Journal of Chromatography B (v.877, #24)

High throughput screening of mixed-mode sorbents and optimisation using pre-packed lab-scale columns for the purification of the recombinant allergen rBet v 1a by Virginie Brenac Brochier; Henri Chabre; Aurélie Lautrette; Vincent Ravault; Marie-Noëlle Couret; Alain Didierlaurent; Philippe Moingeon (2420-2427).
Mixed-mode chromatography was investigated for the purification of the recombinant allergen rBet v 1a expressed in Escherichia coli (E. coli) and used as an active principle for specific immunotherapy (SIT) treatment against birch pollen allergy. The screening of micro-volumes of three mixed-mode sorbents established that rBet v 1a could be captured without any pre-treatment of the crude feedstock on HEA or PPA HyperCel sorbents equilibrated in “physiological-like” conditions. On a mini-column pre-packed with PPA HyperCel sorbent, rBet v 1a was recovered at pH 4, partially separated from a major methionine Bet v 1 contaminant and purified approximately 9-fold in a single step (85% purity).
Keywords: Recombinant allergen; rBet v 1a; High-throughput screening; Mixed-mode sorbents; HEA HyperCel; PPA HyperCel; MEP HyperCel; Pre-packed columns;

Rapid screening of purification strategies for the capture of a human recombinant F(ab′)2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS by J. Pezzini; V. Brenac Brochier; M.P. Barrouillet; M. Cerruti; G. Clofent-Sanchez; A. Schapman; A. Topol; R. Robert; C. Cabanne; P. Cerruti; X. Santarelli (2428-2434).
The development of a capture step of a human recombinant F(ab′)2 produced and expressed in baculovirus-infected cells was investigated by screening three mixed-mode chromatography sorbents (HEA HyperCel™, PPA HyperCel and MEP HyperCel) and two ion exchangers (Q Ceramic HyperD® F, S Ceramic HyperD F) sorbents using a 96-well plate format and SELDI-MS. HEA HyperCel gave the best separation performance therefore the conditions tested in micro-plate were transferred to laboratory scale chromatographic experiments, confirming that the recombinant F(ab′)2 was effectively captured on the mixed-mode sorbent without any pre-treatment of the crude extract with a 82% recovery and a 39-fold purification.
Keywords: F(ab′)2; Screening; Mixed-mode; Ion exchanger; Chromatography sorbents; HEA HyperCel; PPA HyperCel; MEP HyperCel; Q Ceramic HyperD F; S Ceramic HyperD F;

Analysis of free and bound NADPH in aqueous extract of human placenta used as wound healer by Debashree De; Piyali Datta Chakraborty; Debasish Bhattacharyya (2435-2442).
NADPH is an important biomolecule involved in cellular regeneration. The distribution of free and bound NADPH in aqueous extract of human placenta used as a potent wound healer has been analyzed. Quantification from fluorescence and immuno-affinity chromatography indicates that 75.1 ± 2.2% of NADPH present in the extract exists as free nucleotide or bound to very small peptides or amino acids whereas the rest remains bound to large peptides. Inability to dissociate the bound form of the nucleotide from the large peptides using urea or guanidium hydrochloride indicates that the binding is covalent. Identification of a fragmented mass of m/z 382.94 (nicotinamide + sugar + phosphate) from the NADPH-peptide conjugates supported the intactness of the nicotinamide moiety. Glutathione reductase assay indicated that 95.2 ± 3.5% of the total NADPH pool of the extract can act as cosubstrate of the enzyme. This indicates that while a major fraction of free NADPH of the extract is easily available for cellular processes, the rest can also function locally where the conjugated peptides are deposited.
Keywords: Human placental extract; Placental peptides; Free and bound NADPH; Nicotinamide moiety; Mass analysis; Glutathione reductase assay;

Comparative study of strong anion exchangers: Structure-related chromatographic performances by Jerome Pezzini; Charlotte Cabanne; Xavier Santarelli (2443-2450).
Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure and allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we suggested an independent evaluation of major anion exchangers to facilitate media selection, and investigated the relationship between (i) surface modification and (ii) chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained mainly high capacities even with extremely high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity and could contribute to cost reduction.
Keywords: Comparative study; Dynamic binding capacity; Anion exchanger; Ligand accessibility; Surface modification;

An improved liquid chromatography–mass spectrometry method for the determination of pemetrexed in human plasma was developed and validated using a simple quadrupole LC–MS and a new SPE cartridge (Plexa® Bond Elut). The analysis was achieved with a C18 analytical column using a mobile phase consisting of formic acid/acetonitrile and isocratic flow for 7 min. The linear ranges (r 2  > 0.99) were found from 5 to 5000 ng/mL. The lower limit of detection was 2.5 ng/mL. Within-day and between-day precisions were less than 7.2% and inaccuracy did not exceed 2.8%. This new method is suitable to support pharmacokinetic studies and drug monitoring.
Keywords: Pemetrexed; Liquid chromatography/mass spectrometry; Plasma; Pharmacokinetic studies;

We have developed a liquid chromatography tandem mass spectrometric (LC–MS/MS) method for the simultaneous quantitative analysis of several free forms of steroid hormones in bovine serum [pregnenolone (P5), progesterone (P4), 17hydroxyP5, 17hydroxyP4, testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (A), estrone (E1), 2, 4 and 16 hydroxyE1, 2 and 4 methoxyE1]. Deuterated analogs were used as internal standards. Serum proteins were eliminated with acetonitrile. Oxime derivatives of steroids were extracted with tert-butylmethylether and analyzed in positive MRM mode. Methodology was validated in accordance with the European Commission Decision 2002/657/EC. Performance characteristics permit the use of this methodology for steroid determination in animal serum samples.
Keywords: Hormone; LC–MS/MS; Bovine serum;

Comparative determination of methyl mercury in whole blood samples using GC–ICP-MS and GC–MS techniques by J. Hippler; H.W. Hoppe; F. Mosel; A.W. Rettenmeier; A.V. Hirner (2465-2470).
Two methods for the determination of methyl mercury (MeHg) in whole blood samples based on different mass spectrometric detection techniques are compared. The methods were employed in two studies in which the internal exposure of a group of mercury-exposed workers to total mercury and MeHg was investigated. Blood samples of these workers were analysed for MeHg independently from each other in two laboratories using similar extraction procedures but different detection techniques, viz. coupled GC–EI-MS/ICP-MS and GC–MS using D3-MeHg as internal standard. MeHg was detected in all blood samples in concentrations ranging from 0.3 to 9.0 μg/L. Though different detection techniques were employed, the results obtained by the two laboratories were in relatively good agreement.
Keywords: Methylmercury; Biological monitoring; GC–MS; ICP-MS;

A capillary zone electrophoresis (CZE) method was validated for the analysis of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and performed on a fused-silica capillary, with detection at 195 nm. The background electrolyte solution consisted of 50 mM sodium tetraborate solution at pH 9. The method was linear in the concentration range of 1–200 μg/mL and the limit of quantitation (LOQ) was 1 μg/mL, with acceptable validation parameters. The method was applied for the analysis of pharmaceutical formulations, and the results were correlated to the reversed-phase HPLC method (RP-HPLC), size-exclusion HPLC method (SE-HPLC) and in vitro bioassay method.
Keywords: Capillary zone electrophoresis; GNFS-60 cell culture assay; Liquid chromatography; Recombinant human granulocyte colony-stimulating factor; Validation;

Rapid and easy method for monitoring oxidative stress markers in body fluids of patients with asbestos or silica-induced lung diseases by Kamila Syslová; Petr Kačer; Marek Kuzma; Věra Najmanová; Zdeňka Fenclová; Štěpánka Vlčková; Jindriška Lebedová; Daniela Pelclová (2477-2486).
Sensitive assay method was developed for a parallel, rapid and precise determination of the most prominent oxidative stress biomarkers: 8-iso-prostaglandin F, malondialdehyde and 4-hydroxynonenal. The method consisted of a pre-treatment part a solid-phase extraction, for rapid and effective isolation of biomarkers from body fluids (exhaled breath condensate, plasma and urine) and the detection method LC–ESI-MS/MS, where the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the imprecision was below 14.3%, the mean inaccuracy was determined to be lower than 13.1%. The method was tested on samples obtained from patients diagnosed with asbestosis, pleural hyalinosis or silicosis, i.e. occupational lung diseases caused by fibrogenic dusts, inducing oxidative stress in the respiratory system, and then compared to samples from healthy subjects. The difference in concentration levels of biomarkers between the two groups was perceptible in all the body fluids (the difference observed in an exhaled breath condensate was statistically most significant).
Keywords: 8-iso-Prostaglandin F; 4-Hydroxynonenal; Malondialdehyde; Exhaled breath condensate; Solid-phase extraction; LC–MS/MS;

Isolation and purification of three flavonoid glycosides from the leaves of Nelumbo nucifera (Lotus) by high-speed counter-current chromatography by Shengguo Deng; Zeyuan Deng; Yawei Fan; You Peng; Jing Li; Dongmei Xiong; Rong Liu (2487-2492).
Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of flavonoid glycosides from the leaves of Nelumbo nucifera (Lotus) by using a two-phase-solvent system composed of n-hexane–ethyl acetate–methanol–water (1:5:1:5, v/v/v/v). The targeted compounds isolated, collected and purified by HSCCC were analyzed by high performance liquid chromatography (HPLC). A total of 4.6 mg of isoquercitrin, 9.1 mg of hyperoside and 3.0 mg of astragalin with the purity of 95.8%, 97.5% and 98.3%, respectively, were obtained in one-step separation and less than 6 h from 80 mg of crude extract from the leaves of N. nucifera. The chemical structures of all the three compounds were identified by MS, 1H NMR, 13C NMR. Astragalin was obtained from N. nucifera for the first time.
Keywords: Counter-current chromatography; Flavonoid glycosides; Isoquercitrin; Hyperoside; Astragalin;

Determination of the antifungal agent posaconazole in human serum by HPLC with parallel column-switching technique by Werner Neubauer; Armin König; Richard Bolek; Rainer Trittler; Monika Engelhardt; Manfred Jung; Klaus Kümmerer (2493-2498).
Posaconazole is a new broad-spectrum antifungal agent that is currently only available as an oral suspension and shows high intra- and inter-individual differences in oral bioavailibility. Pre-existing methods for the determination of the substance involve the use of internal standards or require a quite complicated and time-consuming sample pre-treatment. Our HPLC method is fast and fully-automated and there is no need for any manual sample pre-treatment. On-line transfer of posaconazole from the extraction column was followed by chromatographic separation on a C18 column and fluorescence detection (λ ex: 261 nm, λ em: 357 nm). Retention time of posaconazole was about 11.7 min, the lower limit of quantification was found to be 0.1 mg/l. A linear calibration curve was obtained over the concentration range of 0.1–5 mg/l using a 50 μl sample (r 2  = 0.999). The relative standard deviations of intra-day variations ranged from 2.3% to 9.4%, intra-day accuracy from 88.8% to 114.8%.
Keywords: Posaconazole; Bioavailability; Serum; HPLC; Column-switching; On-line sample preparation;

Mitragynine is the primary active alkaloid extracted from the leaves of Mitragyna speciosa Korth, a plant that originates in South-East Asia and is commonly known as kratom in Thailand. Kratom has been used for many centuries for their medicinal and psychoactive qualities, which are comparable to that of opiate-based drugs. Kratom abuse can lead to a detectable content of mitragynine residue in urine. Ultra trace amount of mitragynine in human urine was determined by a high performance liquid chromatography coupled to an electrospray tandem mass spectrometry (HPLC-ESI/MS/MS). Mitragynine was extracted by methyl t-butyl ether (MTBE) and separated on a HILIC column. The ESI/MS/MS was accomplished using a triple quadrupole mass spectrometer in positive ion detection and multiple reactions monitoring (MRM) mode. Ajmalicine, a mitragynine's structure analog was selected as internal standard (IS) for method development. Quality control (QC) performed at three levels 0.1, 1 and 5 ng/ml of mitragynine in urine gave mean recoveries of 90, 109, and 98% with average relative standard deviation of 22, 12 and 16%, respectively. The regression linearity of mitragynine calibration ranged from 0.01 to 5.0 ng/ml was achieved with correlation coefficient greater than 0.995. A detection limit of 0.02 ng/ml and high precision data within-day and between days analysis were obtained.
Keywords: Kratom; Mitragynine; Urine drug test; Forensic; HPLC-MS/MS;

Two-dimensional-HPLC procedures have been established for the sensitive and selective determination of d-serine (d-Ser) and d-alanine (d-Ala), and their amounts in the tissues and physiological fluids of mice with various d-amino-acid oxidase (DAO) activities have been demonstrated. These two d-amino acids are modulators of the N-methyl-d-aspartate receptor mediated neurotransmission, and the alterations in their amounts following the changes in the DAO activity are matters of interest. After pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the d-amino acids were determined by the 2D-HPLC system with fluorescence detectors. As the first dimension, a microbore-monolithic-ODS column (750 mm × 0.53 mm I.D.) was adopted and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm × 1.5 mm I.D.) was selected for the second dimension. The lower limits of quantitation of d-Ser and d-Ala were 500 amol, and the within-day and day-to-day precisions were less than 6.8%. Using these methods, the amounts of d-Ser and d-Ala in 6 brain tissues, 4 peripheral tissues, serum and urine of mice having various DAO activities were determined; the amounts of these d-amino acids were drastically increased with a lowering of the DAO activity except for the cases of d-Ser in the frontal brain regions. The present micro-2D-HPLC procedures are powerful tools for the determination of small amounts of d-Ser and d-Ala in mammalian samples, and the obtained results would be useful for developing novel drugs that modulate the DAO activity, such as DAO inhibitors, against neuronal diseases.
Keywords: d-Serine; d-Alanine; d-Amino-acid oxidase; 2D-HPLC; Enantiomer separation;

Comparison between high performance liquid chromatography and capillary zone electrophoresis for the diagnosis of congenital disorders of glycosylation by Ester Quintana; Raquel Montero; Mercedes Casado; Aleix Navarro-Sastre; María A. Vilaseca; Paz Briones; Rafael Artuch (2513-2518).
Transferrin isoelectric focusing (IEF) is the most widely used method to screen for congenital disorders of glycosylation (CDG). Our aim was to compare high performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) procedures for serum sialotransferrin analysis. 58 serum samples were processed both by CZE and HPLC: 35 were from paediatric controls, 18 from patients with an altered sialotransferrin IEF pattern and 5 were transferrin variant samples. HPLC analysis was performed with an anion-exchange column with spectrophotometric detection at 470 nm. CZE analysis was done using the commercial CEofix-CDT kit with spectrophotometric detection at 200 nm. Passing-Bablok regression analysis showed good agreement for tri-, tetra- and penta-sialotransferrin by both procedures. But for disialotransferrin, higher values were observed by the HPLC procedure. The HPLC and CZE methods allowed reproducible separation and analysis of single transferrin glycoforms with similar peak patterns. All patients presented values outside the range established in our control population either by HPLC or by CZE, even in patients with moderate forms of CDG that had been difficult to detect by IEF. In conclusion, measurement of sialotransferrin isoforms and interpretation using method-specific reference values may offer some advantages for the diagnosis of CDG as compared with the standard IEF procedure.
Keywords: Congenital disorders of glycosylation; High performance liquid chromatography; Capillary zone electrophoresis; Isoelectric focusing; Paediatric patients;

The development and validation of a bioanalytical assay is described for the simultaneous analysis in human serum of tamoxifen, four of its main metabolites and three flavonoids, which are known constituents in alternative medicine and dietary supplements often used by breast cancer patients. The method has been fully validated at linear ranges covering steady-state serum concentrations in patients who receive therapeutic dosages of tamoxifen. The wide range also allows for quantification of large inter-patient fluctuations of flavonoid concentrations. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of a protein precipitation with acetonitrile using only 50 μL serum. The described method is simple, robust and reproducible with inter- and intra-assay accuracies within 85–115%. The applicability of the assay was demonstrated and it is now successfully used to study the in vivo pharmacokinetics of tamoxifen, its main metabolites and flavonoids in human serum of patients receiving tamoxifen.
Keywords: Tamoxifen; Endoxifen; Flavonoids; Metabolites; Liquid chromatography; Tandem mass spectrometry;

A method for determination of the plasmid DNA concentration with subsequent analysis of the ratio supercoiled to open circular form is presented. The method is suitable for samples from all steps of the manufacturing process, from fermentation to final product. The analysis consists of size exclusion chromatography, followed by analytical thiophilic aromatic chromatography. In the first step, the plasmid DNA concentration is determined by group separation, including removal of RNA and other impurities, within less than 2 min. The limit of detection and quantification was 0.28 and 0.83 μg/ml, respectively. The precision of the method is high, providing a coefficient of variation as low as below 2%. In the second step, the ratio of open circular to supercoiled plasmid DNA is determined following separation of the two plasmid DNA isoforms with a linear salt gradient. The precision of the second step was evaluated using serial injections of aliquots of a sample stock solution. In comparison with the two most commonly used methods, the developed analysis was found to be significantly more accurate than agarose gel electrophoresis and equivalent to capillary gel electrophoresis. The combined methods for quantification and control of homogeneity of plasmid DNA presented here enable reliable and precise analysis at all steps of the manufacturing process.
Keywords: Concentration determination; Supercoiled plasmid DNA; Analytical group separation; Thiophilic aromatic chromatography;

This work was performed in order to study the possibilities in using molecularly imprinted polymers (MIPs) as sorbent material in solid-phase extraction (MISPE) for the sample clean-up technique for the determination of metoclopramide (MCP) in biological fluids. The effective factors influencing the bulk polymerization have been studied. Molecular recognition properties, binding capability and selectivity of the molecularly imprinted polymers (MIPs) were evaluated and the results revealed the obtained MIPs have high affinity for MCP in aqueous environment. The optimal conditions for solid-phase extraction (SPE) consisted of conditioning with 1 mL of methanol and 1 mL of deionized water at neutral pH, loading with 1 mL of the water sample (50 μg L−1) at pH 8.5, washing using 1 mL of acetone and elution with 2× 1 mL methanol/acetic acid (10/1, v/v). After optimization of SPE procedure, the MIP was then directly used to selectively extract the target drug from human serum and urine with an extraction recovery of more than 90%. Chromatograms of the eluate solutions show an efficient clean-up, which supports the potential of MISPE for clean-up of trace amounts of MCP from serum and urine. The limits of detection of MCP in human serum and urine were 3 and 1.2 μg L−1, respectively.
Keywords: Molecularly imprinted polymer; Solid-phase extraction; Metoclopramide; Biological fluids; Human urine;

On-line strategies for the identification of unknown flavone glycosides in Dracocephalum tanguticum Maxim by Guozhu Liu; Zhonghai Xu; Jitao Chen; Gesangsuo Lang; Qingqing Tian; Yao Shen; Bo Chen; Shouzhou Yao (2545-2550).
In this text we present a series of LC–MS/UV-based strategies for on-line structure identification of unknown flavone glycosides in Dracocephalum tanguticum. The aglycone portion, the glycosylation position, the order of sugars and the identity of sugars of each glycoside were unambiguously identified. The new strategy—identifying the partial structure of a molecule by comparison with a different compound possessing the same assumed partial structure—is quite effective for structure identification of the aglycone of 14; the unusual non-radical [Y0–H–CH2] ion observed for the methoxylated flavonoid glycoside 8 is quite useful for structure elucidation of the aglycone of 58; the UV data obtained on-line with post-column addition of UV-shift reagents are powerful for determination of the glycosylation and methoxylation positions for each flavone glycoside. Finally, isolation and subsequent individual analysis by NMR were performed to provide complementary information for the configurations of each sugar and the linkage types of each disaccharide. Thus, the structures of the eight detected flavone glycosides, which all have never been reported in D. tanguticum before, were positively identified.
Keywords: Dracocephalum tanguticum Maxim; Flavone glycosides; On-line identification; HPLC–MS n ; HPLC–UV; Post-column derivatization;

Breath acetone analysis with miniaturized sample preparation device: In-needle preconcentration and subsequent determination by gas chromatography–mass spectroscopy by Ikuo Ueta; Yoshihiro Saito; Masahiko Hosoe; Mitsuyoshi Okamoto; Hironobu Ohkita; Shingoro Shirai; Hiroshi Tamura; Kiyokatsu Jinno (2551-2556).
A new approach to the determination of human breath acetone with particle-packed sample preparation needle was developed. The extraction needle was packed with a copolymer of methacrylic acid and ethylene glycol dimethacrylate as the extraction medium. For the analysis of breath sample, exhaled breath was collected in a sampling bag, and 50 mL of the breath sample was extracted with the needle-type sample preparation device followed by analysis using gas chromatography–mass spectrometry (GC–MS). After the optimization of several basic extraction conditions for standard acetone samples, breath acetone concentration taken from controlled type-2 diabetic patients was determined. Furthermore, time variations of breath and urine acetone of four healthy individuals under fasting conditions were measured. Urine sample was collected in glass vial, and urine acetone concentration was also determined with the extraction needle by analyzing the corresponding headspace gas. The results demonstrated that the particle-packed extraction needle showed an excellent extraction performance for acetone in both breath and urine headspace samples, and that there is a clear correlation between the concentration of breath acetone and HbA1c level of controlled type-2 diabetic patients. The breath acetone level in controlled diabetic patients was in the range between 0.19 and 0.66 ppmv, where its concentration in medically untreated type-2 patient was 0.92 and 1.20 ppmv. The breath acetone concentration in healthy male was increased to 5.66 ppmv under the 24 h of fasting test, and a high correlation between the breath and urine acetone concentration was also observed. On the basis of the above results, the potential applications of the proposed method to the diagnosis of diabetes and/or ketoacidosis were suggested.
Keywords: Breath analysis; Breath acetone; Urine acetone; Fasting; Needle;

The process of drug metabolite identification is extremely important for drug efficacy, safety and pharmacokinetics. The traditional method usually involves using a drug with a radioactive labeled nuclei and/or isolating major drug metabolites by HPLC before applying MS and NMR analyses, which requires trained specialists to handle the radioactive compounds and is time consuming for offline-HPLC separation. A method using mass spectrometry-based metabonomics combined with multivariate statistical analysis was applied to rapidly identify metabolite profiles of tolcapone, a catechol-O-methyl transferase inhibitor for Parkinson's disease treatment. The tolcapone metabolites were identified based on the accurate mass measurement (<3 ppm) and MS2 mass spectrum. In total, 16 tolcapone metabolites were detected and identified, 6 of which have not been reported previously. Our results indicate that the method has the capability to accelerate the process of identifying drug metabolites, ultimately reduce drug development costs, and make the process safer without requiring a drug with radioactive nuclei. Most importantly, the assay can detect the major and minor drug metabolites in a global view. Furthermore, since tolcapone has been associated with idiosyncratic drug induced liver toxicity the rapid detection of tolcapone-related metabolites can provide mechanistic toxicity information related to drug metabolism and the formation of reactive drug metabolites.
Keywords: UPLC/MS; Metabonomics; Tolcapone; Metabolite/metabolism; Urine;

A selective, rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the first time to determine pidotimod in human plasma and applied to a pharmacokinetic study. Diphenhydramine was used as the internal standard (I.S.). Sample pretreatment involved in one-step protein precipitation (PPT) with methanol of 0.1 mL plasma. The analysis was carried out on an Ultimate™ XB-C8 column with mobile phase of methanol–water containing 0.5% formic acid (65:35, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. Each plasma sample was chromatographed within 4.5 min. The linear calibration curves were obtained in the concentration range of 0.05–20.00 μg/mL (r 2  ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.0500 μg/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 15% and accuracy (relative error, R.E.) was from −5.1% to 3.9% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of pidotimod in healthy volunteers after oral administration.
Keywords: Pidotimod; HPLC–MS/MS; Human plasma; Pharmacokinetic study;

A new and systematic application for separation and online identification of coumarins from Peucedanum praeruptorum Dunn by preparative high-speed counter-current chromatography coupled with electrospray ionization multi-stage mass spectrometry (prep-HSCCC/ESI-MS n ) was established. The procedure of separation was guided by the chromatogram of ion current. The structures of acquisitions were deduced by MS information. The hyphenation between prep-HSCCC/ESI-MS n was designed to keep the split ratio from 1:20 to 1:200 exactly. Seven compounds were obtained and two new compounds were detected. It was proved that prep-HSCCC/ESI-MS n was an effective method for sensitive detection, rapid identification and separation of natural products.
Keywords: prep-HSCCC/ESI-MS n ; Coumarins; Peucedanum praeruptorum Dunn;

Thermodynamic studies of partitioning behavior of lysozyme and conalbumin in aqueous two-phase systems by Rita de Cássia Superbi de Sousa; Jane Sélia dos Reis Coimbra; Luis Henrique Mendes da Silva; Maria do Carmo Hespanhol da Silva; Edwin Elard Garcia Rojas; Antonio António Augusto Vicente (2579-2584).
The objective of this study was to determine the thermodynamic parameters (Δ tr G, Δ tr H and Δ tr S) associated with lysozyme and conalbumin partitioning in aqueous two-phases systems (ATPS). Influence of salt type and polyethylene glycol (PEG) concentrations on the partition coefficient of lysozyme and conalbumin from egg white was studied. The evaluated ATPS were composed of PEG 1500 and inorganic salts (sodium citrate and sodium sulfate) at a temperature of 25 °C and pH 7.0, with PEG 1500 g mol−1 concentrations of 14%, 16% and 18% (mass basis). Partitioning of lysozyme in PEG–citrate ATPS was enthalpically driven, however the PEG–sulfate ATPS was entropically driven. The tested systems can be employed for the separation of these two proteins in egg white, due to the fact that lysozyme migrates toward the polymeric phase and conalbumin to the saline phase in both ATPS. A high recovery of conalbumin in the saline phase of the PEG–sulfate ATPS was determined to be enthalpically driven.
Keywords: Aqueous two-phase systems; Egg white protein; Conalbumin; Lysozyme; Thermodynamic parameters;

A novel and simple method to determine pharmacokinetics of pefloxacin mesylate (PM) in urine of seven healthy adults was developed. The proposed methodology was based on the electrochemiluminescence (ECL) of tris(2,2′-bipyridine)ruthenium (II) at a platinum electrode. The ECL intensity was found greatly enhanced in the presence of PM, which could directly participate in the light-emitting reaction as the reductant (in former/unchanged form). Under optimised conditions, the calibration curve was linear from 0.02 to 12 mg/L with a detection limit of 0.004 mg/L (σ  = 3). The RSD of the peak height was less than 2.4% (n  = 6). The recoveries in human urine were 96.2% to 98.3%. The highest excretion rate in urine was observed during the period 1.5–2 h after oral administration. The urinary excretion ratio of PM was 13.6% within 48 h.
Keywords: Pefloxacin mesylate; Pharmacokinetics; Capillary electrophoresis; Electrochemiluminescence; Human urine;

Time resolved analysis of risperidone and 9-hydroxy-risperidone in hair using LC/MS-MS by Serge Schneider; Estelle Sibille; Michel Yegles; Hugo Neels; Robert Wennig; Annette Mühe (2589-2592).
Risperidone (RSP) is a second generation anti-psychotic drug used for the treatment of schizophrenia and anxiety disorders. In the last decades, clinical applications of hair analysis have received an increasing attention, because of its wide surveillance window. In this work, we describe a simple and fast method for detection and quantification of RSP and its major metabolite, 9-OH-risperidone (9-OH-RSP), in human hair. The validated method (cv of interday precision, intraday precision and accuracy < 15%, r 2 of the calibration curves > 0.98, limit of detection (LOD) was 0.90 pg/mg hair (RSP) and 1.52 pg/mg hair (9-OH-RSP), the lower limit of quantification (LLOQ) were 1.8 and 4.56 pg/mg, respectively, extraction yield were 86.9% for RSP and 86.7% for 9-OH-RSP) was successfully applied to quantify both substances in the hair of psychiatric patients treated with RSP. After washing, pulverisation, incubation in an ultrasound bath and liquid/liquid extraction of the hair samples, quantification was performed using LC/MS-MS in selected reaction monitoring mode with methaqualone as internal standard. Concentrations for RSP and its major metabolite ranged from 36 to 4765 pg/mg and from 14 to 57 pg/mg, respectively in the different hair segments. These preliminary results indicate a better relationship between the administered dose and hair concentration for 9-OH-RSP than for the parent drug. Furthermore, the RSP/9-OH-RSP ratio varied from 1 to 83.
Keywords: Hair analysis; RSP; 9-OH-RSP; LC/MS-MS;

Determination of mitragynine in rat plasma by LC–MS/MS: Application to pharmacokinetics by Natália Valadares de Moraes; Raquel Alves Corrêa Moretti; Edward B. Furr; Christopher R. McCurdy; Vera Lucia Lanchote (2593-2597).
This study used for the first time LC–MS/MS for the analysis of mitragynine (MIT), a μ-opioid agonist with antinociceptive and antitussive properties, in rat plasma. Mitragynine and the internal standard (amitriptyline) were extracted from plasma with hexane-isoamyl alcohol and resolved on a Lichrospher® RP-SelectB column (9.80 and 12.90 min, respectively). The quantification limit was 0.2 ng/mL within a linear range of 0.2–1000 ng/mL. The method was applied to quantify mitragynine in plasma samples of rats (n  = 8 per sampling time) treated with a single oral dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration: 424 ng/mL; time to reach maximum plasma concentration: 1.26 h; elimination half-life: 3.85 h, apparent total clearance: 6.35 L/h/kg, and apparent volume of distribution: 37.90 L/kg.
Keywords: Mitragynine; Mitragyna speciosa; LC–MS/MS; Rats; Pharmacokinetics;