Journal of Chromatography B (v.907, #C)

Determination of glutamate uptake by high performance liquid chromatography (HPLC) in preparations of retinal tissue by Edinaldo Rogério da Silva Moraes; Alan Barroso Araújo Grisolia; Karen Renata Matos Oliveira; Domingos Luiz Wanderley Picanço-Diniz; Maria Elena Crespo-López; Caio Maximino; Evander de Jesus Oliveira Batista; Anderson Manoel Herculano (1-6).
► Michaelis–Menten kinetics was observed in the glutamate uptake by HPLC analysis. ► Glutamate uptake determination by HPLC was Na+ and temperature-dependent. ► GLAST inhibitor evokes a decrease concentration-dependent on the glutamate uptake.The present study describes a simple and efficient method utilizing high performance liquid chromatography (HPLC) coupled to fluorescence detection for the determination of kinetic parameters of glutamate uptake in nervous tissue. Retinal tissue obtained from 7-day-old chicks was incubated with known concentrations of glutamate (50–2000 μM) for 10 min, and the levels of the o-phtaldehyde (OPA)-derivatized neurotransmitter in the incubation medium were measured. By assessing the difference between initial and final concentrations of glutamate in the medium, a saturable uptake mechanism was characterized (K m  = 8.2 and V max  = 9.8 nmol/mg protein/min). This measure was largely sodium- and temperature-dependent, strongly supporting that the mechanism for concentration decrements is indeed uptake by high-affinity transporters. Added to this, our results also demonstrated that zinc chloride (an inhibitor of glutamate/aspartate transporters) evoked a concentration-dependent decrease in glutamate uptake, demonstrating the specificity of our methodology. Overall, the present work characterizes an alternative methodology to evaluate glutamate uptake in nervous tissue using HPLC. This approach could be an important tool for studies associated to the characterization of minute alterations in glutamate transport related with central nervous system injury.
Keywords: Glutamate uptake; HPLC; Retina; Transport;

► Urinary metabolomics method was used to investigate the effect of ginseng polysaccharide on T2DM rats. ► Eight potential biomarkers were found and identified. ► Significant changes of the biomarkers indicated the therapy effect mechanism of ginseng polysaccharide on T2DM rats.Ginseng polysaccharide is known to have anti-hyperglycemic and anti-hyperlipidemic effects in vivo and its precise mechanism of action is not clear. A urinary metabolomics method based on rapid-resolution liquid chromatography/mass spectrometry (RRLC/MS) was developed to investigate the effect of water-soluble ginseng polysaccharide (WGP) on type 2 diabetes in rats. Principal component analysis (PCA) was carried out for pattern recognition and a clear separation between type 2 diabetic rats and those treated with WGP was achieved. Eight potential biomarkers were found and identified. Significantly increased inosine, serotonin, phenylpropionylglycine and dodecanedioic acid showed the effect of WGP on purine metabolism, tryptophan metabolism, fatty acid metabolism and energy metabolism. 1-Methyladenine, 4-deoxyerythronic acid, 5-hydroxyhexanoic acid and tetrahydrocortisol were significantly decreased which indicated that WGP can regulate DNA metabolism, organic acids metabolism and steroid hormone metabolism. This work is helpful in the effect mechanism study of ginseng polysaccharide.
Keywords: Type 2 diabetes; Metabolomics; RRLC/MS; Water-soluble ginseng polysaccharide;

► A LC–MS/MS for measuring thymidine kinase activity in human serum is described. ► Phosphorylation of 3′-deoxy-3′-fluorothymidine indicates activity. ► The monophosphorylated product is measured quantitatively. ► The method uses protein precipitation and on-line column switching. ► The method was FDA validated and applied to hepatocellular carcinoma patients.Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis whose activity in serum is indicative of tumor proliferation and the severity of blood malignancies. 3′-deoxy-3′-fluorothymidine (FLT), a specific exogenous substrate for TK1, is phosphorylated by TK1 in the presence of a phosphorylating buffer, therefore the conversion of FLT to 3′-deoxy-3′-fluorothymidine monophosphate (FLT-MP) can be measured to assess serum TK1 activity. Here we describe a liquid chromatography–MS/MS (LC–MS/MS) method for quantification of FLT and FLT-MP from serum using protein precipitation and column switching followed by detection on an Applied Biosystems SCIEX API 4000 QTrap mass spectrometer. The method was linear over the range of 0.5–500 ng/mL for FLT and 2.5–2000 ng/mL for FLT-MP with a mean correlation coefficient of 0.9964 and 0.9935 for FLT and FLT-MP, respectively. The lower limit of quantification was 0.5 ng/mL for FLT and 2.5 ng/mL for FLT-MP. Intra-assay accuracy and inter-assay accuracy was within ±12% for both FLT and FLT-MP. Intra-assay precision was 2.8% to 7.7% for FLT and 3.3% to 5.8% for FLT-MP. Inter-assay precision was 4.6% to 14.9% for FLT and 4.9% to 14.6% for FLT-MP. Serum TK1 activity was measured in serum from hepatocellular carcinoma patients and age-matched controls under standardized conditions. Elevated TK1 activity was detected in 26.3% of hepatocellular carcinoma samples compared to controls. This method provides a robust alternative to radiometric and immunochemical assays of serum TK1 activity.
Keywords: Serum thymidine kinase 1 (sTK1) assay; 3′-Deoxy-3′-fluorothymidine (FLT); 3′-Deoxy-3′-fluorothymidine monophosphate (FLT-MP); LC–MS/MS; Column switching;

► This UPLC/Q-TOF-MS method is rapid, simple and suitable for pharmacokinetic analysis. ► The absorption promoter C10 could enhance the oral absorption of daidzein. ► It has been identified the pharmacokinetic behavior of three daidzein formulations.An ultra-performance-liquid-chromatography–quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method was developed and validated for the quantitation of daidzein (DZ) in rat plasma. Ethylparaben was chosen as internal standards (IS). DZ was linear over the range of 0.001–5 μg/mL. The lower limit of quantification (LLOQ) was 0.001 μg/mL and the limit of detection (LOD) was 0.0005 μg/mL. The intra-day and inter-day relative standard deviations (RSDs) were ranged from 3.59% to 6.43% and 5.35% to 7.25%, respectively. This UPLC/Q-TOF-MS method provided good specifity, highly sensitivity, accurate and high-speed detection (6 min), applicable to the pharmacokinetics study in rats in vivo after oral administration of free daidzein solution, daidzein-loaded poly (lactide-co-glycolide) (PLGA) nanoparticles (D-NPs) suspension and D-NPs co-administered with sodium caprate (C10) which as the oral absorption promoter. It was shown that the pharmacokinetics behavior was significantly improved after the oral administration of D-NPs suspension co-administered with absorption promoter C10 by the fact that the relative bioavailability were enhanced about 4.24-fold, compared to that of DZ suspension.
Keywords: Ultra-performance-liquid-chromatography–quadrupole-time-of-flight mass-spectrometry; Daidzein; Nanoparticles; Pharmacokinetics; Sodium caprate;

Simultaneous determination of esculin and its metabolite esculetin in rat plasma by LC–ESI-MS/MS and its application in pharmacokinetic study by Ying-yi Li; Ye-ying Song; Chang-hui Liu; Xiao-tao Huang; Xia Zheng; Neng Li; Mei-li Xu; Sui-qing Mi; Ning-sheng Wang (27-33).
► A LC–MS/MS method for determination of esculin and esculetin in rat plasma is firstly reported. ► The LLOQ of esculin and its metabolite esculetin is 0.25 and 1.25 ng/mL, respectively. ► The evaluation of esculetin and aesculin with a total running time is 2.5 min per sample.A new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method operated in the negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of esculin and its metabolite esculetin in rat plasma. After addition of internal standards scopoletin, the plasma sample was pretreated by solid-phase extraction (SPE), and separated on a reversed phase C18 column with a mobile phase of 0.01% formic acid in water (solvent A) and methanol (solvent B) using isocratic elution (A:B = 20:80, v/v). The detection of target compounds was done in multiple reaction monitoring (MRM) mode. The MRM detection was operated in the negative ESI mode using the transitions of m/z 339.1 ([M−H]) → 176.7 for esculetin, m/z 176.9 ([M−H]) → 133.0 and m/z 191.0 ([M−H]) → 175.9 for scopoletin. The standard curves, which ranged from 25 to 3200 ng/mL for esculin with the lowest limit of quantification (LLOQ) of 0.25 ng/mL and from 1.25 to 160 ng/mL for esculetin with the LLOQ of 1.25 ng/mL, were fitted to a 1/x weighted quadratic regression model. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD < 8.73%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to study the pharmacokinetics of esculin and its metabolite esculetin in rat plasma after oral administration of esculin at a dose of 100 mg/kg.
Keywords: Esculin; Esculetin; LC–ESI-MS/MS; Pharmacokinetic; Rat plasma;

► Simultaneous analysis of mannitol, lactulose and sucrose. ► Rapid sample preparation. ► Estimation of upper digestive tract and intestinal permeability at single analysis. ► Comparison of three analytical columns.A new analytical procedure was described for the simultaneous determination of lactulose, mannitol and sucrose in urine, in which HILIC chromatography and tandem mass spectrometry detection are used. Sugars are orally administered for the estimation of intestinal permeability in children digestive tract. Samples were purified by dispersive solid phase extraction (d-SPE) using Amberlite MB150 resin. Raffinose was selected as an internal standard. The chosen chromatographic separation was carried out on ZIC®-HILIC column in 10 min at a flow rate of 0.3 mL/min, using mixture of acetonitrile (ACN) and ammonium acetate (NH4Ac) in water (H2O) as the mobile phase. Within-run precision (CV) measured at three concentrations was 1.08%, 0.32% and 0.49% for lactulose; 1.88%, 0.47% and 0.75% for mannitol, 2.95%, 1.31% and 0.6% for sucrose. Between-run CVs were 0.75%, 1.1% and 1.2% for lactulose; 1.1%, 1.02% and 1.01% for mannitol; 1.17%, 1.4% and 1.05% for sucrose. Analytical recovery of all three sugar probes was 95.06–99.92%. The detection limits were: 15.94 ng/mL for lactulose, 17.10 ng/mL for sucrose and 11.48 ng/mL for mannitol. The proposed method is rapid, simple, sensitive and suitable for the determination of intestinal permeability of the sugar derivatives in children.
Keywords: HILIC; Intestinal permeability; Tandem mass spectrometry; Carbohydrate analysis; Urine;

► An LC/MS/MS method was developed for determination of MTXPGs in erythrocyte. ► It's the first study using LC/MS/MS with liquid–liquid extraction to detect MTXPGs. ► The levels of MTXPGs are generated indirectly. ► The method is sensitive, simple, low cost and can be widely applied in many labs. ► The method has been applied to determine the concentration of MTX in patients with RA.Methotrexate (MTX) is currently one of the most widely used drugs for treatment of rheumatoid arthritis (RA) through polyglutamation of methotrexate polyglutamates (MTXPGs), a process attaching sequential γ-linked glutamic residues to MTX. A new and sensitive LC/MS/MS method was developed and validated for determination of whole-blood MTX and total MTX (MTX + MTXPGs), and then concentration of MTXPGs was calculated. To determine whole-blood MTX, whole blood was precipitated with 50% trifluoroacetic acid, and extraction was performed using ethyl acetoacetate. Analytes were subjected to LC/MS/MS analysis using positive electrospray ionization. To determine whole-blood total MTX, whole blood was incubated with ascorbic acid (200 mM) at 37 °C for 3 h to enzymatically convert the MTXPGs to MTX, and then processed with the same method mentioned above. Recoveries of spiked MTX at ppb (ng/mL) level were between 26.2% and 37.8% with intra- and inter-day precision less than 15.8% and 11.8%, respectively. The lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were 0.5 ng/mL and 1 ng/mL, respectively. The sensitive LC/MS/MS method was fully validated with high selectivity and acceptable accuracy and precision, which was successfully applied to determine the erythrocyte methotrexate polyglutamates in patients with RA.
Keywords: Methotrexate (MTX); Methotrexate polyglutamates (MTXPGs); LC/MS/MS; Liquid–liquid extraction; Rheumatoid arthritis;

► First MLC method for determination of zopiclone and 2-amino-5-chloropyridine (ACP). ► The method is highly sensitive for ACP down to 2.5 ng/mL. ► The method was applied for purity testing of zopiclone within the BP limit. ► The method was extended to determine ACP in urine as a marker for zopiclone intake. ► No pretreatment steps are required due to the solubilizing ability of the micelles.A simple and reliable HPLC method was developed and validated for the simultaneous determination of the hypnotic drug, zopiclone (ZPC) and its degradation product and main impurity, 2-amino-5-chloropyridine (ACP). The analyses were carried out on BDS Hypersil phenyl column (4.6 mm × 250 mm, 5 μm particle size) using micellar mobile phase consisting of 0.15 M SDS, 10% n-propanol, 0.3% triethylamine, and 0.02 M orthophosphoric acid (pH 3.5) with timed programmable fluorescence detection. The proposed method was found to be rectilinear over the concentration ranges of 0.5–10.0 μg/mL for ZPC and 2.5–50 ng/mL for ACP. Moreover, the method was applied for the determination of ZPC in commercial tablets with mean percentage recovery of 99.06 ± 1.49. The results of the proposed method were statistically compared with those obtained by the comparison method revealing no significance differences in the performance of the two methods regarding accuracy and precision. Furthermore, the proposed method was applied for the detection and determination of ACP in human urine as a marker for ZPC intake.
Keywords: Zopiclone; MLC; Stability-indicating assay; Human urine;

► Simple, rapid, sensitive and environmentally benign method for quantitative determination of amino acids in biological and food samples. ► Simultaneous extraction, concentration and sample introduction in one single solvent free step. ► In-matrix derivatization in aqueous media without any lyophilization and pre-purification steps. ► Application of the developed method for the simultaneous determination of 20 amino acids in hair, soybean and urine samples.Amino acids play a vital role as intermediates in many important metabolic pathways such as the biosynthesis of nucleotides, vitamins and secondary metabolites. A sensitive and rapid analytical method has been proposed for the first time for the simultaneous determination of twenty amino acids using solid-phase microextraction (SPME). The protein samples were hydrolyzed by 6 M HCl under microwave radiation for 120 min. Then the amino acids were derivatized by ethyl chloroformate (ECF) and the ethoxy carbonyl ethyl esters of amino acids formed were extracted using SPME by direct immersion. Finally the extracted analytes on the SPME fiber were desorbed at 260 °C and analyzed by gas chromatography–mass spectrometer (GC–MS) in electron ionization mode. Factors which affect the SPME efficiency were screened by Plackett–Burmann design; most significant factors were optimized with response surface methodology. The optimum conditions for SPME are as follows: pH of 1.7, ionic strength of 733 mg, extraction time of 30 min and fiber of divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The recovery of all the amino acids was found to be in the range of 89.17–100.98%. The limit of detection (LOD) of all derivatized amino acids in urine, hair and soybean was found to be in the range of 0.20–7.52 μg L−1, 0.21–8.40 μg L−1 and 0.18–5.62 μg L−1, respectively. Finally, the proposed technique was successfully applied for the determination of amino acids in complex biological (hair, urine) and food samples (soybean). The method can find wide applications in the routine analysis of amino acids in any biological as well as food samples.
Keywords: Amino acids; Direct immersion-solid-phase microextraction; Ethyl chloroformate derivatization; Gas chromatography–mass spectrometry;

Dried blood spot analysis of an iron chelator – Deferasirox and its potential application to therapeutic drug monitoring by Ramakrishna Nirogi; Devender Reddy Ajjala; Vishwottam Kandikere; Raghupathi Aleti; SuryaRao Srikakolapu; Himabindu Vurimindi (65-73).
► Deferasirox is an iron-chelator used in treating the transfusional iron overload. ► Therapeutic drug monitoring of deferasirox was required to titrate dosage for its efficacy/toxicity. ► Quantification of deferasirox was performed by LC–MS/MS using dried blood spot (DBS) approach. ► Pharmacokinetic parameters obtained in DBS method were compared with that of the whole blood method. ► Feasibility to translate method developed in rat DBS to human DBS was checked. ► Fragmentation pathway of deferasirox was described similar to 1,2,4-Triazoles.Deferasirox is an iron chelating agent for the treatment of transfusional iron over load in patients with chronic anemia. These anemic patients require close monitoring of the deferasirox exposures for ensuring its therapeutic efficacy. Dried blood spot (DBS) sampling methodology has the advantages of low volume of blood withdrawal and ease of transportation and storage over liquid blood methods. A LC–MS/MS based analytical method was developed using reversed phase column with gradient elution program and quantitated in MRM mode. Linearity range for the liquid blood was 1–1000 ng/mL and for DBS was 5–5000 ng/mL under similar mass spectrometry conditions. The method was validated with respective (M−H) ions, m/z 372→118 for deferasirox and m/z 410→348 for fluvastatin (internal standard). The validated method was applied for the analysis of DBS samples from a rat pharmacokinetic study and results were compared against liquid blood samples from the same animal. The mean C max from DBS sample (1121 ng/mL) was comparable to mean C max found in blood samples (1015 ng/mL) at 2 h after oral dose of deferasirox. All the other calculated pharmacokinetic parameters were quite comparable for both liquid blood and DBS samples.
Keywords: Deferasirox; Iron chelator; Iron overload; Liquid chromatography–tandem mass spectrometry; Dried blood spot (DBS); FTA-DMPK-C;

Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection by Thi Thanh Thuy Pham; Hong Heng See; Réjane Morand; Stephan Krähenbühl; Peter C. Hauser (74-78).
► CE-C4D allows detection of the non-UV absorbing valproic acid without requiring derivatization. ► Differentiation between free and protein-bound valproic acid was achieved by simple ultrafiltration. ► An extraction procedure allows the determination without matrix interference. ► The method is suitable for pediatric patients because only small sample volumes are required.A new approach for the determination of free and total valproic acid in small samples of 140 μL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid–liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100 μL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40 μL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10 mM histidine, 10 mM 3-(N-morpholino)propanesulfonic acid and 10 μM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4–300 μg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08 μg/mL, and the reproducibility of the peak area was excellent (RSD = 0.7–3.5%, n  = 3, for the concentration range from 1 to 150 μg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.
Keywords: Dispersive liquid–liquid microextraction; Capillary electrophoresis; Contactless conductivity detection; Valproic acid; Human plasma;

Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis by Kelong Wang; Dechen Jiang; Christopher E. Sims; Nancy L. Allbritton (79-86).
► Capillary electrophoresis was used to separate phosphoinositides and sphingolipids. ► A separation buffer based on NaH2PO4 and 1-propanol was optimized. ► Theoretical plate numbers were up to 2 × 105 for the lipids. ► Detection limits for the six lipid analytes were between 10−18 and 10−20  mol. ► Lipid metabolites from single cells were detected.Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2 × 105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10−18–10−20  mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.
Keywords: Capillary electrophoresis; Sphingolipid; Phosphoinositide lipid; Phospholipid separation;

LC–MS analysis of glycopeptides of recombinant monoclonal antibodies by a rapid digestion procedure by Yi Du; Fengqiang Wang; Kimberly May; Wei Xu; Hongcheng Liu (87-93).
► A rapid digestion procedure was established to digest antibodies for glycopeptide analysis. ► The rapid digestion procedure can be used in combination with dimethyl labeling. ► In-source fragmentation of glycopeptides was highly dependent on the charge state.N-glycan analysis of recombinant monoclonal antibodies (mAbs) usually requires the removal of oligosaccharides by PNGase F followed by 2-AB labeling, normal phase high performance liquid chromatography (NP-HPLC) separation and fluorescence detection. Alternatively antibodies can be completely digested by trypsin to generate glycopeptides for analysis by liquid chromatography–mass spectrometry (LC–MS). Here, we report the development of a rapid digestion procedure to generate glycopeptides for quantitative LC–MS analysis. Recombinant monoclonal antibodies were digested using a combination of Lys-C and trypsin at 37 °C for 15 min. The glycan profiles from this rapid digestion procedure are in good agreement with those from LC–MS analysis of glycopeptides from completely digested antibodies and those from NP-HPLC analysis of 2-AB labeled PNGase F released oligosaccharides. This rapid digestion procedure was applied to the comparison of oligosaccharides of two different antibodies. Glycopeptides from the two antibodies were differentially labeled with stable isotopes and analyzed simultaneously after a 1:1 mixing. The combination of the rapid digestion procedure and differential stable isotope labeling significantly reduced the turnaround time.
Keywords: Monoclonal antibody; Glycopeptide; Mass spectrometry;

Determination of unbound fraction of imatinib and N-desmethyl imatinib, validation of an UPLC–MS/MS assay and ultrafiltration method by Cécile Arellano; Peggy Gandia; Thierry Lafont; Rutchanna Jongejan; Etienne Chatelut (94-100).
► Unbound imatinib and N-desmethyl imatinib are separated by ultrafiltration. ► Ultrafiltration process is optimized to give reproducible values for free fractions. ► Total and unbound fractions of imatinib and desmethyl imatinib were assayed by UPLC–MS. ► The validation for the UPLC–MS/MS quantification method is reported.Imatinib is a small-molecule tyrosine kinase inhibitor with large inter-individual but low intra-individual pharmacokinetic variability with consistent concentration–efficacy and concentration–toxicity relationships. For these reasons imatinib therapeutic drug monitoring is based on total plasma concentrations. However, since a significant impact of unbound imatinib concentrations on clinical response and/or toxicity evaluation has been suggested, the quantification of free fraction of imatinib and its active metabolite are of interest for therapeutic monitoring. Hence a reliable method for both separation and assay of the free fraction is needed. Using plasma samples spiked with imatinib (from 1000 to 7500 ng/mL) and its metabolite (from 1000 to 2500 ng/mL), an ultrafiltration procedure and an UPLC assay which give reproductive values for unbound fractions of imatinib (mean 3.0 ± 1.0%) and metabolite N-desmethyl imatinib (3.6 ± 1.8%) have been developed. The validation of the analytical UPLC–MS/MS method associated to ultrafiltration for quantification of imatinib and N-desmethyl imatinib was reported. The LOQ was set at 10 ng/mL for imatinib and 20 ng/mL for N-desmethyl imatinib, intraday CV (%) ranged from 2.7 to 4.8% for imatinib and from 5.4 to 12.4% for N-desmethyl imatinib and interday CV (%) ranged from 5.6 to 6.5% for imatinib and from 5.4 to 16.1% for N-desmethyl imatinib. Methodological modifications were attempted to overcome non specific binding (NSB) on the ultrafiltration device. Two types of devices previously used for unbound determination of drugs were tested. Our results clearly showed that the methodology and the features of devices used for ultrafiltration could totally compromise the determination of unbound concentrations of a drug.
Keywords: Imatinib; N-desmethyl imatinib; Unbound fraction; Ultrafiltration; UPLC–MS/MS;

Liquid chromatography–tandem mass spectrometry method for determination of five antidepressants and four atypical antipsychotics and their main metabolites in human serum by Romana Uřinovská; Hana Brozmanová; Pavel Šištík; Petr Šilhán; Ivana Kacířová; Karel Lemr; Milan Grundmann (101-107).
► The determination of nine antipsychotics in human serum. ► simultaneous measurement of parent drugs and main metabolites. ► This method can be used for fenotyping. ► Method was fully validated and can be successfully applied for routine analyses.The rapid and simple ultra performance liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous determination parent drugs: sertraline, fluoxetine, citalopram, paroxetine, venlafaxine, clozapine, olanzapine, quetiapine, risperidone, and their active and nonactive metabolites N-desmethylsertraline, norfluoxetine, desmethylcitalopram, didemethylcitalopram, N-desmethylvenlafaxine, O-desmethylvenlafaxine, N-desmethylclozapine, N-desmethylolanzapine, 2-hydroxyolanzapine and 9-hydroxyrisperidone in human serum. Precipitation of serum proteins was performed with a precipitation reagent consisting of 0.05% solution of ZnSO4·7H2O in acetonitrile/methanol (40:60, v/v). Alprenolol was used as an internal standard. Chromatographic separation was carried out on a BEH C18 column using gradient elution mobile phase A (2 mmol/L ammonium acetate, 0.1% formic acid in 5% acetonitrile, v/v/v) and B (2 mmol/L ammonium acetate, 0.1% formic acid in 95% acetonitrile, v/v/v). Electrospray in positive mode was used for ionization. Detection was performed on a triple–quadrupole tandem mass spectrometer by multiple reaction monitoring. Analysis time was 5 min. Drugs were separated into three groups with low, medium and high levels. Correlation coefficients of calibration curves were in the range 0.995–1.000. Coefficients of variation were 4.2–9.5% for intra-assay and 3.0–11.9% for inter-assay. Recoveries were 87.1–110% for intra-assay and 88.1–108.2% for inter-assay. The method was fully validated and can be successfully applied for routine analyses.
Keywords: Psychotropic drugs; Therapeutic drug monitoring; High performance liquid chromatography; Mass spectrometry;

A rapid validated UHPLC–PDA method for anthocyanins quantification from Euterpe oleracea fruits by A.L.S. Dias; E. Rozet; G. Chataigné; A.C. Oliveira; C.A.S. Rabelo; Ph. Hubert; H. Rogez; J. Quetin-Leclercq (108-116).
► A first EtOAc extraction of EOF allows a better anthocyanins extraction by MeOH/HCl. ► 7 anthocyanins were separated in only 10 min using UHPLC–PDA. ► cy3glu and cy3rut quantification method was validated: dosing range 1–48 μg/mL. ► Calibrations in the matrix were used for the quantifications. ► Accuracy profiles and total errors were used as validation criteria.The aim of this work is to develop the first validated UHPLC–PDA method for major anthocyanins quantification in Euterpe oleracea fruits after fast extraction procedures and samples preparation. The separation was performed on HSS C18 column (1.8 μm) using a gradient elution with acetonitrile and 5% formic acid in a total run time of only 17 min. Total error and accuracy profiles were used as criteria for the validation process. Calibration in the matrix was found to be more accurate than calibration without matrix. Trueness (<6.76% relative bias), repeatability (<4.6% RSD), intermediate precision (<5.3% RSD), selectivity, response function and linearity for major anthocyanins, cyanidin-3-glucoside and cyanidin-3-rutinoside, were evaluated. The concentration range validated was 1–48 μg/mL for both compounds. In addition two cyanidin-di-O-glycosides were detected for the fist time in this fruit. We also showed that a first extraction of the fruits with ethyl acetate removes the lipophilic compounds and allows an easier extraction by methanol and quantification of anthocyanins in this extract.
Keywords: Euterpe oleracea; Major anthocyanins; UHPLC; Quantification; Method validation;

► Development of HILIC tandem MS method. ► Optimization of extraction process for multiple analytes. ► Validation of method. ► Application of method in rat model.In this work, we developed a sensitive method to quantify cotinine (COT), norcotinine (NCOT), trans-3′-hydroxycotinine (OHCOT) and cotinine-N-oxide (COTNO) in rat plasma and brain tissue, using solid phase extraction (SPE), hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The linear range was 1–100 ng/mL for each analyte in rat plasma and brain homogenate (3–300 ng/g brain tissue). The method was validated with precision within 15% relative standard deviation (RSD) and accuracy within 15% relative error (RE). Stable isotope-labeled internal standards (IS) were used for all the analytes to achieve good reproducibility, minimizing the influence of recovery and matrix effects. This method can be used in future studies to simultaneously determine the concentrations of COT and three major metabolites in rat plasma and brain tissue.
Keywords: Cotinine; Norcotinine; Trans-3′-hydroxycotinine; Cotinine-N-oxide; Plasma; Brain; Hydrophilic interaction chromatography; Tandem mass spectrometry;

► Mouse urinary metabolic profile of thiol-conjugated [6]-shogaol was established. ► Sixteen phase II metabolites of thiol-conjugated [6]-shogaol were identified. ► Thiol conjugated [6]-shogaol are the substrates for glucuronidation and sulfation.Ginger is frequently consumed as a spice and has numerous medicinal properties. Extensive research has characterized the anti-inflammatory, antioxidant, and antitumor activities of ginger. Previously, we reported the mercapturic acid pathway as a major metabolic route of [6]-shogaol in mice and the thiol conjugates of [6]-shogaol existed in the glucuronidated and sulfated forms in mouse urine. However, their structures are still unknown. In the present study, we further investigated the phase II metabolism of thiol-conjugated [6]-shogaol in mouse urine, in which we identified sixteen phase II metabolites of thiol-conjugated [6]-shogaol: 5-cysteinyl-[6]-shogaol glucuronide (9), 5-N-acetylcysteinyl-[6]-shogaol glucuronide (10), 5-cysteinylglycinyl-[6]-shogaol glucuronide (11), 5-methylthio-[6]-shogaol glucuronide (12), 5-cysteinyl-M6 glucuronide (13 and 14), 5-cysteinyl-M6 sulfate (15 and 16), 5-N-acetylcysteinyl-M6 glucuronide (17 and 18), 5-cysteinylglycinyl-M6 glucuronide (19 and 20), 5-cysteinylglycinyl-M6 sulfate (21 and 22), and 5-methylthio-M6 glucuronide (23 and 24) using liquid chromatography/electrospray ionization tandem mass spectrometry. The structures of these metabolites were confirmed by analyzing their MS n (n  = 1–4) spectra as well as comparing with the tandem mass spectra of authentic standards. To the best of our knowledge, this is the first report involving identification of phase II urinary metabolites of [6]-shogaol in mice.
Keywords: [6]-Shogaol; Thiol-conjugated phase II metabolites; HPLC–MS/MS; Mouse urine;

► We developed two chiral RP-HPLC methods to quantify naftopidil enantiomers in plasma. ► Both of the methods were validated and met the demands of bioanalysis. ► The CSPs method was more suitable for pharmacokinetics study of (+)- and (−)-NAF. ► We determined (+)- and (−)-NAF after intravenous injection of (±)-NAF by CSPs HPLC.Two bioanalytical HPLC methods (chiral solid phases (CSPs) HPLC and pre-column derivatization HPLC) were developed and validated for the determination of naftopidil enantiomers in rat plasma. Analytes were extracted from biomaterials by liquid–liquid extraction. The pre-column derivatization HPLC method employed (+)-diacetyl-l-tartaric anhydride (DATAN) as the pre-column derivatization reagent, and subsequent separation of diastereomers was conducted on an Agilent Hypersil ODS column with a mixture of methanol–acetonitrile–phosphate buffer (pH 4.1; 20 mM) (40:30:30, v/v/v) flowing at 1 mL/min as the mobile phase. The CSPs HPLC method utilized a Chiralpak IA column with a mobile phase of methanol–acetonitrile–acetate buffer (pH 5.3; 5 mM) (50:25:25, v/v/v) flowing at 0.5 mL/min. In both methods, the analytes were monitored using a fluorescence detector with an excitation wavelength of 290 nm and an emission wavelength of 340 nm. Both methods were consistent (RSD < 15% by the derivatization method and < 10% by the CSPs method) and linear (r  > 9950). Compared to the pre-column derivatization method, the CSPs method had lower quantification limits (10.6/9.6 ng/mL of (+)-/(−)-naftopidil by derivatization method and 1.1/1.8 ng/mL of (+)-/(−)-naftopidil by CSPs method), and was simpler to carry out. The validated CSPs method was successfully applied in a pharmacokinetic study of naftopidil enantiomers in rats, which showed that pharmacokinetic parameters of (+)- and (−)-NAF after intravenous administration of (±)-NAF were similar.
Keywords: Naftopidil; Pre-column derivatization; Chiral column; Enantioselective pharmacokinetics;

► XCMS and UPLC–MS were used to discover the metabolites of helicidum in rat urine. ► XCMS is open source software and easily customized for different data analysis. ► Nine metabolites were identified and quantified. ► Oxidation was the major bio-reaction occurred in vivo.
Keywords: Helicidum; Metabolites; Urine; UPLC/TOF MS; XCMS;

A routine method for cholesterol and 7-dehydrocholesterol analysis in dried blood spot by GC–FID to diagnose the Smith–Lemli–Opitz syndrome by Monica Gelzo; Stefano Clericuzio; Rosalba Barone; Oceania D’Apolito; Antonio Dello Russo; Gaetano Corso (154-158).
► A fast and simple method to quantify CHOL, DHC, in DBS to diagnose SLOS is reported. ► We developed and validated a method to analyze underivatized sterols by GC–FID. ► Analysis of CHOL, 7-DHC, and 8-DHC, has been performed on DBS disc of 6 mm. ► BHT-treated paper for QC preparation permitted to stabilize sterols in DBS for 8 weeks.This work was aimed to implement a fast and simple method to quantify cholesterol (CHOL) and 7-dehydrocholesterol (7-DHC) in dried blood spot (DBS) to diagnose the Smith–Lemli–Opitz syndrome (SLOS), an inborn error of CHOL biosynthesis. We developed and validated a GC–FID method for separation and quantification of underivatized CHOL and 7-DHC using a DBS disc of 6 mm with a run time of 9 min. Correlation coefficients (r) of calibration curves ranged from 0.998 to 0.999 for CHOL and from 0.997 to 0.998 for 7-DHC. Within-day and between-day imprecision (CV%), accuracy (%), carry-over, and extraction efficacy (%) were also evaluated for validation. CHOL and 7-DHC were analyzed in DBS and plasma samples from 8 SLOS patients and 30 unaffected subjects. In SLOS patients, 7-DHC/CHOL ratios in DBS and plasma samples ranged from 0.035 to 1.448 and from 0.012 to 0.926, respectively. Results from calibration curves, quality controls and patient samples reveal that the method is suitable to analyze DBS to screen patients affected by SLOS.
Keywords: DBS; GC–FID; SLOS;

► SUMO protease was fused with a poly lysine tag at the C-terminus. ► Lysine tagged SUMO protease was purified by weak cation-exchangers. ► Target protein purification by magnetite particles had high purity. ► Magnetic particles had a higher adsorption loading than classical cation-exchanger.A fusion tag that can be purified by the cheap ion-exchanger based on the ionic binding force may provide a cost-effective scheme over other affinity fusion tags. Small ubiquitin-like modifier (SUMO) protease derived from Saccharomyces cerevisiae was fused with a poly lysine tag containing 10 lysine residues at its C-terminus and then expressed in Escherichia coli. The ionic binding force provided by the ploy lysine tag allowed the selective recovery of the small ubiquitin-like modifier protease from recombinant E. coli cell extracts. A preliminary comparative study of the adsorption and elution of poly lysine tagged SUMO protease on Amberlite Cobalamion and magnetite carboxymethyl chitosan nanoparticles was performed. Amberlite Cobalamion and magnetite nanoparticles had the similar elution profile due to the common functional groups – carboxyl groups. The maximum dynamic adsorption capacity of Amberlite Cobalamion and magnetite nanoparticles reached 36.8 and 211.4 mg/g, respectively. The lysine-tagged protease can be simply purified by magnetite nanoparticles from cell extracts with higher purity than that by Amberlite Cobalamion. The superparamagnetic nanoparticles possess the advantages of highly specific, fast and excellent binding of a larger amount of lysine tagged SUMO modifier protease, and it is also easier to separate from the crude biological process liquors compared with the conventional separation techniques of polycationic amino acids fusion proteins.
Keywords: Poly-lysine tag; Ion exchange; Magnetic particles; Small ubiquitin-like modifier protease; Carboxymethyl chitosan;

Quantification of limonin in human urine using solid-phase extraction by LC–MS/MS by Shijia Liu; Jun Zhang; Ling Zhou; Boyang Yu; Changyin Li; Zixiu Liu; Wenzheng Ju (163-167).
► This new method has been successfully applied to the evaluation of limonin in human urine after oral administration for the first time. ► The lower limit of quantification (LLOQ) of developed LC–MS/MS method was lower than the previous report. ► The method is simple, sensitive and specific, with a short analysis time (5 min). ► The work presented here used a solid-phase (SPE) extraction.A highly sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of limonin in human urine using podophyllotoxin as internal standard. The analyte and IS were extracted with solid-phase extraction and separated by a rapid isocratic elution with 1% formic acid/methanol (v:v, 40:60) on an C18 column (150 mm × 2.1 mm I.D.). The detection was performed by mass spectrometry in the multi-reaction-monitoring mode. The precursor to product ion transitions of m/z 471.3 → 161.2 and m/z 397.2 → 313.1 were used to measure the analyte and the IS. The assay was linear over the concentration range of 0.0783–10 ng/mL for limonin in human urine. The lower limit of quantification was 0.0783 ng/mL and the extraction recovery was larger than 76.7% for limonin. The inter- and intra-day precision of the method at three concentrations was less than 7.4%. The method was successfully applied to pharmacokinetic study of limonin in humans.
Keywords: Limonin; LC–MS/MS; SPE; Electrospray ionization; Human urine;

Analytical methodology for the study of endosulfan bioremediation under controlled conditions with white rot fungi by Anisleidy Rivero; Silvina Niell; Verónica Cesio; M. Pía Cerdeiras; Horacio Heinzen (168-172).
► Endosulfan dissipation/metabolites formation determined in a single analytical step. ► The validated method ensures the accurate evaluation of the bioremediation technique. ► Bjerkandera adusta showed 83% of endosulfan bioconversion after 27 days. ► The most toxic metabolite (endosulfan sulfate) was produced at a minimum level. ► Other metabolites (endosulfan diol and endosulfan ether) were detected.A general procedure to study the biodegradation of endosulfan under laboratory conditions by white rot fungi isolated from native sources growing in YNB (yeast nitrogen base) media with 1% of glucose is presented. The evaluation of endosulfan biodegradation as well as endosulfan sulfate, endosulfan ether and endosulfan alcohol production throughout the whole bioremedation process was performed using an original and straightforward validated analytical procedure with recoveries between 78 and 112% at all concentration levels studied except for endosulfan sulfate at 50 mg L−1 that yielded 128% and RSDs < 20%. Under the developed conditions, the basidiomycete Bjerkandera adusta was able to degrade 83% of (alpha + beta) endosulfan after 27 days, 6 mg kg−1 of endosulfan diol were determined; endosulfan ether and endosulfan sulfate were produced below 1 mg kg−1 (LOQ, limit of quantitation).
Keywords: Basidiomycete; Endosulfan; Bioremediation; Method validation;

► ASB, not andrographolide, in dog plasma was firstly determined by LC–MS/MS. ► The LC–MS/MS method was fully validated. ► The method was applied to a pharmacokinetic study in dogs.A sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated for the determination of andrographolide sodium bisulphite (ASB) in dog plasma using dehydroandrographolide (DAG) as an internal standard. Chromatographic separation was achieved on a Hypersil Gold C18 column (50 mm × 2.1 mm, 1.9 μm) with gradient elution that consisted of methanol and water at a flow rate of 0.2 mL/min. Quantification was done using selected reaction monitoring (SRM) mode to monitor precursor–product ion transitions of m/z 413.2→287.2 for ASB and 331.2→303.3 for DAG at negative ionization mode. Good linearity was obtained over the range of 10–1000 ng/mL and the correlation coefficient was better than 0.99. The intra- and inter-day accuracies ranged from 97.2% to 107.8% and precisions (RSD) were within 13.9%. ASB was found stable under three freeze–thaw cycles, short-term temperature, post-preparative and long-term temperature conditions. The method was successfully applied to a pharmacokinetic study of ASB intravenously administered to Beagle dogs.
Keywords: Andrographolide sodium bisulphite; LC–MS/MS; Pharmacokinetics;

► Clinical imperative to foster research in the area of optimising antibiotic dosing. ► Determined the free (unbound) concentration of ten beta-lactam antibiotics. ► Validated method used in pathology laboratory for TDM in critically ill patients. ► Suggest as preferred assay due to antibiotic protein binding variability. ► Method is highly advantageous from a laboratory and clinical perspective.With the clinical imperative to further research in the area of optimising antibiotic dosing in the intensive care setting, a simple high performance liquid chromatography method was developed and validated for routinely determining the free (unbound) concentration of ten beta-lactam antibiotics in 200 μL of human plasma. Antibiotics determined include three cephalosporins (ceftriaxone, cephazolin and cephalotin); two carbapenems (meropenem and ertapenem); and five penicillins (ampicillin, piperacillin, benzylpenicillin, flucloxacillin and dicloxacillin). There was a single common sample preparation method involving ultracentrifugation and stabilisation. Chromatography was performed on a Waters XBridge C18 column with, depending on analytes, one of four acetonitrile-phosphate buffered mobile phases. Peaks of interest were detected via ultraviolet absorbance at 210, 260 and 304 nm. The method has been validated and used in a pathology laboratory for therapeutic drug monitoring in critically ill patients. The significant variability in the level of protein binding that is common with antibiotics traditionally considered to have high protein binding (e.g. ceftriaxone, cephazolin, ertapenem, flucloxacillin and dicloxacillin) suggests that this assay should be preferred for measuring the pharmacologically active concentration of beta-lactam antibiotics in a therapeutic drug monitoring programme.
Keywords: Free; Unbound; Beta-lactam; Antibiotics; HPLC; TDM;