl-benvitimod was used as internal standard (I.S.). Chromatographic separation was performed on an Ultra C18 column (150 mm × 2.1 mm, 5.0 μm). The mixed mobile phase delivered at 300 μl/min was CH3CN/H2O, 76.65:23.35 (v/v), containing 0.2 mmol/L NH4COOH. Detection and quantitation was performed by electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M−H] MRM transition of benvitimod at m/z 253.1→211.0 was used for benvitimod quantitation and the transition at m/z 270.9→229.2 was used to monitor I.S. The calibration curve was linear within the concentration range of 0.1–10.0 ng/mL (r  > 0.99). The lower limit of quantification (LLOQ) was 0.1 ng/mL. The extraction recovery was above 80%. The accuracy expressed as relative error (RE) was less than 1.03%. The intra- and inter-day precisions were less than 11.81%. The freeze–thaw stability was also investigated and it was found that both benvitimod and the I.S. were quite stable. This method is especially useful for the pharmacokinetic study of benvitimod.
Keywords: Benvitimod; LC/ESI/MS/MS; Human plasma; Pharmacokinetics;