Journal of Chromatography B (v.877, #7)

Solid-phase microextraction using poly(pyrrole) film and liquid chromatography with UV detection for analysis of antidepressants in plasma samples by Andréa Rodrigues Chaves; Glaico Chiericato Júnior; Maria Eugênia Costa Queiroz (587-593).
Poly(pyrrole) (PPY) coating was prepared on a stainless-steel (SS) wire for solid-phase microextraction (SPME) by electrochemical deposition (cyclic voltammetric). The PPY was evaluated by analyzing new-generation antidepressants (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline) in plasma sample by SPME and liquid chromatography with UV detection (LC-UV). The effect of electrolyte solution (lithium perchlorate or tetrabutylammonium perchlorate) and the number of cycles (50, 100 or 200) applied during the polymerization process on the SPME performance was evaluated. Important factors in the optimization of SPME efficiency such as extraction time, temperature, pH, influence of plasma proteins on sorption mechanisms, and desorption conditions are discussed. The SPME–PPY/LC method showed to be linear in concentrations ranging from the limit of quantification (LOQ) to 1200 ng mL−1. The LOQ values range from 16 to 25 ng mL−1. The inter-day precision of the SPME–PPY/LC method presented coefficient of variation (CV) lower than 15%. Based on analytical validation results, the SPME–PPY/LC methodology showed to be adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the SPME–PPY/LC method was applied to the analysis of plasma samples from elderly depressed patients.
Keywords: Antidepressants; Poly(pyrrole); SPME; Liquid chromatography;

A highly efficient and low-cost affinity chromatography strategy for lysozyme (LZM) purification is reported. Using tris(hydroxymethyl)aminomethane (Tris) as ligand and macroporous silica spheres as matrix, a novel affinity column was prepared. The high specificity, stability and repeatability of this Tris immobilized affinity column were proved by LZM separations from protein mixture solutions for 20 circles and 6 months test. LZM purified from chicken egg white on the Tris affinity column had even higher purity than the commercial standard and well-maintained activity of 8287 U/mg (activity of commercial LZM was 8171 U/mg). The efficient affinity process avoiding expensive or fragile ligand would bring advantages to the routine production of LZM from chicken egg white.
Keywords: Lysozyme; Tris(hydroxymethyl)aminomethane; Affinity chromatography; Protein purification;

Extraction of PCR-ready DNA from Staphylococcus aureus bacteriophages using carboxyl functionalized magnetic nonporous microspheres by Jana Kahánková; Alena Španová; Roman Pantůček; Daniel Horák; Jiří Doškař; Bohuslav Rittich (599-602).
Magnetic microspheres P(HEMA-co-EDMA) were used for PCR-ready phage DNA isolation from lysogenic strains of Staphylococcus aureus, including two new clinical isolates. The conditions of phage particle lysis were optimized. The quality of eluted phage DNA was evaluated by PCR. It was demonstrated that PCR-ready phage DNA can be isolated from small volumes of phage lysates (150 μl) by magnetic microspheres. The reported method is very expeditious without using toxic compounds such as phenol or chloroform. It can be used for phage identification and phage gene detection.
Keywords: Magnetic microspheres; P(HEMA-co-EDMA); Staphylococcus aureus; Bacteriophage DNA; Polymerase chain reaction (PCR);

Plasma tryptophan, kynurenine and 3-hydroxykynurenine measurement using automated on-line solid-phase extraction HPLC–tandem mass spectrometry by Wilhelmina H.A. de Jong; Reinier Smit; Stephan J.L. Bakker; Elisabeth G.E. de Vries; Ido P. Kema (603-609).
Tryptophan metabolism plays a key role in several (patho)physiological conditions. In order to study the clinical importance of tryptophan and its predominant metabolites (kynurenines), it is important to be able to measure large series of samples with high accuracy and reliability. We aimed to develop a high-throughput on-line solid-phase extraction-liquid chromatographic–tandem mass spectrometric (XLC–MS/MS) method that enables the measurement of tryptophan and its metabolites kynurenine and 3-hydroxykynurenine in plasma. Fifty microliters plasma equivalent was pre-purified by automated on-line solid-phase extraction, using strong cation exchange (PRS, propylsulphonic) cartridges. Chromatographic separation of the analytes and deuterated analogues occurred by C18 reversed phase chromatography. Mass spectrometric detection was performed in the multiple reaction-monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Total run-time including sample clean-up was 8 min. Intra- and inter-assay analytical variations were less than 9%. Linearity in the 0.11–1200 (tryptophan) and 0.050 and 0.023–45 μmol/L (kynurenine and 3-hydroxykynurenine, respectively) calibration range was excellent (R  > 0.99). Detection limits were 30 nmol/L for tryptophan, 1 nmol/L for kynurenine and 5 nmol/L for 3-hydroxykynurenine. Reference intervals for 120 healthy adults were 45.5–83.1 μmol/L (tryptophan), 1.14–3.02 μmol/L (kynurenine), <0.13 μmol/L (3-hydroxykynurenine) and 19.0–49.8 for tryptophan-to-kynurenine ratio. Blood sampling for tryptophan and tryptophan-to-kynurenine ratio should be performed before breakfast, due to biological variation during the day. This study describes how plasma tryptophan, kynurenine and 3-hydroxykynurenine can be measured accurately and precisely by automated high-throughput XLC–MS/MS.
Keywords: Tryptophan; Kynurenine; Tryptophan-to-kynurenine ratio; IDO; 3-hydroxykynurenine; On-line SPE; Mass spectrometry; XLC–MS/MS; Plasma;

Measurement of pentosidine in human plasma protein by a single-column high-performance liquid chromatography method with fluorescence detection by Jean L.J.M. Scheijen; Marjo P.H. van de Waarenburg; Coen D.A. Stehouwer; Casper G. Schalkwijk (610-614).
A rapid and sensitive single-column high-performance liquid chromatography method and application for the detection of protein bound pentosidine is described. Pentosidine, a cross-link between arginine and lysine, is a well-characterized advanced glycation endproduct. In order to detect protein-bound pentosidine, plasma proteins were hydrolysed in 6 N HCl. Detection of pentosidine is done based on its own fluorescence characteristics using fluorimetric detection (E x  = 325 nm, E m  = 385 nm). Separation is done, with a run-to-run time of 30 min, on a C18 Allspehere ODS-II column with a citric acid acetonitrile buffer. This detection enables sensitive and specific determination of protein bound pentosidine in plasma with a detection limit of 2.2 nmol/l or 0.02 pmol/mg protein (signal-to-noise: 6). The intra-assay coefficient variation is 6.5% at a plasma pentosidine concentration of 0.47 pmol/mg protein and 2.0% at a concentration of 1.27 pmol/mg protein. The inter-assay coefficient variation is 3.1% at a plasma pentosidine concentration of 0.43 pmol/mg protein and 1.6% at a concentration of 1.40 pmol/mg protein. Linearity is tested in 4 different plasma samples and showed linearity (0–200 nmol/l, r 2  > 0.99). Recovery of pentosidine in 4 different plasma samples at different concentration levels is 102 ± 10% (mean ± SD). Using this method protein bound pentosidine concentration is investigated in healthy controls (n  = 24, age 67 ± 9 years) and patients with end stage renal disease (n  = 24, age 65 ± 10 years). Higher plasma concentrations of protein bound pentosidine are measured in the patient group as compared with the control group 3.05 (2.03–3.92) pmol/mg protein and 0.21 (0.19–0.33) pmol/mg protein, respectively (median (interquartile range), p  < 0.00001). These results are consistent with previously reported results.
Keywords: HPLC; Fluorescence; Pentosidine; Peritoneal dialysis;

Artificial neural networks (ANNs) were used in conjunction with an experimental design to optimise a gradient HPLC separation of nine benzodiazepines. Using the best performing ANN, the optimum conditions predicted were 25 mM formate buffer (pH 2.8), 10% MeOH, acetonitrile (ACN) gradient 0–15 min, 6.5–48.5%. The error associated with the prediction of retention times and peak widths under these conditions was less than 5% for six of the nine analytes. The optimised method, with limits of detection (LODs) in the range of 0.0057–0.023 μg/mL and recoveries between 58% and 92%, was successfully applied to authentic post-mortem samples. This method represents a more flexible and convenient means for optimising gradient elution separations using ANNs than has been previously reported.
Keywords: Artificial neural networks (ANNs); Gradient elution; Optimisation; HPLC; Benzodiazepines;

Simultaneous determination of androstenedione, 11β-hydroxyandrostenedione, and testosterone in human plasma by stable isotope dilution mass spectrometry by Akitomo Yokokawa; Kazuhiro Yamamoto; Yuhei Omori; Hiromi Shibasaki; Yoshihiko Shinohara; Yasuji Kasuya; Takashi Furuta (621-626).
This study describes a GC–MS method for the simultaneous determination of androstenedione (AD), 11β-hydroxyandrostenedione (11β-OHAD), and testosterone (TS) in human plasma. [19,19,19-2H3]Androstenedione (AD-2H3), 11β-hydroxy-[1,2,4,19-13C4]androstenedione (11β-OHAD-13C4), and [1,16,16,17-2H4]testosterone (TS-2H4) were used as internal standards. Pentafluoropropionic (PFP) derivatization with good GC behavior was employed for the GC–MS analysis of the three steroids. The detection limit of the present GC–MS–SIM method was found to be 1 pg per injection for AD (S/N ratio = 4.5), 5 pg for 11β-OHAD (S/N ratio = 5.0), and 1 pg for TS (S/N ratio = 4.4), respectively. Calibration curves were linear from 0.22 to 2.80 ng/mL (r  = 0.9998) for AD, from 0.56 to 3.19 ng/mL (r  = 0.9996) for 11β-OHAD, and from 2.05 to 10.3 ng/mL (r  = 0.9996) for TS. The intra- and inter-day assay reproducibilities in the amounts of the three androgens determined were in good agreement with the actual amounts added, the relative errors (R.E.) were −3.1 to 2.4%. The inter-assay relative standard deviation (R.S.D.) was less than 5.3%. The present method provides a sensitive and reliable technique for the simultaneous determination of AD, 11β-OHAD, and TS in plasma. The method can be applied to pharmacokinetic and metabolic studies of androgens with a particular interest in evaluating the conversion of AD to 11β-OHAD and the interconversion of AD and TS in humans.
Keywords: GC–MS; Stable isotope; Androstenedione; 11β-Hydroxyandrostenedione; Testosterone;

Analysis of the protein complex associated with 14-3-3 epsilon by a deuterated-leucine labeling quantitative proteomics strategy by Shufang Liang; Yanbao Yu; Pengyuan Yang; Sheng Gu; Yan Xue; Xian Chen (627-634).
By using an unambiguous in vivo deuterated-leucine labeling quantitative proteomic approach, at close to the physiologically relevant level, we systematically profiled multiple proteins interacting with 14-3-3ɛ, the isoform with least characterized protein interactions in 14-3-3 family in mammalian cells. Among the 19 proteins interacting with 14-3-3ɛ identified, 6 of them including SKb1Hs, p54nrb, serine/threonine kinase 38, MEP50, 14-3-3θ and cofilin 2 were the previously unknown interacting partners with 14-3-3ɛ. The newly identified interactor cofilin 2 was also validated in co-transfection and co-immunoprecipitation. In contrast, with the same stringent criteria only three known partners were identified by conventional tandem affinity purification (TAP) approach. Therefore the ‘in-spectra’ quantitative marker of deuterated-leucine assisted to precisely identify those genuine interacting partners with minimum requirement of validation using other molecular approaches.
Keywords: 14-3-3ɛ interacting proteins; Epitope affinity tag; Deuterated-leucine; Stable isotope labeling;

A liquid chromatography tandem mass spectrometry (LC/MS n ) method for the determination of 12 corticosteroids in bovine liver has been optimized and validated in accordance with the European Commission Decision 2002/657/EC. A bovine liver sample was deconjugated with β-glucuronidase/sulfatase enzyme, extracted with diethyl ether and further cleaned up with Solid Phase Extraction (SPE) before analysis with LC/MS n . Two different enzyme extracts (originating from Helix Pomatia and Keyhole Limpet) and three SPE elution solvents (ethyl acetate, acetonitrile and methanol) were compared during the optimization. Helix Pomatia is generally known as the enzyme most being used for enzymatic hydrolysis purposes. Nevertheless, when detecting corticosteroids in the low μg kg−1 concentration range, the Helix Pomatia extract may lead to interferences in the final LC/MS n chromatogram. When using the Keyhole Limpet enzyme extract, no interferences were observed and therefore, this extract was the best choice for enzymatic hydrolysis tested in this case. Ethyl acetate was used as elution solvent during the validation procedure since SPE elution with acetonitrile resulted in higher chromatographic backgrounds, while elution with methanol showed less reproducible results. Validation of the optimized method was carried out for 10 of the 12 corticosteroids, giving mean recoveries between 91 and 109%, and repeatability and reproducibility coefficients of respectively maximum 13.7 and 18.0%. The working ranges for the linear calibration curves were 5–20 μg kg−1 for prednisolone, methylprednisolone and prednisone and 0.5–4 μg kg−1 for the other compounds (coefficients of determination R 2  ≥ 0.97). Specificity, decision limit (CCα) and detection capability (CCβ) were for all compounds within the EC specified limits.
Keywords: Corticosteroids; Liquid chromatography tandem mass spectrometry; Spectrometry; Bovine liver; Keyhole Limpet; Validation;

A high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of rosiglitazone and gemfibrozil in human plasma using α-asarone as an internal standard (IS). Plasma samples were pretreated by protein precipitation. The analytes were separated on a Macherey–Nagel Nucleodur C18 (250 mm × 4.6 mm, 5 μm) column using acetonitrile and 30 mmol/l ammonium acetate solution (including 0.1% methanoic acid) as mobile phase which was delivered at 1.2 ml/min. A gradient elution program was adopted to adjust proportion of solvent in mobile phase. A time program was used to regulate conditions of fluorescence detection. The method was validated over the concentration range of 5.0–751.3 ng/ml for rosiglitazone and 0.5–75.4 μg/ml for gemfibrozil with acceptable accuracy, precision and extraction recoveries. This method is suitable for routine therapeutic drug monitoring and pharmacokinetic interaction study of the two drugs.
Keywords: Rosiglitazone; Gemfibrozil; HPLC;

Rapid and sensitive hydrophilic interaction chromatography/tandem mass spectrometry method for the determination of glycyl-sarcosine in cell homogenates by Yongbing Sun; Jin Sun; Jianfang Liu; Shiliang Yin; Ying Chen; Peng Zhang; Xiaohui Pu; Yinghua Sun; Zhonggui He (649-652).
A rapid, selective and sensitive hydrophilic interaction chromatography/tandem mass spectrometry (HILIC/MS/MS) was developed and validated for the determination of glycyl-sarcosine (Gly-Sar) in Caco-2 cell homogenates. After a simple protein precipitation with acetonitrile, the analyte was separated on a HILIC column and detected by a triple quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear among the concentration range of 1–2000 ng/mL for Gly-Sar and the lower limit of quantification (LLOQ) was 1 ng/mL using as little as 50 μL of cell homogenates. The intra-day and inter-day relative standard deviations (RSD) were less than 15% and the relative errors (RE) were all within ±15%. The validated method was successfully employed in the study of Gly-Sar uptake inhibition in Caco-2 cells by valcytarabine, a potential substrate of the peptide transporter 1 (PEPT1).
Keywords: Hydrophilic interaction chromatography/tandem mass spectrometry; Gly-Sar; Caco-2 cell homogenates;

A rapid and sensitive method for the determination of visnagin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS). The plasma samples were processed by protein precipitation with methanol:acetonitrile (25:75) and warfarin was used as the internal standard. After vortex mixing and centrifugation, the supernatants were diluted with water (1:5) and then directly injected onto a Phenomenex Synergi Max RP column (75 mm × 2.0 mm ID, 4 μm) with isocratic elution at a flow rate of 0.2 mL/min. The mobile phase consisted of water and methanol (15:85, v/v) containing 0.1% formic acid and 5 mM ammonium acetate. The total run time (injection to injection) was less than 4 min. Detection of the analytes was achieved using positive ion electrospray in the selected reaction monitoring (SRM) mode. The linear standard curve ranged from 1.0 to 5000 ng/mL and the precision and accuracy (inter- and intra-run) were within 4.5% and 4.3%, respectively. The method, which is rapid, simple, and precise, was successfully used to support a visnagin pharmacokinetics study.
Keywords: Visnagin; LC–MS; Pharmacokinetics; Ammi visnaga;

An improved analytical assay was developed and validated for the quantification of milrinone concentrations in patients undergoing cardiac surgery. A solid-phase extraction was optimized to isolate milrinone from a plasma matrix followed by HPLC using UV detection. Plasma samples (1 ml) were extracted using a C18 solid-phase cartridge. Milrinone was separated on a strong cation exchange analytical column maintained at 23.4 °C. The mobile phase consisted of a gradient (10:90 to 45:55), 0.05 M phosphate buffer (pH 3):acetonitrile. Calibration curves were linear in the concentration range of 1.25–320 ng/ml. Mean drug recovery and accuracy were respectively ≥96% and ≥92%. Intra- and inter-day precisions (CV%) were ≤6.7% and ≤7.9%, respectively. This method proved to be reliable, specific and accurate. Using different types of column for extraction and separation of milrinone proved to be necessary to achieve the sensitivity and specificity required when milrinone is given by inhalation.
Keywords: Milrinone; HPLC; Assay; Validation; Inhalation; Cardiac surgery;