Journal of Chromatography B (v.877, #29)

Separation of diastereoisomers of Ara-C phosphotriesters using solid phase extraction and HPLC for the study of their decomposition kinetic in cell extracts by C. Foulon; J. Tedou; S. Peyrottes; C. Perigaud; J.P. Bonte; C. Vaccher; J.F. Goossens (3475-3481).
Separations of the diastereoisomers of three nucleoside 5′-phosphotriester derivatives of Ara-C (tBuSATE, hydroxy tBuSATE and bishydroxy tBuSATE phenylphosphotriester derivatives; pronucleotides) were performed by HPLC using derivatized cellulose and amylose chiral stationary phases. An optimal baseline separation (Rs > 1.5) was readily obtained with an amylose based chiral column (AD-H) used in normal phase mode. This stereospecific HPLC method has been associated to a solid phase extraction step using a C18 cartridge and an internal standard for the quantification of one nucleoside 5′-phosphotriester derivative in cell extracts. After optimization, this method was validated in terms of specificity, recovery, linearity, precision and accuracy and detection limit. It was applied to the determination of the apparent rate constants of disappearance and half-lives of each diastereoisomer. This enabled us to conclude that the enzymatic activity involved in the first step of the decomposition pathway of the hydroxyl tBuSATE phenylphosphotriester of Ara-C is stereoselective and is related to the nature of the pyrimidic base.
Keywords: Ara-C phosphotriesters; SPE; Chiral HPLC; Cell extracts; Kinetic;

A sensitive method was developed and validated for simultaneous measurement of an investigational antiviral nucleoside, Amdoxovir (DAPD), its deaminated metabolite 9-(β-d-1,3-dioxolan-4-yl)guanine (DXG), and Zidovudine (ZDV) in human plasma. This method employed high-performance liquid chromatography–tandem mass spectrometry with electrospray ionization. DXG and DAPD separation with sufficient resolution was necessary since they differ in only one mass to charge ratio, which increases the risk of overlapping MS/MS signals. However, the new method was observed to have functional sensitivity and specificity without interference. Samples were purified by ultrafiltration after protein precipitation with methanol. The total run time was 29 min. A linear calibration range from 2 to 3000 ng mL−1 and 2 to 5000 ng mL−1 was achieved for DAPD and DXG, and ZDV, respectively. Precisions and accuracies were both ±15% (±20% for the lower limit of quantification) and recoveries were higher than 90%. Matrix effects/ion suppressions were also investigated. The analytes were chemically stable under all relevant conditions and the method was successfully applied for the analysis of plasma samples from HIV-infected persons treated with combinations of DAPD and ZDV.
Keywords: Nucleoside analogs; Pharmacokinetics;

Two HPLC methods are described for the enantioselective analysis of R- and S-propafenone in plasma. In a column switching approach, centrifuged plasma was injected onto a silica-based strong acid cation-exchanger and the fraction containing propafenone was switched on-line onto an enantioselective Chiralcel column for separation of the enantiomers. In another approach, propafenone was extracted from plasma by liquid–liquid extraction at pH 11.4. The extracted components were transferred into aqueous medium and separated on a Chiralcel ODR. Both methods were validated and showed comparable performance. Within-day and between-day precision was better than 5% for both methods. Linear calibration functions were obtained (r 2  > 0.999), and the limit of detection for each enantiomer was 0.2 μg/mL for column switching and 0.55 μg/mL for liquid–liquid extraction. The analysis methods were applied for the determination of the effect of physical exercise on the enantiomeric ratio of R- and S-propafenone in plasma of healthy volunteers. During exercise, the concentration of both enantiomers reached a maximum, followed by a significant decrease during recovery.
Keywords: Propafenone; Enantioselective analysis; HPLC; Column switching; Liquid–liquid extraction;

Characterization of in vitro modified human high-density lipoprotein particles and phospholipids by capillary zone electrophoresis and LC ESI-MS by Chung-Yu Wu; Yu-Nong Peng; Jing-Huei Chiu; Yu-Ling Ho; Chin-Pong Chong; Ying-Ling Yang; Mine-Yine Liu (3495-3505).
A simple capillary zone electrophoresis (CZE) method was used to characterize native, in vitro oxidized and glycated human high-density lipoprotein (HDL) particles. Both native and in vitro oxidized HDL capillary electrophoresis (CE) profiles showed a major peak, but the oxidized HDL particles had higher effective mobilities. The in vitro glycated HDL particles showed a major peak and one or two minor peaks. The effective mobility of the major peak of glycated HDL was similar to that of the major peak of native HDL, whereas the effective mobilities of the two minor peaks were much lower. For the analysis of HDL phospholipids, a solid phase extraction procedure was optimized and a LC ESI-MS method was developed. Several possible HDL phospholipid molecular species including phosphatidylcholine (PC 16:0/18:2, 16:0/18:1, 18:0/18:2 and 18:0/18:1), sphingomyelin (SM 16:0) and lyso-phosphatidylcholine (lysoPC 16:0 and 18:0) were found. It appeared that the ion intensity ratios of hydroperoxy-PC or epoxyhydroxy-PC (16:0/hydroperoxy-18:2 or 16:0/epoxyhydroxy-18:2, m/z 790.4) and trihydroxy-PC (16:0/trihydroxy-18:2, m/z 808.3) relative to PC (C16:0/C18:2, m/z 758.5) were higher for oxidized HDL than for native and glycated HDL. It should be helpful to use both CZE and LC ESI-MS methods for analyzing high-density lipoproteins from patients of cardiovascular disease. Their combination may be also useful for further studies concerning the role of oxidized and glycated HDLs in the development of atherosclerosis.
Keywords: High-density lipoprotein; Phospholipids; Capillary zone electrophoresis; Electrospray ionization mass spectrometry; Solid phase extraction; In vitro oxidation; In vitro glycation;

Drug-eluting stents are sustained-release intra-coronary devices that are usually coated with a few hundred micrograms of drug. Measuring the drugs that are released over weeks in order to assess human pharmacokinetics is a challenge that requires assays with high sensitivity. We developed and validated a semi-automated LC–MS/MS assay for the quantification of Biolimus A9, a proliferation signal inhibitor that was specifically developed for coating on drug-eluting stents in human EDTA blood. The only manual step was the addition of a zinc sulfate/methanol protein precipitation solution which included the internal standard. Samples were injected into the HPLC and extracted online. The assay had the following performance characteristics: range of reliable response 0.01–100 ng/mL (r 2  > 0.99), inter-day accuracy (0.033 ng/mL): 111.7%, and inter-day precision: 8.6%. There was no ion suppression, matrix interferences or carry-over. Extracted samples were stable in the autosampler at +4 °C for at least 24 h and could undergo three freeze–thaw cycles. The assay, with a lower limit of detection of 333 fg/mL and a lower limit of quantitation of 10 pg/mL, was sufficiently sensitive and robust for quantifying Biolimus A9 in clinical trials after IV injection and after stent implantation.
Keywords: Biolimus A9; LC–MS/MS; Column switching; Drug-eluting stents; Human whole blood;

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to extract and quantify the androgen concentration in the rat prostate. This method introduced a novel 96-well plate format for the extraction and derivatization of testosterone (T) and dihydrotestosterone (DHT) from rat prostatic tissue that greatly simplified the sample preparation procedure. Due to the difficulty to obtain reproducible specimens with non-detectable level of androgen, a matrix-free standard solution was used for method validation. Both T and DHT calibration curves were linear over the calibration range (12.5–2500 pg) with correlation coefficient values greater than 0.9900. The intra-day and inter-day accuracy, reported as %bias, and precision, reported as %CV, of T and DHT were within ±10%. The lower limit of detection (LLOD) and lower limits of quantification (LLOQ) for both T and DHT were determined to be 5 and 12.5 pg. The validation results demonstrated the selectivity, sensitivity, accuracy, precision, linearity and ruggedness of the method, as well as the suitability of the method for simultaneous detection of T and DHT in rat prostatic tissues. The validated method was successfully applied to determine the physiological T and DHT level in rat prostatic tissues. Similarly to the serum concentration profile pattern, T and DHT intraprostatic levels peaked 2 h after lights-on and decreased after lights-off with DHT level approximately 4-fold greater than T.
Keywords: Testosterone; Dihydrotestosterone; Rat prostatic tissue; Liquid chromatography–tandem mass spectrometry; 96-well plate; Validation;

A novel method to detect seven microcystins in hard clam and corbicula fluminea by liquid chromatography–tandem mass spectrometry by Bo Yang; Jin-Zhong Xu; Tao Ding; Bin Wu; Su Jing; Shu-jing Ding; Hui-Lan Chen; Chong-Yu Sheng; Yuan Jiang (3522-3528).
A simple and reliable method to detect seven microcystins in hard clam and corbicula fluminea, based on liquid chromatography with electrospray ionization and tandem mass spectrometry (LC–ESI-MS/MS), was developed and validated. The sample preparation procedure includes extraction of tissue by methanol, followed by cleanup on a reversed-phase solid phase extraction (SPE) cartridge. With the optimized method, recoveries were between 43.7% and 92.3% for hard clam, 54.3% and 93.8% for corbicula fluminea, the relative standard deviations (RSD) were less than or equal to 16.2% and 15.7% in hard clam and corbicula fluminea at spiking levels of 1 μg/kg, 2 μg/kg and 5 μg/kg for MC-RR, MC-YR, MC-LR, and MC-LY, and 2 μg/kg, 5 μg/kg and 10 μg/kg for MC-LA, MC-LW and MC-LF, respectively, the limits of quantitation (LOQ) of this method were ranged from 0.7 μg/kg to 2.0 μg/kg.
Keywords: Microcystins; Shellfish; Liquid chromatography; Tandem mass spectrometry;

Purification and partial characterization of Cu/Zn superoxide dismutase from Kluyveromyces marxianus yeast by Trayana Nedeva; Pavlina Dolashka-Angelova; Vesela Moshtanska; Wolfgang Voelter; Ventzislava Petrova; Anna Kujumdzieva (3529-3536).
A new thermostable Cu/Zn SOD from a thermotolerant yeast strain Kluyveromyces marxianus NBIMCC 1984 has been purified and characterized. The purification procedure comprises thermal treatment and dialysis, ion-exchange chromatography and chromatofocusing. The methodology is a rapid, efficient and highly specific, generating pure preparation (specific activity 996 U mg of protein−1) with a yield of 53%. The purified enzyme is a homodimer with Mw of 34 034 Da and has high N-terminal homology with other yeasts’ Cu/Zn SOD enzymes. The protein is characterized with some unique features such as—thermostability (t 1/2 at 70 °C = 30 min), pH stability in the alkaline range (7.5–8.5) and resistance to inhibitors and variety of chemicals. These characteristics reveal possibilities for wide practical application of K. marxianus Cu/Zn SOD enzyme.
Keywords: Cu/Zn superoxide dismutase; K. marxianus; Purification; Thermostability; Glycoprotein;

An LC-MS/MS method for the simultaneous quantification of nicotine, cotinine, trans-3′-hydroxycotinine and norcotinine in human plasma was developed and fully validated. Potential endogenous and exogenous interferences were extensively evaluated and limits of quantification were determined by decreasing analyte concentration. Analytical ranges were 1–500 ng/mL for nicotine and cotinine, 5–500 ng/mL for trans-3′-hydroxycotinine and norcotinine. Mean intra- and inter-assay analytical recoveries were between 101.9 and 116.8%, and intra- and inter-assay imprecision were less than 11% RSD for all analytes: parameters were evaluated at three different concentrations across the linear range of the assay. Extraction efficiency was ≥70% for all analytes. This validated method is useful for the determination of nicotine and metabolites in human plasma to support research on the role of nicotine biomarkers on neuronal systems mediating cognitive and affective processes and to differentiate active, passive and environmental exposure.
Keywords: Nicotine; Cotinine; trans-3′-Hydroxycotinine; Norcotinine; Plasma; LC-MS/MS;

Validation of an LC–MS/MS method for the determination of epirubicin in human serum of patients undergoing Drug Eluting Microsphere-Transarterial Chemoembolization (DEM-TACE) by Cristina Sottani; Emanuela Leoni; Benedetta Porro; Benedetta Montagna; Alessio Amatu; Federico Sottotetti; Pietro Quaretti; Guido Poggi; Claudio Minoia (3543-3548).
Drug Eluting Microsphere-Transarterial Chemoembolization (DEM-TACE) is a new delivery system to administrate drugs in a controlled manner useful for application in the chemoembolization of colorectal cancer metastases to the liver. DEM-TACE is focused to obtain higher concentrations of the drug to the tumor with lower systemic concentrations than traditional cancer chemotherapy. Therefore a specific, precise and sensitive LC–ESI-MS/MS assay procedure was properly designed to detect and quantify epirubicin at the concentrations expected from a transarterial chemoembolization with microspheres. Serum samples were kept acidic (pH approximately of 3.5) and sample preparation consisted of a solid phase extraction (SPE) procedure with HLB OASIS® cartridges using a methylene chloride/2-propanol/methanol mixture solution to recover epirubicin. The analyses consisted of reversed-phase high-performance liquid chromatography (rp-HPLC) coupled with tandem mass spectrometry (MS/MS). Accuracy, precision and matrix effect of this procedure were carried out by analyzing four quality control samples (QCs) on five separate days. The validation parameters were assessed by recovery studies of spiked serum samples. Recoveries were found to vary between 92 and 98% at the QC levels (5, 40, 80 and 150 μg/L) with relative standard deviation (RSD) always less than 3.7%. The limit of detection (LOD) was set at 1 μg/L. The developed procedure has been also applied to investigate the different capability of two types of commercially available microspheres to release epirubicin into the human circulatory system.
Keywords: Epirubicin; LC–MS/MS; Validation; Drug eluting microspheres; DC-Beads; Hepaspheres;

A simple, selective and sensitive reversed phase liquid chromatography method utilizing ultraviolet detection has been developed and validated for the simultaneous determination of the prodrug AZD1152 and its active product AZD1152-hydroxyquinazoline pyrazol anilide (hQPA) in human and mouse plasma and mouse tissues. Isocratic separation was achieved using an 5 μm UptiSphere HDO C-18 column (150 mm × 4.6 mm) with guard column in combination with a mobile phase comprised of phosphate buffered water (50 mM; pH 3.4) and acetonitrile (81.5: 18.5; v/v). UV detection at 318 nm was used. Sample preparation involved a single-step protein precipitation with ethanol. Ex vivo conversion of AZD1152 by endogenous phosphatases was prevented by immediate cooling of the samples in ice-water and addition of sodium fluoride and EDTA. The validation parameters included specificity, recovery, accuracy, precision, sensitivity and stability. The lower limit of quantification in human plasma for AZD1152 and hQPA was 25 ng/ml. The applicability of the method was demonstrated by successful determination of AZD1152 and hQPA in human plasma and in plasma, brain, liver, kidney and ileum samples from mice dosed with AZD1152.
Keywords: AZD1152; AZD1152-hydroxyquinazoline pyrazol anilide; hQPA; HPLC; Mouse; Human; Tissues; Plasma;

The aim of the present work was to develop and validate a simple RP-HPLC method with UV detection to quantify peptide dendrimers in skin permeation experiments. Six dendrimers of varying positive charges (4+, 8+ and 16+) containing either histidine or arginine as terminal aminoacids were prepared by solid phase peptide synthesis. Mobile phase containing 0.02% (v/v) heptafluorobutyric acid in 90% acetonitrile–water was capable of separating all dendrimers from interfering peaks of receptor fluid. For the calibration of each dendrimer, a different dendrimer from the same class was selected as the internal standard. The results of preliminary human skin permeation studies showed that the developed analytical method can be successfully used for the quantification of cationic poly(aminoacid)-based dendrimers in skin permeation experiments.
Keywords: Peptide dendrimers; HPLC; Ion-pairing; Transdermal delivery; Iontophoresis;

Liquid chromatography–tandem mass spectrometry method for the quantification of dimebon in rat plasma and brain tissue by Ramakrishna Nirogi; Vishwottam Kandikere; Koteshwara Mudigonda; Prashanth Komarneni; Rajeshkumar Boggavarapu (3563-3571).
A sensitive high-performance liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of dimebon in rat plasma and brain tissue. Following liquid–liquid extraction, the analyte was separated using a gradient mobile phase on a reversed phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 320–277 for dimebon and m/z 407–100 for the internal standard. The assay exhibited a linear dynamic range of 0.25–250 ng/mL for dimebon in rat plasma and brain tissue. Acceptable precision (<11%) and accuracy (100 ± 7%) were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 250 samples per day. The method was successfully applied to quantify dimebon concentrations in a rodent pharmacokinetic study. Moreover, it can be believed that the assay method in rat plasma would facilitate the ease of adaptability of dimebon quantification in human plasma for clinical trials.
Keywords: Dimebon; Liquid chromatography–tandem mass spectrometry; Plasma; Brain tissue; Clinical trials;

Joint GC–MS and LC–MS platforms for comprehensive plant metabolomics: Repeatability and sample pre-treatment by Ruben t’Kindt; Kris Morreel; Dieter Deforce; Wout Boerjan; Jan Van Bocxlaer (3572-3580).
Metabolomics nowadays mostly comprises the application of both LC–MS and GC–MS based approaches. Here we investigate different extraction set-ups for these two established analytical platforms in the field of plant metabolomics. Six extraction approaches for Arabidopsis thaliana leaves, varying in extraction solvent composition, extraction temperature and order of solvent addition within the extraction sequence, were analyzed on the two platforms. Our aim was to establish the most suitable analysis protocol, practicable for both LC–MS and GC–MS analysis, in order to obtain as extensive as possible metabolome coverage. One single sample preparation procedure would save time and valuable sample while still offering the complementary datasets generated by GC–MS and LC–MS. All extraction approaches were evaluated based on the following criteria: number of detected m/z-retention time pairs, heat maps of the detected peaks, and residual enzymatic activity of invertase and phosphatase in the plant leaf extracts. Unsupervised principal component analysis (PCA) was used to evaluate grouping trends between the different extraction approaches. Quality controls, a blend of aliquots of the different extracts, were used to establish a paired evaluation of the repeatability performance of the GC–MS and LC–MS analysis. We conclude that the use of chloroform in the extraction solvent is counterproductive in an untargeted LC–MS metabolomics approach as is heating. Below room temperature (instead of heated) extraction does not significantly degrade GC–MS performance but one should be more cautious with respect to residual enzymatic activity in the plant extract.
Keywords: Repeatability; Metabolite extraction; GC–MS; LC–MS; Plant metabolomics; Enzymatic activity;

To develop an optimal quantitative LC/MS method with high sensitivity, high selectivity and robustness in a limited time period can be very challenging, especially for methods in which many analytes are to be quantified. In this study the relevant options are reviewed and a simple screening strategy of mass spectrometric and chromatographic conditions is presented. The strategy is divided into two stages, mass spectrometric ionisation screening and reversed phase LC column screening. The objective of the first stage is to find out how sensitivity is affected by ionisation technique, ionisation polarity and buffer. The compounds are dissolved in different buffers covering a broad pH range. Thereafter they are injected using flow injection analysis without LC column, evaluating both electrospray and atmospheric pressure chemical ionisation (APCI). In the second stage the buffers yielding the best sensitivity and selectivity in the ionisation screening stage are used as mobile phase buffers to LC column screening with different stationary phases applying a shallow gradient. The aim is to find the combinations of column(s) and buffer(s) that give symmetric peaks, adequate retention and selectivity. Finally the retention is adjusted using isocratic or gradient elution. The strategy provides a simple and practical experimental design that allows fast screening a large range of ionisation and chromatographic conditions especially for multiple compounds. The examples included in this study demonstrate that optimal buffer, ionisation technique, ionisation polarity and column cannot be predicted from compound properties such as structure and pK a.
Keywords: Method development; LC/MS; Interfaces; HPLC columns; Mobile phase buffer effects; Ionisation; Optimisation; Bioanalysis; Quantitative;

A sensitive, rapid and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay has been established for the quantitation of catalpol in rat plasma. Plasma samples were treated by precipitating protein with methanol and were chromatographed by a Diamonsil C18 column (150 mm × 4.6 mm I.D., 5 μm) with the mobile phase consisting of methanol and 10 mM ammonium formate (30:70, v/v). The selected reaction monitoring (SRM) transitions were performed at m/z 380.1 → 183.0 for catalpol and m/z 364.3 → 167.0 for aucubin (IS) in the positive ion mode with electrospray ionization (ESI) source. Calibration curve was linear over the concentration range of 10–20,000 ng/mL. The mean recovery was 76.5 ± 5.2% and the matrix effect ranged from −5.1 to 13.0%. The intra- and inter-day precisions were less than 6.3 and 14.6%, respectively, and the accuracy was within ±5.6%. Catalpol was stable in possible conditions of storing and handling. The validated method has been successfully applied to determine the plasma concentration of catalpol for a pharmacokinetic study of catalpol after oral administration of 50 mg/kg to rats.
Keywords: Catalpol; LC/MS/MS; Pharmacokinetics;

A simple and sensitive method based on high-performance liquid chromatography (HPLC) coupled with chemiluminescence detection was developed for the quantitation of kaempferol (KA) in rat plasma. Isorhamnetin (IS) was used as an internal standard. Plasma samples were prepared only by acidification with 20% phosphoric acid and protein precipitation using methanol. Good separations of kaempferol and internal standard were achieved with an isocratic elution using a mobile phase consisting of methanol and aqueous 0.4% phosphoric acid (47:53, v/v) within 25 min. The detection limit for kaempferol was 1.0 × 10−9  g/ml. The mean accuracy was within 80.0–100.2%, and the intra- and inter-day precision had RSD (%) < 5.0. The sample preparation method was able to produce high recovery (≥80.0%). The proposed method with wide linear range has been successfully applied to a pharmacokinetic study in SD rats after oral administration at a dose of 2500 or 1250 mg/kg bodyweight (BW) kaempferol. Kaempferol concentration was detectable in plasma up to 24 h post-dosing, and the pharmacokinetic parameters of T max, C max, AUC0−∞, MRT0−∞, and T 1/2 of kaempferol were reported.
Keywords: Kaempferol; High-performance liquid chromatography; Chemiluminescence; Cerium(IV); Rhodamine 6G; Rat plasma; Pharmacokinetics;

A novel and sensitive method for ethinylestradiol quantification in human plasma by high-performance liquid chromatography coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometry: Application to a comparative pharmacokinetics study by Ney Carter Borges; Rafael Barrientos Astigarraga; Carlos Eduardo Sverdloff; Paulo Rabelo Galvinas; Washington Moreira da Silva; Vinícius Marcondes Rezende; Ronilson Agnaldo Moreno (3601-3609).
In the present study, a novel, fast, sensitive and robust method to quantify ethinylestradiol in human plasma using 17α-ethinylestradiol-d4 as the internal standard (IS) is described. The analyte and the IS were extracted from acidified plasma by liquid–liquid extraction (LLE) using diethyl ether–hexane followed by online solid phase extraction (SPE) using online C18 cartridges. Extracted samples were analyzed by high-performance liquid chromatography coupled to atmospheric pressure photoionization tandem mass spectrometry (HPLC–APPI-MS/MS). Chromatography was performed isocratically on a C18, 5 μm analytical column. The method had a chromatographic run time of 2.50 min and a linear calibration curve over the range 5–500 pg ml−1 (r 2  > 0.9992). The lowest concentration quantified was 5 pg ml−1, demonstrating acceptable accuracy and precision. The intra-assay precisions ranged from 2.1 to 14.6%, while inter-assay precisions ranged from 4.4 to 11.4%. The intra-assay accuracies ranged from 94.6 to 103.8%, while the inter-assay accuracies ranged from 98.9 to 101.6%. The recovery of ethinylestradiol was determined as part of the assay validation process and was 73.1 and 79.0% for the concentrations 15 and 375 pg ml−1, respectively. Short-term stability showed that ethinylestradiol was stable in plasma for at least 19 h at room temperature or for at least 385 days when stored at −20 °C. In the study of bioequivalence conducted in Brazil, healthy volunteers received two ethinylestradiol 0.035 mg tablet formulations using an open, randomized, two-period crossover design with a 2-week washout interval. Since the 90% confidence interval for C max and area under the curve ratios were all inside the 80–125% interval proposed by the US Food and Drug Administration, it was concluded that the two ethinylestradiol formulations are bioequivalent with respect to both the rate and the extent of absorption.
Keywords: Ethinylestradiol; Bioequivalence; Plasma; Pharmacokinetics; APPI; Mass spectrometry;

Discriminatory protein binding by a library of 96 new affinity resins: A novel dye-affinity chromatography tool-kit by Sunil Kumar; Darshan B. Dalvi; Masana Moorthy; Shilpa S. Korde; Kamalesh Pai Fondekar; Suhas D. Sahasrabudhe; Hans-Thomas Schacht; Vadiraj S. Ekkundi; Christine Halik; Rajarshi Choudhury; Awanit Kumar; Narayan S. Punekar (3610-3618).
Initial acceptance of Cibacron Blue 3G-A® based matrices has made dye-ligand affinity chromatography an attractive proposition. This prompted the synthesis and search for new dye structures. A systematic library of 96 affinity resins was generated using novel analogs of Cibacron Blue 3G-A® and also by varying spacer lengths for immobilization. The library was tested in a batch binding and elution mode using seven different proteins – four Aspergillus enzymes namely, NADP-glutamate dehydrogenase, laccase, glutamine synthetase and arginase, bovine pancreatic trypsin and the two serum proteins human serum albumin and immunoglobulin G. Unique binding patterns were observed for each of them indicating that the library displayed discriminatory interactions. The significance of spacer length in the interaction with proteins was discernable. Trypsin interacted best with affinity resins that had no spacer. It was possible to resolve IgG and HSA from a mixture using a combination of resins. There was a good spread of HSA binding capacity in the 96 affinity resins. While some showed better HSA binding capacity than the commercial CB3GA-based matrix, a few with lower capacity were also observed. Subsequent to an initial screen, one affinity resin (CR-017) could be used to enrich Aspergillus terreus NADP-GDH from crude cell extracts. The efficacy of this dye-affinity resin was rationalized by characterizing NADP-GDH inhibition kinetics with the corresponding free dye ligand. In the sum, the library provides a set of dye-ligand affinity matrices with a potential for use in high throughput screening for protein purification.
Keywords: Cibacron Blue 3G-A®; Affinity resin; Dye-ligand chromatography; Aspergillus enzymes; Human serum albumin; Immunoglobulin G; NADP-glutamate dehydrogenase;

Mitiglinide is a new insulinotropic agent of the glinide class and its precise mechanism is not very clear yet. In this study, a urinary metabonomics method based on ultra-performance liquid chromatography–tandem mass spectrometry was developed to study the effect mechanism of mitiglinide on type 2 diabetes mellitus. With pattern recognition analysis of urinary metabolite profiles, a clear separation between Streptozotocin-induced type 2 diabetic rats and those treated with mitiglinide was achieved. Some significantly changed metabolites like citrate, creatinine, phenylalanine and bile acids (cholic acid, chenodeoxycholic acid and deoxycholic acid) have been identified and used to explain the mechanism. This work shows that metabonomics method is a valuable tool in drug mechanism study.
Keywords: Metabonomics; Type 2 diabetic rats; UPLC/MS; Mitiglinide; Biomarkers;

A validated assay for the quantitative analysis of vatalanib in human EDTA plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry by A.G. Lankheet; M.J.X. Hillebrand; M.H.G. Langenberg; H. Rosing; A.D.R. Huitema; E.E. Voest; J.H.M. Schellens; J.H. Beijnen (3625-3630).
A sensitive and accurate method for the determination of vatalanib in human EDTA plasma was developed using high-performance liquid chromatography and detection with tandem mass spectrometry. Stable isotopically labeled imatinib was used as internal standard. Plasma proteins were precipitated and an aliquot of the supernatant was directly injected onto a Phenomenex Gemini C18 analytical column (50 mm × 2.0 mm ID, 5.0 μm particle size) and then compounds were eluted with a linear gradient. The outlet of the column was connected to a Sciex API 365 triple quadrupole mass spectrometer and ions were detected in positive multiple reaction monitoring mode. The lower limit of quantification was 10 ng/mL (S/N ≈ 10, CV ≤ 8.4%). This method was validated over a linear range from 10 to 2500 ng/mL, and results from the validation study demonstrated a good intra- and inter-assay accuracy (inaccuracy ≤9.57%) and precision (CV ≤ 8.81%). This method has been used to determine plasma vatalanib concentrations in patients with advanced solid tumor, enrolled in a phase I pharmacokinetic trial with the drug.
Keywords: Vatalanib; Liquid chromatography; Mass spectrometry;

Detonation nanodiamond (dND) was firstly employed as adsorbent for pretreatment of peptides in dilute/contaminated sample solution. Detonation nanodiamond showed high efficiency for isolating and enriching peptides in a wide pH range. Remarkably, good tolerance capability toward salts and detergents could be achieved by using dNDs. Due to the inherent specificities of dNDs, dND-bound peptides could be directly analyzed by MALDI-TOF MS, so as to avoid the elution step and reduce sample loss. This pretreatment method also exhibited a better performance for protein identification compared to solvent evaporation and Ziptip pretreatment approach.
Keywords: Detonation nanodiamond; Sample pretreatment; Low-abundance peptides; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry;

[13C] GC–C-IRMS analysis of methylboronic acid derivatives of glucose from liver glycogen after the ingestion of [13C] labeled tracers in rats by Catherine Luengo; Dalila Azzout-Marniche; Claire Fromentin; Julien Piedcoq; Sophie Lemosquet; Daniel Tomé; Claire Gaudichon (3638-3644).
We developed a complete method to measure low [13C] enrichments in glycogen. Fourteen rats were fed a control diet. Six of them also ingested either [U-13C] glucose (n  = 2) or a mixture of 20 [U-13C] amino acids (n  = 4). Hepatic glycogen was extracted, digested to glucose and purified on anion–cation exchange resins. After the optimization of methylboronic acid derivatization using GC–MS, [13C] enrichment of extracted glucose was measured by GC–C-IRMS. The accuracy was addressed by measuring the enrichment excess of a calibration curve, which observed values were in good agreement with the expected values (R  = 0.9979). Corrected delta values were −15.6 ± 1.6 δ 13C (‰) for control rats (n  = 8) and increased to −5 to 8 δ 13C (‰) ‰ and 12–14 δ 13C (‰) ‰ after the ingestion of [U-13C] amino acids or [U-13C] glucose as oral tracers, respectively. The method enabled the determination of dietary substrate transfer into glycogen. The sequestration of dietary glucose in liver glycogen 4 h after the meal was 35% of the ingested dose whereas the transfer of carbon skeletons from amino acids was only 0.25 to 1%.
Keywords: Low 13C enrichment; Hepatic glycogen; Glucose; Methylboronic acid derivatization; GC–C-IRMS;

A new method is reported for the simultaneous derivatization and extraction of free cyanide in biological samples using home-made hollow fiber-protected headspace liquid-phase microextraction (HF-HS-LPME) followed by capillary electrophoresis (CE) determination. The acceptor phase containing Ni(II)-NH3 (as derivatization agent), sodium carbonate and ammonium pyromellitate (as internal standard) is held within a hollow fiber membrane, affixed to a syringe needle and immersed in the headspace of sample container. The extracted cyanide from the neutral samples forms a stable Ni(CN)4 2− complex which is determined by CE. Parameters affecting extraction efficiency were investigated and optimized. For optimum peak shapes, the capillary was coated with cetyltrimethylammonium bromide (CTAB). The calibration curve was linear for concentrations of CN in the range from 0.1 to 20 μmol L−1 (R 2  = 0.9987). The LOD (S/N = 3) was estimated to be 0.01 μmol L−1 of CN concentration. Such a detection sensitivity is high enough for free cyanide determination in common environmental and biological samples. Excellent repeatability of the extraction (RSD ≤ 5.6%, n  = 5) was achieved. The feasibility of this method was demonstrated by analyzing human urine and saliva samples with spiked recoveries in the range of 92–103.4%. This work provided an efficient alternative to the present headspace microextraction techniques such as headspace solid-phase microextraction (HS-SPME) and headspace single-drop microextraction (HS-SDME).
Keywords: Headspace liquid-phase microextraction; Home-made hollow fiber membrane; Free cyanide; Capillary electrophoresis; Urine; Saliva;

Polar volatile organic compounds (PVOCs) such as aldehydes and alcohols are byproducts of normal human metabolism and thus are found in blood and exhaled breath. Perturbation of the normal patterns of such metabolites may reflect exposures to environmental stressors, disease state, and human activity. Presented herein is a specific methodology for assaying PVOC biomarkers in exhaled breath condensate (EBC) samples with application to a series of samples from a controlled chamber exposure to dilute diesel exhaust (DE) or to purified air. The collection/analysis method is based on condensation of normal (at rest) exhaled breaths for 10 min (resulting in 1–2 ml of liquid) with subsequent analyte adsorption onto Tenax® cartridges followed by thermal desorption and analysis by gas chromatography/mass spectrometry (GC/MS). Analytical data have linearity of response (R 2  > 0.98) across a range of 0–160 ng/ml with a detection limit ranging from 0.2 to 7 ng/ml depending on the compound. Statistical analyses of the results of the controlled exposure study indicate that metabolism, as reflected in simple breath-borne oxygenated species, is not affected by exposure to ambient airborne levels of DE. Linear mixed-effects models showed that PVOC biomarker levels are affected by gender and vary significantly among nominally healthy subjects. Differences among PVOCs analyzed in clinic air, purified chamber air, and chamber air containing dilute DE confirm that most of the compounds are likely of endogenous origin as the exogenous exposure levels did not perturb the EBC measurements.
Keywords: Biomarkers; Exhaled breath condensate; Diesel exhaust;

On-line coupling of anion exchange and ion-pair chromatography for measurement of intracellular triphosphate metabolites of reverse transcriptase inhibitors by Zsuzsanna Kuklenyik; Amy Martin; Chou-Pong Pau; Angela Holder; Ae S. Youngpairoj; Qi Zheng; Mian-Er Cong; J. Gerardo Garcia-Lerma; Walid Heneine; James L. Pirkle; John R. Barr (3659-3666).
We developed an automated on-line weak anion exchange (WAX) solid-phase extraction (SPE) method coupled with ion-pair (IP) chromatography–tandem mass spectrometry (MS/MS) detection for quantitatively measuring triphosphorylated metabolites of three reverse transcriptase inhibitors (RTI). The administered pro-drugs were Tenofovir disoproxil fumarate (TDF), Emtricitabine (FTC) and Lamivudine (3TC). Their intracellular metabolites Tenofovir-diphosphate (TFV-DP), Emtricitabine-triphosphate (FTC-TP), and Lamivudine-triphosphate (3TC-TP) were measured in peripheral blood mononuclear cells (PBMC). We coupled the WAX and IP chromatography systems using a combination of 6-port and 10-port switching valves, and we mixed the WAX elute with 1,5-dimethyl-hexyl-amine before IP chromatography separation. Multiple waste outlets allowed for eliminating potential matrix components interfering with MS/MS detection. Limits of detection were 9, 200 and 75 pg per sample for TFV-DP (448/176 m/z), FTC-TP (488/130 m/z) and 3TC-TP (468/119 m/z), respectively.
Keywords: Anion exchange; Ion-pair; Mass spectrometry; Liquid chromatography; quadrupole; Metabolism; Metabolites; On-line analysis;

Quantitative determination of free glycerol and myo-inositol from plasma and tissue by high-performance liquid chromatography by Ryan A. Frieler; Dane J. Mitteness; Mikhail Y. Golovko; Heidi M. Gienger; Thad A. Rosenberger (3667-3672).
A high-performance liquid chromatographic method that accurately measures glycerol and myo-inositol from plasma and tissue is described. The method incorporates a pre-column derivatization reaction using aqueous extracts with benzoyl chloride as a modifying agent. The benzoylated derivatives are isolated by HPLC using reversed-phase gradient chromatography and quantified via absorbance detection at 231 nm. The benzoylated derivatives of glycerol and myo-inositol are well resolved from other known carbohydrates, internal standard and other contaminants encountered within samples and during incubation. The benzoylation of these analytes reach a maximum between 3.5 and 6 h of incubation and are stable for at least 24 days at 4 °C. The limit of quantization (LOQ) of glycerol was equal to 2.5 nmol/ml plasma and 6.4 nmol/g tissue and the LOQ of myo-inositol was 1.8 nmol/ml plasma and 3.6 nmol/g tissue. Incubation of known standards and samples with benzoyl chloride at 40 °C for 4 h showed fully benzoylated products as determined by mass spectral analysis. Calibration curves were linear between 2.7 and 174 nmol for glycerol and 1.4–89 nmol for myo-inositol. Comparison of tissue and plasma concentrations of glycerol and myo-inositol found using this method are in good agreement with other reported values using other techniques.
Keywords: Glycerol; myo-Inositol; Carbohydrates; Plasma; Tissue; High-performance chromatography;

A new method development and validation approach is proposed in order to develop a reliable method for the simultaneous quantitation of ramipril and ramiprilat in the presence of numerous labile metabolites. This new approach involves the usage of a synthesized labile acyl glucuronide of ramipril as well as individual and pooled incurred (study) samples in the development and validation process. Following the method validation and prior to its application to a large clinical study, a mini pilot study was performed to evaluate the performance of the method. When the samples from the mini pilot study were analyzed by two different scientists, 100% of the results from incurred sample reanalysis (ISR) matched within 8% of difference and the mean differences were 0.21% and 1.40% for ramipril and ramiprilat, respectively. The validated concentration range reported in this article is 0.2–80 ng/mL for both analytes. Various stabilities, such as bench-top, autosampler, freeze/thaw, and long-term, were also successfully evaluated. The key to the success were low sample processing temperature (4 °C), proper choice of sample extraction procedure, and adequate chromatographic conditions to obtain good peak shape without the need of derivatization and baseline separation between the analytes and their glucuronide metabolites.
Keywords: Ramipril; Ramiprilat; Metabolite back-conversion; Method development and validation; Incurred samples; LC–MS/MS;

A hybrid LC/MS/MS and proteomics method was developed for the assessment of multiple pesticide exposure. The methodology was based on the analysis of tryptic peptides resulting from inhibited butyrylcholinesterase (BChE) after exposure to pesticides including organophosphates (OPs) and carbamates (CBs). The primary advantage of the assay was its ability to simultaneously examine multiple pesticide exposures in a single analytical experiment. Application of tandem and MS3 techniques provided identities of the inhibiting pesticide, confirmation and localization of the site of inhibition and relative quantification of phosphorylated peptides present in tryptic digests of equine BChE (eBChE).
Keywords: LC/MS/MS; Butyrylcholinesterase; Organophosphate; Carbamate; Proteomics;

Electrospun polyethersulfone affinity membrane: Membrane preparation and performance evaluation by Zuwei Ma; Zhengwei Lan; Takeshi Matsuura; Seeram Ramakrishna (3686-3694).
Non-woven polyethersulfone (PES) membranes were prepared by electrospinning. After heat treatment and surface activation, the membranes were covalently functionalized with ligands to be used as affinity membranes. The membranes were characterized in terms of fiber diameter, porosity, specific area, pore size, ligand density and binding capacities. To evaluate the binding efficiency of the membrane, dynamic adsorption of bovine serum albumin (BSA) on the Cibacron blue F3GA (CB) functionalized PES membrane was studied. Experimental breakthrough curves were fitted with the theoretical curves based on the plate model to estimate plate height (H p ) of the affinity membrane. The high value of H p (1.6–8 cm) of the affinity membrane implied a poor dynamic binding efficiency, which can be explained by the intrinsic microstructures of the material. Although the electrospun membrane might not be an ideal candidate for the preparative affinity membrane chromatography for large-scale production, it still can be used for fast small-scale protein purification in which a highly efficient binding is not required. Spin columns packed with protein A/G immobilized PES membranes were demonstrated to be capable of binding IgG specifically. SDS-PAGE results demonstrated that the PES affinity membrane had high specific binding selectivity for IgG molecules and low non-specific protein adsorption. Compared with other reported affinity membranes, the PES affinity membrane had a comparable IgG binding capacity of 4.5 mg/ml, and had a lower flow through pressure drop due to its larger pore size. In conclusion, the novel PES affinity membrane is an ideal spin column packing material for fast protein purification.
Keywords: Affinity membrane; Frontal analysis; Breakthrough; Electrospinning; Polyethersulphone (PES); IgG purification; Spin column; Protein A/G; Plate model;

A sensitive, simple and rapid assay based on hydrophilic interaction liquid chromatography (HILIC) with tandem mass spectrometry was developed and validated for the quantitative analysis of metformin in human plasma using protein precipitation. Plasma samples were prepared using a protein precipitation solution containing acetonitrile, 0.5% formic acid and the internal standard, metformin-D6. The analytes were separated on a GL Sciences Inertsil HILIC column using an isocratic mobile phase consisting of water/acetonitrile (30:70, v/v) and 0.1% formic acid. Metformin and internal standard were recorded using multiple reaction monitoring in positive ion electrospray mode with transitions of m/z 130–71 and m/z 136–77, respectively. No endogenous components in plasma were found to interfere with metformin measurements. The lower limit of quantification (LLOQ) was 0.5 ng/mL (0.1 pg on-column). The linear range was 0.5–500 ng/mL with an average correlation coefficient of 0.999 using weighted (1/x 2) linear least-squares regression. Dilutional linearity was evaluated up to 5000-fold dilution and the results indicate no influence on the accuracy of analysis. The absolute extraction recovery was 81% for metformin. Intra-day and inter-day precision (CV, %) ranged from 0.73% to 7.18%, and accuracy within ±10.98% from nominal. The analyte was found to be stable for at least 38 days at −20 and −80 °C, 24 h at room temperature, and stable for four freeze–thaw cycles. The processed extracts were stable for 88 h at 4 °C.
Keywords: Metformin; HILIC; LC–MS/MS; GLP; Method validation;

Determination of amphetamine and methamphetamine in umbilical cord using liquid chromatography–tandem mass spectrometry by Joseph Jones; Rosemarie Rios; Mary Jones; Douglas Lewis; Charles Plate (3701-3706).
The use of meconium as a drug-screening matrix for newborns has been the gold standard of care for the past two decades. A recent study using matched pairs of meconium and umbilical cord demonstrated a high degree of agreement. The use of liquid chromatography–tandem mass spectrometry as a means to confirm amphetamines presumptive positive umbilical cord specimens for amphetamine and methamphetamine is described here for the first time. The limit of detection for both compounds was 0.2 ng/g. The limit of quantitation for both compounds was 0.6 ng/g. The assay was linear for both compounds up to 100 ng/g.
Keywords: Umbilical cord; Newborn drug screening; Amphetamine; Methamphetamine; Liquid chromatography–tandem mass spectrometry; LCMSMS;

Malondialdehyde (MDA) has been proposed as a useful biomarker of lipoperoxidation in biological samples, and more developed analytical methods are necessary. A simple and sensitive gas chromatography–mass spectrometry (HS-SPME-GC–MS) was described for the determination of malondialdehyde (MDA) in blood. Acetone-d6 was used as internal standard. MDA and acetone d6 in blood reacted for 40 min at 50 °C with 2,2,2-trifluoroethylhydrazine in headspace vial and simultaneously the formed TFEH derivatives were vaporized and adsorbed on polydimethylsiloxane-divinylbenzene (PDMS-DVB). The compounds were desorbed for 1 min at 240 °C and injected in GC–MS. The reaction solution showed good recoveries at pH 4.0. In the established condition, the method detection limit (MDL) was 0.4 μg/L in 0.1 mL blood sample and the relative standard deviation was less than 8% at the concentration of 25.0 and 50.0 μg/L. The mean concentrations of MDA in normal human blood (n  = 20) were measured to be 187.9 μg/L (2.61 μmol/L).
Keywords: Malondialdehyde; 2,2,2-Trifluoroethylhydrazine; Headspace-solid phase micro-extraction; Gas chromatography–mass spectrometry;

Separation of Leishmania-infected macrophages by step-SPLITT fractionation by Mauricio Hoyos; Andrea Niño; Manuel Camargo; Juan Carlos Díaz; Sonia León; Marcela Camacho (3712-3718).
After a primary infection protocol of macrophages with Leishmania amazonensis, the percentage of infection drops as infection progresses and the uninfected population of macrophages mask the effects of infection for electrophysiological studies. In order to increase or maintain the infection percentage, we introduce an enrichment process after primary infection, which increases the possibility of following the infection longer times than any known process. A membraneless separation technique, step-SPLITT fractionation, implying flow and transverse gravity field in a ribbon-like channel, was used for enriching samples of macrophages infected with particles and with L. amazonensis. We demonstrate the capability of the s-SPLITT of generating, from a mixture resulting from a primary infection, an enriched and a depleted fraction with infected cells, without using any selective labeling pre-processing. It is also shown that a continuous sorting is possible without damaging cells and the losses of matter into the separation chamber is minimal.
Keywords: SPLITT fractionation; Macrophages; Leishmania; Bio-separation; Sedimentation;

Two simple, rapid, sensitive and economic chromatographic methods have been developed for determination of gemifloxacin mesylate in human plasma by using internal standard. First method depends on reverse phase high performance liquid chromatography. The plasma sample was extracted using chloroform:acetic acid (5.4:0.1, v/v). A concentration range from 30 to 600 ng/ml was used for calibration curve. The percent recovery of gemifloxacin mesylate was found to be 80.06–84.88. The mobile phase used consist of methanol:sodium acetate (1%):ortho phosphoric acid (65:35:0.5, v/v/v) with pH 2.1 and flow rate 0.8 ml/min in isocratic mode. The separation was carried out by UV-detector at wavelength 263 nm. Second method depends on high performance thin layer chromatography. The plasma sample was extracted using chloroform:acetic acid (5.9:0.1, v/v). A concentration range from 50 to 600 ng/spot was used for calibration curve. The percent recovery of gemifloxacin mesylate was found to be 80.01–86.17. The mobile phase used consists of ethyl acetate:methanol:ammonia (8.0:4.0:3.0, v/v/v). Densitometric analysis was carried out at wavelength 254 nm. The R f values for gemifloxacin mesylate and linezolide were 0.33 ± 0.03 and 0.69 ± 0.03 respectively. The stability of gemifloxacin mesylate in plasma was confirmed during three freeze–thaw cycles (−20 °C), on bench during 12 h. The proposed method was validated statistically and by performing recovery study for determination of gemifloxacin mesylate in human plasma.
Keywords: Gemifloxacin mesylate; RP-HPLC; HPTLC; Human plasma; Liquid–liquid extraction;

Determination of silodosin in human plasma by liquid chromatography–tandem mass spectrometry by Xia Zhao; Yuwang Liu; Junyu Xu; Dan Zhang; Ying Zhou; Jingkai Gu; Yimin Cui (3724-3728).
A rapid, sensitive and specific liquid chromatography–tandem mass spectrometric (LC–MS/MS) method has been developed and validated for the determination of silodosin in human plasma. Silodosin and internal standard (IS) were extracted from human plasma by liquid–liquid extraction using methyl t-butyl ether and analyzed on an Agilent C8 column with the mobile phase of acetonitrile–10 mM ammonium acetate (40:60, v/v) adjusted to pH 4.5 with acetic acid. Detection was carried out by MS/MS using TurboIonSpray (TIS) ionization and multiple reaction monitoring (MRM) in the positive-ion mode. The mass transitions monitored were m/z 496.3 → 261.4 and m/z 440.4 → 259.3 for silodosin and IS, respectively. The standard curve was linear in the range of 0.50–50.0 ng/ml with intra- and inter-day precision of 3.2–7.2% and 2.0–7.5%, respectively. The lower limit of quantification (LLOQ) for silodosin was 0.50 ng/ml using 500 μl plasma sample. This method was successfully applied to the pharmacokinetic study in healthy volunteers.
Keywords: Silodosin; α1A-Adrenergic receptor antagonist; LC–MS/MS;

A rapid HPLC method using a monolithic column with fluorescence detection has been developed for determination of telmisartan in human plasma. Sample preparation was done by protein precipitation with acetonitrile and naproxen was used as IS. The compounds were detected by fluorescence detection, using an excitation wavelength of 300 nm and emission wavelength of 385 nm. Calibration curves of telmisartan were linear in the range of 1–200 ng/mL. The assay was high throughput, sensitive and precise, and it was successfully applied to a bioequivalence study of two formulations of telmisartan.
Keywords: Telmisartan; Monolithic column; HPLC; Fluorescence detection; Plasma; Bioequivalence;

Raltegravir is the first antiretroviral agent to target the human immunodeficiency virus-1 (HIV-1) integrase. It is indicated, in association with other antiretrovirals, in the treatment of acquired immunodeficiency syndrome (AIDS) in antiretroviral treatment-experienced adult patients with viral resistance. To evaluate the feasibility of raltegravir therapeutic drug monitoring, we developed a rapid and specific analytical method for the quantification of raltegravir in human plasma by online sample clean-up liquid chromatography–tandem mass spectrometry (LC–MS/MS).After protein precipitation (with 100 μL of acetonitrile/methanol (50/50)) of 25 μL of plasma, fast online matrix-clean-up was performed using a column switching program. The chromatographic step was optimized to separate raltegravir and its glucuronide metabolite (G-raltegravir). Multiple reaction monitoring (MRM) was used for detection of raltegravir and G-raltegravir. In the absence of G-raltegravir standard, G-raltegravir identification was confirmed by β-glucuronidase pre-treatment.A total analysis of 3.8 min was needed to separate raltegravir to G-raltegravir. The method was linear between 10 and 3000 ng/mL for raltegravir. Analytical recovery was 94 ± 1%. Variation coefficients ranged between 5% and 8.4%. Pre-treatment of plasma from a patient under raltegravir treatment with β-glucuronidase suppressed G-raltegravir peak.We describe a fast online LC–MS/MS assay that is valid and reliable for the quantification of raltegravir, despite the lack of specificity that could occur in MRM scanning mode experiments.
Keywords: Raltegravir; Raltegravir glucuronide; Mass spectrometry; In-source transformation;

The levels of lysophosphatidic acid (LPA) or lysophosphatidylcholine (LPC) in plasma have been shown to be markers for several human diseases, including cancers. Here we show that the presence of LPC or other lysophospholipids (LPLs) in lipids extracted from biological samples affects accurate measurement of endogenous LPA in biological samples. We report for the first time the artificial conversion of LPC and lysophosphatidylserine (LPS) to LPA at the ion source of electrospray ionization tandem mass spectrometry (ESI-MS/MS). To avoid the interference of LPC with the quantification of LPA, a method based on high-performance liquid chromatography (HPLC) separation of LPA from LPC has been developed.
Keywords: High-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS); Lysophosphatidic acid (LPA); Lysophosphatidylcholine (LPC);

Direct determination of N-methyl-2-pyrrolidone metabolites in urine by HPLC-electrospray ionization-MS/MS using deuterium-labeled compounds as internal standard by Yoshihiro Suzuki; Yoko Endo; Masanori Ogawa; Shinobu Yamamoto; Akito Takeuchi; Tomoo Nakagawa; Nobuhiko Onda (3743-3747).
N-Methyl-2-pyrrolidone (NMP) has been used in many industries and biological monitoring of NMP exposure is preferred to atmospheric monitoring in occupational health. We developed an analytical method that did not include solid phase extraction (SPE) but utilized deuterium-labeled compounds as internal standard for high-performance liquid chromatography-electrospray ionization-mass spectrometry using a C30 column. Urinary concentrations of NMP and its known metabolites 5-hydoxy-N-methyl-2-pyrrolidone (5-HNMP), N-methyl-succinimide (MSI), and 2-hydroxy-N-methylsuccinimide (2-HMSI) were determined in a single run. The method provided baseline separation of these compounds. Their limits of detection in 10-fold diluted urine were 0.0001, 0.006, 0.008, and 0.03 mg/L, respectively. Linear calibration covered a biological exposure index (BEI) for urinary concentration. The within-run and total precisions (CV, %) were 5.6% and 9.2% for NMP, 3.4% and 4.2% for 5-HNMP, 3.7% and 6.0% for MSI, and 6.5% and 6.9% for 2-HMSI. The method was evaluated using international external quality assessment samples, and urine samples from workers exposed to NMP in an occupational area.
Keywords: N-Methyl-2-pyrrolidone; 5-Hydoxy-N-methyl-2-pyrrolidone; 2-Hydroxy-N-methylsuccinimide; Electrospray ionization-MS/MS; Deuterium-labeled compound; Biological monitoring;

A sensitive, simple and rapid high performance liquid chromatographic–tandem mass spectrometric (HPLC–MS/MS) method was developed for simultaneous quantitation of epothilone D and its major metabolite, the hydrolytic metabolite, epothilone C as internal standard in human plasma. Plasma samples were precipitated with acetonitrile, the analysis used a Venusil ASB C18 analytical column. A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive-ion mode. Selected reaction monitoring (SRM) using the precursor to product ion pairs of m/z 492.3 → 304.1 (epothilone D), m/z 510.3 → 492.3 (metabolite), m/z 478.3 → 290.1 (internal standard) was used for quantification. The analytical method was validated in terms of specificity, precision, accuracy, extraction recovery, stability, matrix effect and dilution effect. The linear calibration curves of epothilone D and metabolite were obtained over the concentration range of 0.2–1000 ng/ml and 5.0–1000 ng/ml, respectively. Lower limits of quantification (LLOQ) of epothilone D and metabolite were 0.2 ng/ml and 5.0 ng/ml, respectively. Due to its high sensitivity, specificity and simplicity, the method could be used for pharmacokinetic studies of both epothilone D and its hydrolytic metabolite.
Keywords: Epothilone D; Hydrolytic metabolite; HPLC–MS/MS; Pharmacokinetics;

A laboratory made capillary electrophoresis (CE) system using shorter capillary column was designed and constructed in our research group. This promising CE system was then connected with confocal fluorescence microscopy which was also developed in our research group. In order to show the applicability of this new system on chiral separation of dl-tryptophan (dl-Trp), studies were undertaken using cyclodextrins (CDs) and their derivatives; sulfated (S-CDs) and highly sulfated cyclodextrins (HS-CDs) as chiral selectors. Different selector concentrations were tested at various pH levels (pH 2.5, 4.0, 6.0, 9.0) to optimize the separation conditions. The best results were achieved at lower pH values (e.g. pH 2.5 and pH 4.0). HS-β-CD and HS-γ-CD were found to be the most effective chiral selectors with a shorter retention time at low selector concentrations.
Keywords: Capillary electrophoresis; Chiral separation; dl-Tryptophan; Cyclodextrin; Microfluidics;

A routine feasible HPLC analysis for the anti-angiogenic tyrosine kinase inhibitor, sunitinib, and its main metabolite, SU12662, in plasma by Marie-Christine Etienne-Grimaldi; Nicole Renée; Hassan Izzedine; Gérard Milano (3757-3761).
Sunitinib is an oral inhibitor of multiple tyrosine kinase receptors with antitumor activity in metastatic renal cell carcinoma. So far, published methods for analysis of sunitinib and its active metabolite (SU12662) in plasma are exclusively based on mass spectrometry. In the context of a large-scale feasibility pharmacokinetic analysis, we developed an original, simple, high-performance liquid chromatography (HPLC) assay with UV detection. A stability study of sunitinib and SU12662 in different light exposure conditions is presented. Due to photo-instability of the compounds, blood sampling and the whole handling procedure have to be performed quickly and with minimal light exposure (6–7 lx). Following single organic extraction with tert-butyl methyl ether, HPLC analysis was performed on an ODS column and UV detection was monitored at 369 nm (run time 15 min). This assay was selective and sensitive enough (limit of detection approximately 1 ng/ml) to quantify minimal concentrations at steady state (Css min) of sunitinib and SU12662 in treated patients.
Keywords: Sunitinib; HPLC; Pharmacokinetics;

Concurrent extractions of proteins and RNA from cell-laden scaffolds would be of great help to biomaterialists and tissue engineers. Here we describe a procedure to extract proteins from the discard solution generated during the RNA isolation from polysaccharide-rich hydrogel scaffolds. This approach allows to obtain proteins and RNA from same sample while eliminating the polysaccharide interference.
Keywords: Protein extraction; RNA; Tissue engineering; Biomaterials; Polysaccharide; Mass spectrometry;

Detection in urine of 4-methyl-2-hexaneamine, a doping agent by Laurent Perrenoud; Martial Saugy; Christophe Saudan (3767-3770).
Stimulants are banned in-competition for all categories of sports by the World Anti-Doping Agency. A simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay employing electrospray ionisation in positive mode was developed in that work for the quantification in urine specimens of 4-methyl-2-hexaneamine, a primary amine exhibiting sympathomimetic properties. Following a simple pretreatment procedure, the analyte was separated using a gradient mobile phase on reverse phase C8 column. Selected reaction monitoring m/z 116.2 → 57.3 was specific for detection of 4-methyl-2-hexaneamine and the assay exhibited a linear dynamic range of 50–700 ng/mL. The validated method has been successfully applied to analyze the target compound in food supplements as well as in urine specimens. The administered drug (40 mg) was detected at the level of 350 ng/mL in the urine up to 4 days.
Keywords: Doping; Stimulant; Mass spectrometry; Liquid chromatography; Gas chromatography;

Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography–tandem mass spectrometry: Application in basal and stimulated conditions by Ilias Perogamvros; Laura J. Owen; John Newell-Price; David W. Ray; Peter J. Trainer; Brian G. Keevil (3771-3775).
Immunoassays used for the measurement of salivary cortisol are limited by variable interference from cortisone. Salivary cortisone is a consequence of the salivary glands expressing 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) which converts cortisol to cortisone. We report a combined salivary cortisol and cortisone (SalF and SalE respectively) liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay to address the cortisone cross-reactivity in cortisol immunoassays and as a tool to study 11β-HSD2 activity. The method was linear up to 400 nmol/L for SalF and 200 nmol/L for SalE and the lower limits of quantitation were 0.39 nmol/L (SalF) and 0.78 nmol/L (SalE). No evidence of ion suppression was found and precision, accuracy and recovery were within internationally accepted limits. No interference was identified from 13 structurally related steroids. SalF, SalE and SalF/SalE were significantly greater in the morning than at bed-time and following stimulation of the adrenal glands. As serum cortisol increased, an exponential rise was observed in SalF and a linear increase in SalE which reached a plateau at higher SalF concentrations. We have developed a novel, robust LC–MS/MS assay for the combined measurement of SalF and SalE. Our results confirm the 11β-HSD2 activity of the salivary glands resulting in high SalE concentrations and the enzyme saturation at high substrate concentrations. This method can be used as a simple, non-invasive and highly specific tool to assess the value of salivary cortisol as a surrogate for free serum cortisol and as a potential novel way to assess 11β-HSD2 activity.
Keywords: Liquid chromatography–tandem mass spectrometry (LC–MS/MS); Salivary cortisol; Salivary cortisone; 11β-HSD2;

Thirty-five aliphatic acids were identified by gas chromatography–mass spectrometry from the ether extract of commercial preparations containing lyophilized royal jelly. The article presents linear-programmed retention indices on capillary columns with non-polar and low-polar stationary phases and mass spectra for identified compounds which were not characterized earlier by these parameters. Nine compounds are reported for the first time as royal jelly constituents.
Keywords: Royal jelly; Hydroxy fatty acids; Retention indices; Mass spectra;