Journal of Chromatography B (v.897, #C)

► A new analytical method to determine extracellular levels of lipid-related metabolites. ► Five metabolites are separated in 20 min with lower limit of quantification at 0.5 nM. ► The developed method is successfully applied to monitor the extracellular levels of metabolites in S. cerevisiae.A highly sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed and validated for the quantification of glycerophosphoinositol (GroPIns), glycerophosphocholine (GroPCho), glycerol 3-phosphate (GroP), inositol, and choline in the extracellular medium of Saccharomyces cerevisiae. The media samples were pretreated with a single two-phase liquid extraction. Chromatographic separation was achieved on a Waters Xbridge HILIC (150 mm × 4.6 mm, 5 μm) column under isocratic conditions using a mobile phase composed of acetonitrile/water, 70:30 (v/v) with 10 mM ammonium acetate (pH adjusted to 4.5) at a flow-rate of 0.5 mL/min. Using a triple quadrupole tandem mass spectrometer, samples were detected in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curves were linear (r 2  ≥ 0.995) over the range of 0.5–150 nM, with the lower limit of quantitation validated at 0.5 nM for all analytes. The intra- and inter-day precision (calculated by coefficient of variation, CV%) ranged from 1.24 to 5.88% and 2.46 to 9.77%, respectively, and intra- and inter-day accuracy (calculated by relative error, RE%) was between −8.42 to 8.22% and −9.35 to 6.62%, respectively, at all quality control levels. The extracellular metabolites were stable throughout various storage stability studies. The fully validated method was successfully applied to determine the extracellular levels of phospholipid-related metabolites in S. cerevisiae.
Keywords: HILIC–MS/MS; Glycerophospholipid metabolites; Metabolomics; Saccharomyces cerevisiae; Metabolic footprinting;

Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood by Annamaria Jakab; Serge Winter; Bertrand Guy; Marc Raccuglia; Frank Picard; John M. Kovarik; Jayraj Chudasama; Swati Guttikar; Puran Singhal; Olivier Kretz (10-16).
► Sotrastaurin, an investigational drug for organ transplantation, autoimmune disorders. ► A method to estimate the drug and its active metabolite in human blood is developed. ► This original method employs LC–MS/MS and was validated. ► The limit of quantification was between 3 ng/mL and 1200 ng/mL. ► The acceptance criteria (FDA) for method validation were met.A liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin in human blood. The validation of the analytical procedure was performed according to the latest Food and Drug Administration (FDA) “Guidance for Industry, Bioanalytical Method Validation”. Chromatographic separation was performed using an RP C18 (50 mm × 4.6 mm, 5 μm) column at 40 ± 3.0 °C with a mobile phase consisted of 2 mM ammonium acetate in water (pH 4.5):methanol:acetonitrile (25:15:60, v/v) of a flow rate of 1 mL/min followed by quantification with tandem mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The validated method described in this paper presents high absolute recovery, with a sensitivity of 3.00 ng/mL as lower limit of quantitation using a sample volume of 300 μL, low inter-run bias and variability (for Sotrastaurin, −4.4 to 0.4% and 1.8 to 2.5% and for N-desmethyl-sotrastaurin, ranged from 1.6 to 2.3% and 2.7 to 3.9%, respectively) with a short runtime of 3.5 min. The method was validated using K3EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethyl-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethyl-sotrastaurin in zero samples ≤20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3.00 ng/mL and 1200 ng/mL.
Keywords: Sotrastaurin; N-desmethyl-sotrastaurin; AEB071; Metabolite; LC–MS/MS; Human blood;

LC–ESI–MS method for the monitoring of Abl 1 tyrosine kinase by Hui Chen; Erwin Adams; Ann Van Schepdael (17-21).
► The kinetic constants of Abl 1 are successfully studied. ► With online desalting system, Tris–HCl buffer is used to conduct enzyme reaction. ► Enzyme reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism.A liquid chromatography–electrospray ionization–mass spectrometric (LC–ESI–MS) method was developed and validated to study Abl 1 tyrosine kinase. An online desalting system was adopted, and a transformation of the ratio of product to substrate instead of a deuterated internal standard was introduced to calculate the concentration of product. In this study, the substrate used was Abltide (KKGEAIYAAPFA-NH2). The detection was performed by selected ion monitoring (SIM) mode via positive ESI interface. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The limit of quantification (LOQ) was 10 nM for the product and 25 nM for the substrate. The simple ratios of product to substrate maintained a linear relationship (R 2  = 0.9997) over the ratio of 0–50% product. Intra- and inter-day precision was less than 10% and accuracy was from −1.6 to +5.3%. The validated method was applied to the Abl 1 kinase kinetic study and the K m and V max constants obtained for Abltide were 34.78 μM and 5.563 μmol/mg/min and for adenosine triphosphate (ATP) were 43.61 μM and 5.906 μmol/mg/min. The enzymatic reaction of Abl 1 tyrosine kinase belongs to ternary-complex mechanism.
Keywords: LC–ESI–MS; Abl 1 tyrosine kinase; Abltide; p-Abltide; Enzyme assay;

► Good sensitivity, enabling tracking of urinary metabolites for several weeks. ► Herbal blend analysis showed suppliers tailoring composition, as in Europe. ► Application of method to urine samples showed appropriate biomarkers were selected. ► Several hundred samples processed, with positive rate as expected. ► Samples from single individual produced informative time-course profile.An LC–MS/MS method for the quantitation of urinary metabolites of eight JWH-type synthetic cannabinoids (SCs) has been developed and validated. Urine samples are subjected to deconjugation using β-glucuronidase, followed by a solvent extraction procedure. Compounds are separated on a reverse-phase HPLC column within a 14 min cycle. Low assay limits are required in order to demonstrate prior exposure to SCs. Matrix effects were studied and proved to be significant for selected analytes, and were challenging to circumvent as isotope-labeled internal standards are not available. An elimination profile from a naïve user following a single smoke of “Kronic” was constructed, showing urinary excretion over 2–3 days with peak concentrations of different metabolites 3–16.5 h after smoking. This method has been developed to process several hundred samples within a high-throughput drugs of abuse laboratory, with growing evidence that the use of synthetic cannabinoid blends is common within the Australian workforce.
Keywords: LC–MS/MS; Synthetic cannabinoids; Urine;

► MIP-MSPD–LC–MS/MS method was developed. ► The method was used for determination of imidacloprid in rice. ► The recoveries of imidacloprid in rice samples from 83.8% to 92.5% were obtained. ► The proposed method was proved to be sensitive and rapid.A new method based on matrix solid-phase dispersion (MSPD) coupled with liquid chromatography tandem mass spectrometry has been developed for the determination of imidacloprid in rice. The molecularly imprinted polymers were synthesized and applied as the dispersant of MSPD for selective extraction of imidacloprid from rice, while interferences originated from sample matrices were eliminated simultaneously. The satisfactory recovery of imidacloprid was obtained by the optimized extraction conditions: 1:2 as the ratio of sample to MIPs; 8 min as the dispersion time; 20% aqueous methanol as washing solvent and methanol as elution solvent. Under the optimal conditions, the linearity of imidacloprid in rice sample was achieved in the range of 10–1000 ng/g, and limit of detection was 2.4 ng/g. The relative standard deviations of intra- and inter-day tests ranging from 4.5% to 5.9% and from 4.8% to 7.1% are obtained, respectively. The proposed method was applied to the determination of imidacloprid in eight rice samples with recoveries in the range of 83.8–92.5%.
Keywords: Matrix solid-phase dispersion; Molecularly imprinted polymer; Liquid chromatography–tandem mass spectrometry; Imidacloprid; Rice;

On-fiber furan formation from volatile precursors: A critical example of artefact formation during Solid-Phase Microextraction by An Adams; Fien Van Lancker; Bruno De Meulenaer; Agnieszka Owczarek-Fendor; Norbert De Kimpe (37-41).
► Considerable amounts of furan can be formed on the SPME fiber during thermal desorption of volatile furan precursors. ► The artefactual furan formation increased with increasing desorption time, but especially with increasing desorption temperature. ► The artefactual formation of furan may influence a high amount of literature data and current investigations.For the analysis of furan, a possible carcinogen formed during thermal treatment of food, Solid-Phase Microextraction (SPME) is a preferred and validated sampling method. However, when volatile furan precursors are adsorbed on the carboxen/PDMS fiber, additional amounts of furan can be formed on the fiber during thermal desorption, as shown here for 2-butenal and furfural. No significant increase in furan amounts was found upon heating the furan precursor 2-butenal, indicating that the furan amounts formed during precursor heating experiments are negligible as compared to the additional amounts of furan formed during fiber desorption. This artefactual furan formation increased with increasing desorption time, but especially with increasing desorption temperature. Although this effect was most pronounced on the Carboxen/PDMS SPME-fiber, it was also noted on two other SPME-fibers tested (PDMS and DVB/Carboxen/PDMS). The general impact on furan data from food and model systems in literature will depend on the amounts of volatile precursors present, but will probably remain limited. However, considering the importance of this worldwide food contaminant, special care has to be taken during SPME-analysis of furan. Especially when performing precursor studies, static headspace sampling should preferably be applied for furan analysis.
Keywords: SPME; Furan; Artefact formation; 2-Butenal; Furfural;

► New method for immunosuppressive quantification utilizing MEPS as sample preparation. ► Fully automated method. ► 50 μl sample volume was used. ► Good precision and accuracy results. ► Compassion was made with protein precipitation.In treatment with immunosuppressive drugs, monitoring of blood drug concentration is needed. The aim of this work was to explore micro extraction by packed sorbent (MEPS) as a possible on-line sample preparation method in combination with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for quantification of cyclosporine, everolimus, sirolimus and tacrolimus in whole blood. An automated on-line MEPS system connected with a LC–MS/MS instrument was set up. A C8 sorbent was used for the MEPS extraction. Subsequent analysis was performed with a gradient LC system. The adduct ions [M + NH4]+ of the analytes were monitored in SRM mode for quantification. Ascomycin and cyclosporine D were used as internal standards. The chromatographic run time 2.5 min and the quantification ranges were 3–1500 ng/mL (r 2  ≥ 0.999, n  = 6) for cyclosporine and 0.5–50 ng/mL for everolimus, sirolimus and tacrolimus (r 2  ≥ 0.998, 0.994 and 0.993, respectively, n  = 6). Precision and accuracy were documented at three levels. Accuracy results were between 102% and 109% with precision between 2% and 13% and carry over <0.02%. Matrix effects were characterized and found to be below 20%. The quantifications obtained were in agreement with a reference LC–MS/MS method based on protein precipitation, and results obtained from external proficiency test samples compared with the mean of all other LC–mass spectrometry methods showed good agreement. This method provides an accurate, precise and automated procedure that can be applied for therapeutic drug monitoring of immunosuppressive drugs in clinical laboratories equipped with LC–MS/MS.
Keywords: MEPS; Immunosuppressive drugs; LC–MS/MS; Online-analysis; Whole blood; Micro extraction;

► A LC–ESI-MS/MS method has been developed and validated for the determination of 4-p-anisamidobutyric acid (ABA; or N-anysoyl-γ-aminobutiryc acid, N-anisoyl-GABA) in human plasma. ► The extraction procedure for sample preparation is simple and rapid. ► The method has a short analysis time and high selectivity ► The method has been applied to a pharmacokinetic study in humans.A simple and rapid high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the determination of 4-p-anisamidobutyric acid (ABA; or N-anysoyl-γ-aminobutiryc acid, N-anisoyl-GABA), a major active metabolite of aniracetam, in human plasma. After protein precipitation of plasma sample with methanol, ABA and the internal standard lisinopril were separated on a Venusil ASB C18 column at 25 °C. The mobile phase consisted of methanol–ammonium acetate (10 mmol/L) (30:70, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer with an ESI source in negative ion mode. Multiple reaction monitoring (MRM) using the precursor→product ion combinations of m/z 235.8→m/z 106.6, and m/z 403.8→m/z 113.6 was used to quantify ABA and lisinopril, respectively. This is the first LC–MS/MS method for ABA with advantages of short analysis time (4.5 min per sample run) and high selectivity attributable to the MRM detection and optimized HPLC conditions. The response was linear in a concentration range of 0.0485–19.4 μg/mL in plasma. The extraction recovery of ABA was between 89.1% and 100.7%. The precision (RSD) and accuracy (RE) of the method were evaluated to be within 7.3% and from 2.5% to 6.9%. The validated method has been applied to the pharmacokinetic study after a single oral administration of aniracetam dispersible tablets to human beings.
Keywords: N-anisoyl-GABA; LC–MS/MS;

► We developed a rapid, sensitive and specific HILIC–MS/MS method for the simultaneous analysis of pyroglutamic acid (PGA), cis- and trans-urocanic acid (UCA). ► The method was fully validated and successfully applied to the determination of PGA and UCA in the tape-stripped stratum corneum (SC) samples. ► Their levels were well-correlated with the skin physiology. ► The suggested method will be used for the studies of dermatology and skin physiology.A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC–MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0–250 ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5 ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2 ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3–102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans-urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.
Keywords: Pyroglutamic acid; cis-/trans-Urocanic acid; Human stratum corneum; Natural moisturizing factor (NMF); HILIC–MS/MS;

Determination of 17 macrolide antibiotics and avermectins residues in meat with accelerated solvent extraction by liquid chromatography–tandem mass spectrometry by Yanfei Tao; Gang Yu; Dongmei Chen; Yuanhu Pan; Zhenli Liu; Huimin Wei; Dapeng Peng; Lingli Huang; Yulian Wang; Zonghui Yuan (64-71).
► A rapid method for quantitative determination of 17 macrolide antibiotics and avermectins. ► Parameters of accelerated solvent extraction were investigated by a fractional factorial design. ► The method reduced the analysis time and improved the extraction efficiency.A method has been developed for simultaneous determination of 17 kinds of macrolide antibiotics and avermectins residues in animal origin foods. Samples were extracted with acetonitrile-methanol using accelerated solvent extraction (ASE) instrument. Parameters such as extraction temperature and pressure were investigated by a fractional factorial design (FFD) and the selected extraction (60 °C, 1500 psi for 10 min in two cycles) was most effective. High correlation coefficients (r  > 0.999) of 17 macrolide antibiotics and avermectins were obtained within their respective linear ranges (2–400 μg/kg) using roxithromycin as internal standard. The recoveries of them were above 75% at different spiked levels in various samples. Using ASE the method was featured as short extraction times, reduction use of extraction solvent, high extraction yields, with high level of automation.
Keywords: Macrolide antibiotics; Avermectins; ASE; Animal origin food; LC–MS/MS;

► Accurate mass liquid chromatography–high resolution TOF mass spectrometry method for quantification of atenolol in dried blood spots. ► Atenolol extraction efficiency from dried blood spots was 96 ± 5%. ► The developed LC–HRMS method was linear within the tested calibration range of 25–1500 ng/ml with good accuracy and precision. ► The developed LC–HRMS method was applied to DBS samples obtained from volunteers previously administered a 50 mg atenolol tablet. ► The DBS based assay has the potential to assess adherence to medications.The use of blood spot collection cards was investigated as a means of obtaining small volume samples for the quantification of therapeutic drugs for assessing medication adherence. A liquid chromatography–high resolution TOF mass spectrometry (LC–HRMS) method, based on the measurement at the accurate mass to charge ratio of the target analyte, was used to ensure specificity for atenolol in the dried blood spot (DBS) samples. A working method was developed and validated. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30 μl blood spots on specimen collection cards. A 5 mm disc was cut from the dried blood spot and extracted using methanol:water (60:40, v/v) containing the internal standard, atenolol-d7. Extracts were vortexed, sonicated and then centrifuged. Gradient chromatographic elution was achieved using an Ascentis Express C18 100 mm × 2.1 mm column and a mobile phase flow rate of 0.2 ml/min and the column oven temperature at 30 °C. MS detection was carried out in electrospray positive ion mode for target ions at accurate mass m/z 267.1703 for atenolol and 274.2143 for the IS. Drug extraction efficiency from spiked blood spots was demonstrated to be 96 ± 5% and the drug was stable in DBS for at least 10 weeks. The developed LC–HRMS method was linear within the tested calibration range of 25–1500 ng/ml and validation showed the accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤15% at all concentrations with a limit of quantification of 25 ng/ml. Factors with potential to affect drug quantification measurements such as the matrix effects, volume of blood applied onto the collection card and effect of different sampling cards were investigated. The developed LC–HRMS method was applied to blood spots on sampling card taken from adult healthy volunteers previously administered a 50 mg atenolol tablet and a DBS concentration–time profile was obtained for atenolol. Requiring only a micro volume (30 μl) blood sample for analysis, the developed DBS based assay has the potential to assess patient adherence to atenolol.
Keywords: Dried blood spot (DBS); Atenolol; Accurate mass; Guthrie card; LC–HRMS; Adherence;

► Disulfiram is rapidly metabolized to active metabolites via CYP450 oxidation. ► Metabolites of disulfiram have different therapeutic applications in humans. ► Interaction studies have been done with disulfiram and HIV medication. ► Measurement of the disulfiram metabolite, S-methyl-N,N-diethylthiocarbamate was needed. ► A UPLC–MS method for this metabolite was developed and validated.Disulfiram has been used extensively for alcohol abuse and may have a role in treatment for cocaine addiction. Recent data suggest that disulfiram may also reactivate latent HIV in reservoirs. Disulfiram has complex pharmacokinetics with rapid metabolism to active metabolites, including S-methyl-N,N-diethylthiocarbamate (DET-Me) which is formed from cytochrome P450 (CYP450). Assessing disulfiram in HIV-infected individuals with a CYP450 inducing drug (e.g., efavirenz) or a CYP450 inhibiting drug (e.g., HIV-1 protease inhibitors) requires an assay that can measure a metabolite that is formed directly via CYP450 oxidation. Therefore, an assay to measure concentrations of DET-Me in human plasma was validated. DET-Me and the internal standard, S-ethyldipropylthiocarbamate (EPTC) were separated by isocratic ultra performance liquid chromatography using a Waters Acquity HSS T3 column (2.1 mm × 100 mm, 1.8 μm) and detection via electrospray coupled to a triple quadrupole mass spectrometer. Multiple reaction monitoring in positive mode was used with DET-Me at 148/100 and the internal standard at 190/128 with a linear range of 0.500–50.0 ng/mL with a 5 min run time. Human plasma (500 μL) was extracted using a solid phase procedure. The interassay variation ranged from 1.86 to 7.74% while the intra assay variation ranged from 3.38 to 5.94% over three days. Representative results are provided from samples collected from subjects receiving daily doses of disulfiram 62.5 mg or 250 mg.
Keywords: Disulfiram; S-Methyl-N,N-diethylthiocarbamate; Ultra performance liquid chromatography; Mass spectrometry;

Sensitive and robust method for anabolic agents in human urine by gas chromatography–triple quadrupole mass spectrometry by Miguel A. Delgadillo; Lorena Garrostas; Óscar J. Pozo; Rosa Ventura; Benjamín Velasco; Jordi Segura; Josep Marcos (85-89).
► GC/QqQ instruments allow fast, sensitive and reliable detection of banned anabolics. ► 17α-methyl-5β-androstane-3α,17β-diol is routinely detected at 2 ng/mL in urine. ► Analysis of 1367 samples 2011 in the 2011 Pan American Games gave excellent results.A rapid, sensitive and robust gas chromatography–triple quadrupole mass spectrometry method was developed for the determination of seven anabolic agents in human urine. The selection of analytes includes the main metabolites of all anabolics with higher sensitivity requirements. After optimizing the fragmentation conditions for each compound, a validation procedure for qualitative analysis was performed. The selectivity of the method showed that no interfering peaks were observed at the retention time of the compound. Adequate intermediate precision, below 14%, was observed for all of the compounds at the lower concentration tested. The concentrations assayed were in accordance with the performance limits required by the World Anti-Doping Agency (WADA). Unlike a previously published GC/QqQ method, detection of 17α-methyl-5β-androstane-3α,17β-diol (the main metabolites of methyltestosterone) at 2 ng/mL was accomplished under routine conditions. The qualitative method was applied to the analysis of 1367 samples in the span of 2 weeks, as part of the doping control of the XVI Pan American Games which took place in Mexico (14th–30th October, 2011). The high sensitivity was maintained during the analysis of all analytical batches, proving for the first time the excellent ruggedness of GC/QqQ methods.
Keywords: Doping; Gas chromatography/tandem mass spectrometry; Human urine; Triple quadrupole; Anabolic agents;

Display Omitted► A method for determination of medroxyprogesterone in aquatic products was developed. ► 1,1,1,2-Tetrafluoroethane (R134a) is used as subcritical fluid. ► Response surface methodology (RSM) was employed to optimise the conditions of the extraction. ► High extraction efficiency could be obtained at low pressure and temperature. ► The proposed method was successfully applied to the real samples.A simple, rapid and sensitive method was developed for the determination of medroxyprogesterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) and high performance liquid chromatography (HPLC). A response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure, 3 MPa; temperature, 25 °C; and co-solvent volume, 6 ml. The analysis was carried out on Zorbax SB-C18 column (4.6 mm × 150 mm, 5 μm) with the mobile phase acetonitrile–water (55:45, v/v), flow rate 1.0 ml/min, temperature 30 °C and wavelength 240 nm. Good linearity of detection was obtained for medroxyprogesterone between concentrations of 50–250 ng/ml, r 2  = 0.999. The method was validated using samples fortified with medroxyprogesterone at levels of 10, 30 and 50 ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%.
Keywords: Determination; Subcritical R134a extraction; Medroxyprogesterone; Aquatic products;

Determination of bullatacin in rat plasma by liquid chromatography–mass spectrometry by Yong Chen; Jian-wei Chen; Shi-jia Liu; Chun-lei Xu; Hui-qing Xu; Bao-chang Cai; Xiang Li; Wen-zheng Ju (94-97).
Display Omitted► We developed a LC–MS method for the quantification of bullatacin in rat plasma. ► The method was fully validated. ► This method was successfully applied to pharmacokinetic studies in rats.A liquid chromatography–mass spectrometry method has been developed and validated for the quantification of bullatacin, a bistetrahydrofuran annonaceous acetogenin, in rat plasma. Squamostatin-A was selected as the internal standard. Analytes were extracted from rat plasma by liquid/liquid extraction using ethyl acetate with high efficiency. The chromatographical separation was performed on an Agilent Zorbax SB-C18 column (150 mm × 2.1 mm, 5 μm). The mobile phase consisted of methanol and deionized water (95:5, v/v) containing 0.01% (v/v) formic acid. The chromatographic run time was 7 min per injection and flow rate was 0.2 mL/min. The retention time was 3.22 and 5.23 min for internal standard and bullatacin, respectively. The elutes were detected under positive electrospray ionization and the target analytes quantified by selected ion monitoring mode (645.9  m/z for bullatacin and 661.9  m/z for squamostatin-A). The method was sensitive with the limit of quantitation at 0.5 ng/mL in 100 μL of rat plasma. Good linearity (r 2  = 0.9998) was obtained covering the concentration of 0.5–2000 ng/mL. The intra- and inter-day assay precision ranged from 3.2 to 8.7% and 2.7 to 9.2%, respectively. In addition, the stability, extraction recovery and matrix effect involved in the method were also validated. This method was applied to measure the plasma bullatacin concentrations after a single tail vein intravenous administration of bullatacin in rats.
Keywords: Bullatacin; Rat plasma; Liquid chromatography–mass spectrometry;

A rapid GC–MS method for quantification of positional and geometric isomers of fatty acid methyl esters by Josef Ecker; Max Scherer; Gerd Schmitz; Gerhard Liebisch (98-104).
► Rapid GC–MS method for fatty acid quantification (17 min). ► Separation of positional and geometric isomers including conjugated linoleic acids. ► Stable isotope labeling to profile fatty acid synthesis and metabolism.So far the most frequently used method for fatty acid (FA) analysis is GC coupled to flame ionization detector (FID). However, GC–FID does not allow profiling of FA synthesis and metabolism using stable isotopes. Here we present a rapid and sensitive GC–MS method for determination of fatty acid methyl esters (FAMEs). Fatty acid methylation was carried out by transesterification with acetyl-chloride and methanol. FAME separation applies a short and polar cyano-column resulting in an analysis time of 17.2 min. Separation was achieved for positional and geometrical (cis/trans) isomers with chain lengths between C8 and C28. Partial overlap of FAMEs (e.g. for C20:2 (n−6) and C21:0) could be resolved using selected ion monitoring (SIM). The precisions for human plasma samples were better than 10% coefficient of variation (CV) except for very low abundant FAs and LODs were in the low femtomol range on column. The developed GC–MS method also allows quantification of conjugated FAs such as conjugated linoleic acid (CLA) isomers because lowering the derivatization temperature from 95 °C to room temperature prevented cis to trans double bond isomerization. Finally, profiling of fatty acid synthesis and metabolism was exemplified with stable isotope labeling of macrophages using fatty acid precursors or deuterated fatty acids. In summary, we present a fast and robust GC–MS method for fatty acid profiling of positional and geometrical isomers including CLAs as well as very long chain fatty acids (VLCFAs). The method is suitable for both clinical studies and basic research including application of stable isotope compounds.
Keywords: GC–MS; Fatty acid; Conjugated linoleic acid; Stable isotope; Metabolism;