Journal of Chromatography B (v.877, #11-12)

Highly efficient peptide separations in proteomics by Koen Sandra; Mahan Moshir; Filip D’hondt; Robin Tuytten; Katleen Verleysen; Koen Kas; Isabelle François; Pat Sandra (1019-1039).
Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.
Keywords: Proteomics; Peptides; Multidimensional; Chromatography; Electrophoresis; On-line; Off-line;

A sensitive and selective quantitative method to determine α-fluoro-β-alanine (FBAL), 5-fluorouracil (5-FU), and capecitabine (Cape) from a single human plasma aliquot (50 μL) has been developed and validated. First, 5-FU and Cape were extracted by liquid–liquid extraction (LLE) using a mixture of acetonitrile and ethyl acetate. This was followed by derivatization with dansyl chloride. The dansyl-derivatives from 5-FU and Cape were further purified using LLE with methyl tertiary-butyl ether (MTBE) and analyzed using a reversed-phase analytical column “Primesep D” (2.1 mm × 50 mm; 5 μm) with embedded basic ion-pairing groups. The remaining aqueous phase containing FBAL was treated with dansyl chloride and the dansyl-FBAL was purified by solid phase extraction. Ultra high pressure liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for analysis of dansyl-FBAL. The method was validated over the concentration ranges of 10–10,000, 5–5000, and 1–1000 ng/mL for FBAL, 5-FU, and Cape, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.
Keywords: α-Fluoro-β-alanine (FBAL); 5-Fluorouracil (5-FU); Capecitabine; Derivatization; Dansyl chloride; Mass spectrometry;

HPLC–MS/MS methods for the determination of a Hepatitis C NS3/NS4 protease inhibitor (MK-7009) in human plasma and Tween-treated urine were developed and validated over the concentration range 1–1000 ng/mL and 0.2–100 μg/mL respectively. A stable isotope labeled internal standard (ISTD), D4-MK-7009, was employed. Analytes were chromatographed by reversed phase HPLC and quantified by an MS/MS system. Electrospray ionization in the positive mode was employed. Multiple reaction monitoring of the precursor to product ion pairs m/z 758.6 → 637.4 MK-7009 and m/z 762.5 → 637.4 ISTD was used for quantitation. Analyte and internal standard were extracted from 250 μL of plasma using an automated 96-well liquid–liquid extraction. Plasma pH adjustment prior to extraction minimized ionization suppression in plasma samples from patients with Hepatitis C. The urine method involved direct dilution in the 96-well format of 0.020 mL Tween-treated urine. These methods have supported several clinical studies. Incurred plasma sample reanalysis demonstrated adequate assay reproducibility and ruggedness.
Keywords: MK-7009; NS3/NS4 protease inhibitor; LC–MS/MS; Plasma and urine quantitation; Signal suppression;

A LC–tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir, and etravirine by A. Fayet; A. Béguin; B. Zanolari; S. Cruchon; N. Guignard; A. Telenti; M. Cavassini; H.F. Günthard; T. Buclin; J. Biollaz; B. Rochat; L.A. Decosterd (1057-1069).
Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100 μl) is performed by protein precipitation using 600 μl of acetonitrile, after the addition of 100 μl darunavir-d9 (DRV-d9) at 1000 ng/ml in MeOH/H2O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10 min, vortex-mixed again and centrifuged. An aliquot of supernatant (150 μl) is diluted 1:1 with a mixture of 20 mM ammonium acetate/MeOH 40/60 and 10 μl is injected onto a 2.1 × 50 mm Waters Atlantis™-dC18 3 μm analytical column. Chromatographic separations are performed using a gradient program with 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000 ng/ml, 2.5 to 1000 ng/ml, 25 to 10,000 ng/ml, 10 to 4000 ng/ml, and 5 to 2000 ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1–9.8%, and accurate (range of inter-day deviation from nominal values −3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir–glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.
Keywords: Integrase inhibitors; CCR5 co-receptor antagonists; Protease inhibitors; Non-nucleoside reverse transcriptase inhibitors; HPLC–MS/MS; Raltegravir; Maraviroc; Darunavir; Etravirine; Ritonavir;

Determination of pyrrole–imidazole polyamide in rat plasma by liquid chromatography–tandem mass spectrometry by Takashi Nagashima; Takahiko Aoyama; Akiko Fukasawa; Satoshi Watabe; Noboru Fukuda; Takahiro Ueno; Hiroshi Sugiyama; Hiroki Nagase; Yoshiaki Matsumoto (1070-1076).
Pyrrole (Py)–imidazole (Im) polyamides synthesized by combining N-methylpyrrole and N-methylimidazole amino acids have been identified as novel candidates for gene therapy. In this study, a sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with an electrospray ionization (ESI) source was developed and validated for the determination and quantification of Py–Im polyamide in rat plasma. Py–Im polyamide was extracted from rat plasma by solid-phase extraction (SPE) using a Waters Oasis® HLB cartridge. Separation was achieved on an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 50 mm) column by gradient elution using acetonitrile:distilled water:acetic acid (5:95:0.1, v/v/v) and acetonitrile:distilled water:acetic acid (95:5:0.1, v/v/v). The method was validated over the range of 10–1000 ng/mL and the lower limit of quantification (LLOQ) was 10 ng/mL. This method was successfully applied to the investigation of the pharmacokinetics of Py–Im polyamide after intravenous administration.
Keywords: Pyrrole–imidazole polyamide; Solid-phase extraction; LC–MS/MS; Plasma; Rat;

An improved isocratic and rapid HPLC method was developed for the measurement of carotenoids, retinol and tocopherols in human serum. Vitamins were extracted with hexane. Mobile phase consisted of a mixture acetonitrile:methylene chloride:methanol with 20 mM ammonium acetate. This method used a small bead size (3 μm) Spherisorb ODS2 column with titane frits. Diode array and fluorescence detectors were used respectively for the detection of carotenoids and retinol/tocopherols. Chromatographic separation was complete in 13 min for β-cryptoxanthin, cis–trans-lycopene, α-carotene, β-carotene, cis-β-carotene, retinol, δ-tocopherol, γ-tocopherol and α-tocopherol. Echinenone and tocol were employed as internal standards for diode array and fluorescence detection. Accuracy was validated using standard reference material (SRM) 968C. Intra-assay and inter-assay precision were respectively 0.2–7.3% and 3.6–12.6%. Sensitivity was verified using the ICH recommendations and the limit of detection (LOD) obtained was sufficient for routine clinical application.
Keywords: Fat-soluble vitamins; Retinol; Carotenoids; Tocopherols; HPLC; Diode array detector; Fluorescence detector;

Botulinum neurotoxin types A to G are produced from different strains of Clostridium botulinum. The complex neurotoxins belong to the most toxic substances known and cause botulism both in humans and animals. Botulinum toxin complexes are produced with molecular weights of 300, 500 and 900 kDa. These large protein complexes contain beside the toxic zinc protease of 150 kDa, additional neurotoxin associated proteins, which are responsible for the extreme pH and protease stability. In this study we present for the first time a rugged detection method of botulinum toxins at femtomole levels in complex culture media after peptic sample pre-treatment and 2D-nano-LC–ESI–MS–MS-technique. In contrast to other studies, we used progenitor toxins directly from culture supernatant of C. botulinum strains A, B, E and F without further purification, to simulate complex, protein-containing sample conditions. We were able to demonstrate, that peptic pre-treatment is a great challenge in reducing ubiquitous proteins as well as proteins from suspicious samples. The study also found that multidimensional chromatography leads to significant better peptide differentiation and identification in protein loaded matrices than one dimensional nano-LC–ESI–MS.
Keywords: Botulinum toxins; Nanoscale liquid chromatography; Two dimensional chromatography; Electrospray mass spectrometry; Peptic and tryptic digestion;

Methylglyoxal, protein binding and biological samples: Are we getting the true measure? by Arti Dhar; Kaushik Desai; Jianghai Liu; Lingyun Wu (1093-1100).
Methylglyoxal (MG), a reactive metabolic byproduct and a precursor of advanced glycation endproducts (AGEs), is elevated in diabetes. In the body MG is free or reversibly or irreversibly bound (mostly with proteins). Variable plasma MG values have been reported. MG is commonly measured using high performance liquid chromatography. We tested several protocols on different biological samples, which resulted in significant differences in MG values measured in a given sample. The different values do not appear due to the release and detection of bound MG under assay conditions. Protocols that provide consistent values of MG in biological samples are recommended.
Keywords: Methylglyoxal; Perchloric acid; o-Phenylenediamine; HPLC;

Molecularly imprinted polymer for analysis of zidovudine and stavudine in human serum by liquid chromatography–mass spectrometry by S. Vo Duy; I. Lefebvre-Tournier; V. Pichon; F. Hugon-Chapuis; J.-Y. Puy; C. Périgaud (1101-1108).
A molecularly imprinted polymer (MIP) using zidovudine (AZT) as template and methacrylic acid as monomer was prepared. The synthesis of the MIP was performed in acetonitrile. The synthesized material was then tested for the solid-phase extraction of AZT from different media (pure organic solvents and hydro-organic mixtures). An optimised procedure was developed for the selective extraction of AZT with a recovery of 96% using the MIP and only 3% on a non-imprinted polymer used as control polymer. A specific capacity of 0.2 μmol g−1 was determined. The specificity of the MIP was evaluated by studying the retention behaviour of two others nucleoside analogues. The feasibility of the MIP to selectively extract AZT and stavudine (d4T) from human serum was also demonstrated with recoveries of 80 and 85% respectively. The lower limit of quantification (LLOQ) and the lower limits of detection (LLOD) for AZT were 5.10−7 and 10−7  M respectively.
Keywords: Molecularly imprinted polymer; Zidovudine; Stavudine; Human serum; LC/MS;

Optimization of analytical instrumentation is essential when analyses of persistent organic pollutants in human serum are performed at ultra-trace levels. This research describes the analysis of polychlorinated biphenyls (PCBs) in serum using gas chromatography coupled with a mass selective detector (GC/MSD) in the selected ion monitoring (SIM) mode. We selected PCB-58 and PCB-186 as internal standards (ISs) for the method development, and newborn calf serum (NCS) was chosen as the matrix. The matrix was fortified with PCB congeners and extracted by an automated solid phase extraction system using C18 sorbent. The extracts were analyzed at two ion source temperatures, 230 and 300 °C. The use of high ion source temperature increased the abundances of high-mass ions, and the response factors in SIM mode for PCBs. An excellent linearity from 0.5 to 100 ng/ml at ion source temperature of 300 °C was demonstrated, with a calculated detection limit of 0.1 ng/g serum. Seven replicate fortifications of newborn calf serum, at three spiking levels of 1, 10, and 50 ng/g of serum, gave mean recoveries of 110%, 85%, and 98%, with average relative standard deviation (RSD) values of 5.4%, 7.3%, and 4.7%, respectively.
Keywords: Serum; PCBs; GC/MSD; High ion source temperature; SPE;

Using liquid chromatography–tandem mass spectrometry to quantify monohydroxylated metabolites of polycyclic aromatic hydrocarbons in urine by Frank Onyemauwa; Stephen M. Rappaport; Jon R. Sobus; Dagmar Gajdošová; Ren’an Wu; Suramya Waidyanatha (1117-1125).
We present an assay which employs enzyme digestion and solid phase extraction followed by liquid chromatography–tandem mass spectrometry to simultaneously quantify 16 hydroxylated polycyclic aromatic hydrocarbons (OHPAHs) in 3-ml samples of urine. The analytes consisted of 2-, 3-, and 4-ring OHPAHs, namely, 1- and 2-hydroxynaphthalene (1- and 2-OHNAP), 2-hydroxyfluorine (2-OHFLU), 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, and 9-OHPHE), 1-hydroxypyrene (1-OHPYR), 1- and 2-hydroxybenzo(a)anthracene (1- and 2-OHBAA), 3- and 6-hydroxychrysene (3- and 6-OHCHR) and 3-, 7-, and 9-hydroxybenzo(a)pyrene (3-, 7-, and 9-OHBAP). The method was validated using urine samples from steel workers and control subjects. The coefficients of variation of the method for the particular analytes were between 7% and 27% and the limits of quantitation were between 0.002 and 0.010 μg/l urine. The 2- and 3-ring OHPAHs were easily quantified in all subjects. However, 1-OHPYR was the only representative of the 4- and 5-ring metabolites that could be quantified. Pairwise correlations showed that all OHPAHs were highly correlated with each other (0.553 ≤  r  ≤ 0.910) and with 1-OHPYR (0.614 ≤  r  ≤ 0.910), the metabolite most widely accepted as a short-term biomarker of exposure to PAHs. The analyte, 2-OHNAP exhibited the lowest pairwise correlations with the other OHPAHs (0.542 ≤  r  ≤ 0.628), presumably due to confounding by smoking. Metabolites of phenanthrene, an abundant PAH and the smallest to possess a bay region, are promising OHPAHs for characterizing both exposures to PAHs and the various metabolic pathways.
Keywords: Monohydroxylated metabolites; Liquid chromatography–tandem mass spectrometry; Exposure; Selected reaction monitoring; Urinary biomarkers; Biomonitoring; Phenanthrene; Naphthalene; Polycyclic aromatic hydrocarbons;

Determination of duloxetine in human plasma by capillary electrophoresis with laser-induced fluorescence detection by Alessandro Musenga; Mario Amore; Roberto Mandrioli; Ernst Kenndler; Ludovica de Martino; Maria Augusta Raggi (1126-1132).
A method based on capillary electrophoresis has been developed for the analysis of the novel antidepressant drug duloxetine in human plasma. The method makes use of laser-induced fluorescence detection after derivatisation of the analyte with 5-(4,6-dichlorotriazinyl)aminofluorescein at pH 11. A single step liquid/liquid extraction procedure with a mixture of hexane/2-propanol allows the sample clean-up with extraction yields always ≥84% and interference removal. The electrophoretic separation is achieved using uncoated fused silica capillaries (60.0 cm effective length, 75.0 cm total length, 50 μm internal diameter) and a background electrolyte composed of borate buffer (40 mM, pH 10.3), tetrabutylammonium bromide (10 mM), and acetone (10%, v/v). The applied voltage is 20 kV; the samples are injected by pressure (50 mbar × 8 s). The method has been fully validated in terms of linearity range (2.5–150 ng mL−1), LOD and LOQ (1.0 and 2.5 ng mL−1, respectively), precision (R.S.D. < 6.7%) and accuracy (recovery >78%). Application to samples obtained from patients under treatment with duloxetine gave good results. The method represents the first application of capillary electrophoresis to the analysis of duloxetine in human plasma.
Keywords: Duloxetine; Capillary electrophoresis; Derivatisation; 5-(4,6-Dichlorotriazinyl)aminofluorescein; Human plasma; Laser-induced fluorescence;

A simple, sensitive and rapid method for the analysis of lumefantrine in rat plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The method included a chromatographic run of 5 min using a C18 analytical column and the calibration curve was linear over the concentration range of 2–500 ng/mL with a correlation coefficient (r) of 0.996 or better. The intra- and inter-day assay precision ranged from 1.5 to 7.5% and 5.5 to 7.7%, respectively, and intra- and inter-day assay accuracy was between 91.3–109.7% and 97.0–104.7%, respectively. The method was successfully applied for the pharmacokinetic study in rats.
Keywords: Lumefantrine; Rat plasma; Validation; LC–MS/MS; Pharmacokinetics;

A method for the determination of organotin compounds in urine samples based on liquid–liquid extraction (LLE) in hexane and gas chromatographic separation was developed and optimized. Seven organotin species, namely monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), tetrabutyltin (TeBT), monophenyltin (MPhT), diphenyltin (DPhT) and triphenyltin (TPhT), were in situ derivatized by sodium tetraethylborate (NaBEt4) to form ethylated less polar derivatives directly in the urine matrix. The critical parameters which have a significant effect on the yield of the successive liquid–liquid extraction procedure were examined, by using standard solutions of tetrabutyltin in hexane. The method was optimized for use in direct analysis of undiluted human urine samples and ways to overcome practical problems such as foam formation during extraction, due to various constituents of urine are discussed. After thorough optimization of the extraction procedure, all examined species could be determined after 3 min of simultaneous derivatization and extraction at room temperature and 5 min phase separation by centrifugation. Gas chromatography with a microwave-induced plasma atomic emission detector (MIP-AED) as element specific detector was employed for quantitative measurements, while a quadrupole mass spectrometric detector (MS) was used as molecular specific detector. The detection limits were between 0.42 and 0.67 μg L−1 (as Sn) for the quantitative LLE–GC–MIP-AED method and the precision between 4.2% and 11.7%, respectively.
Keywords: Gas chromatography; Atomic emission detection; Mass spectrometry; Organotin compounds; Liquid–liquid extraction; Urine;

Simultaneous HPLC–UV determination of rhein and aceclofenac in human plasma by Ashwini Ojha; Rajeshwari Rathod; Harish Padh (1145-1148).
Diacerein and aceclofenac are prescribed for reducing the symptoms associated with osteoarthritis. We present a simple HPLC method with UV detection for simultaneous determination of rhein (the immediate metabolite of diacerein) and aceclofenac from human plasma samples. Sample preparation was accomplished through liquid–liquid extraction with ethyl acetate and chromatographic separation was performed on a reversed-phase ODS column. Mobile phase consisted of a mixture of acetate buffer and acetonitrile run under gradient at flow rate of 1.0 ml/min. Wavelength was set at 258 nm. The method was validated for linearity, accuracy, precision and stability. The calibration was linear over the range of 0.1–7.0 μg/ml for rhein and 0.5–20 μg/ml for aceclofenac using 500 μl plasma samples. Extraction recoveries were 85% for rhein and 70% for aceclofenac. The method can easily be adopted for high-throughput clinical and pharmacokinetic studies of above two-drug fixed dose combination formulations.
Keywords: Rhein; Aceclofenac; Human plasma; Quantification; Reversed-phase chromatography; Pharmacokinetics;

Studies of imipramine binding to human serum albumin by high-performance affinity chromatography by Michelle J. Yoo; Quentin R. Smith; David S. Hage (1149-1154).
Binding by the drug imipramine to the protein human serum albumin (HSA) was studied by using high-performance affinity chromatography. The association equilibrium constants and number of binding sites for imipramine with HSA were first estimated by utilizing frontal analysis. Imipramine was found to have one major binding site on HSA with an association equilibrium constant of 1.6 × 105  M−1 at pH 7.4 and 37 °C, as well as a second group of weaker and non-specific binding regions (8–9 mol/mol HSA) with an average association equilibrium constant of 1.5 × 103  M−1. Competition studies based on zonal elution were performed to identify the location of the major binding site for imipramine on HSA. Imipramine was found to have direct competition with L-tryptophan, which indicated that imipramine was interacting with Sudlow site II, or the indole-benzodiazepine site of HSA. No competition or allosteric effects were noted between imipramine and warfarin, a probe for Sudlow site I or the warfarin-azapropazone site of HSA. The association equilibrium constant found for imipramine at its site of competition with L-tryptophan also agreed with the value that was obtained for the major binding site of imipramine in the frontal analysis studies. These results confirmed that Sudlow site II was the location of the major binding site for imipramine on HSA. These results gave good agreement with previous observations made in the literature and should provide a more detailed description of how imipramine is transported in blood and of how it may interact with other drugs in the body.
Keywords: Imipramine; Human serum albumin; Protein binding; High-performance affinity chromatography; Frontal affinity chromatography; Competition studies;

Analysis of relationship between cell cycle stage and apoptosis induction in K562 cells by sedimentation field-flow fractionation by J. Bertrand; B. Liagre; G. Bégaud-Grimaud; M.O. Jauberteau; J.L. Beneytout; P.J.P. Cardot; S. Battu (1155-1161).
Recently, sedimentation field-flow fractionation (SdFFF) was used to study the specific kinetics of diosgenin-induced apoptosis in K562 cells. Here, we propose a new SdFFF cell separation application in the field of cancer research concerning the correlation between induction of a biological event (i.e. apoptosis) and cell status (i.e. cell cycle position). SdFFF isolated subpopulations depending on the cell cycle position allowing the study of apoptosis kinetics and extent. Results showed that cells in G0/G1 phases (F3 cells) underwent significant and earlier apoptosis than cells in the active part of the cell cycle (S/G2/M phases). Results shed light on the correlation between differences in apoptosis kinetics and cell cycle stage when exposure to the inducer began. SdFFF monitoring and size measurement also led to the description of different subpopulations demonstrating complex variations in density between fractions associated with differences in biological processes.
Keywords: K562 cells; Diosgenin; Cell cycle; Apoptosis; Sedimentation field-flow fractionation; Cell sorting;

A sensitive and specific LC–MS/MS method for simultaneous detection of 10 plant-derived psychoactive substances (atropine, N,N-dimethyltryptamine, ephedrine, harmaline, harmine, ibogaine, lysergic acid amide, psilocin, scopolamine and yohimbine) in urine was developed. Direct injection of urine diluted with 3 deuterated internal standards allowed for a readily accessible method suitable for application in clinical intoxication cases. Separation was achieved using reversed phase chromatography and gradient elution with a total analysis time of 14 min. Electrospray ionization was used and ions were monitored in the positive selected reaction monitoring mode. The calibration curves were linear (r 2  > 0.999) and the total imprecision at high (1000 μg/L) and low (50 μg/L) substance concentrations were 4.9–13.8% and 8.3–26%, respectively. Infusing the analytes post column and injecting matrix samples showed limited influence by ion suppression. The multi-component method proved to be useful for investigation of authentic cases of intoxication with plant-derived psychoactive drugs and was indicated to cover the clinically relevant concentration ranges.
Keywords: Hallucinogens; LC–MS; Plant-derived drugs; Psychoactive drugs; Urine;

Determination of procyanidins and their metabolites in plasma samples by improved liquid chromatography–tandem mass spectrometry by Aida Serra; Alba Macià; Maria-Paz Romero; Maria-Josepa Salvadó; Mario Bustos; Juan Fernández-Larrea; Maria-José Motilva (1169-1176).
An off-line solid-phase extraction (SPE) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining procyanidins, catechin, epicatechin, dimer, and trimer in plasma samples. In the validation procedure of the analytical method, linearity, precision, accuracy, detection limits (LODs), quantification limits (LOQs), and the matrix effect were studied. Recoveries of the procyanidins were higher than 84%, except for the trimer, which was 65%. The LODs and LOQs were lower than 0.003 and 0.01 μM, respectively, for all the procyanidins studied, except for the trimers, which were 0.8 and 0.98 μM, respectively. This methodology was then applied for the analysis of rat plasma obtained 2 h after ingestion of grape seed phenolic extract. Monomers (catechin and epicatechin), dimer and trimer in their native form were detected and quantified in plasma samples, and their concentration ranged from 0.85 to 8.55 μM. Moreover, several metabolites, such as catechin and epicatechin glucuronide, catechin and epicatechin methyl glucuronide, and catechin and epicatechin methyl-sulphate were identified. These conjugated forms were quantified, in reference to the respective unconjugated form, showing concentrations between 0.06 and 23.90 μM.
Keywords: Plasma; Procyanidins; Tandem mass spectrometry; UPLC;

Isolation and detection of steroids from human urine by molecularly imprinted solid-phase extraction and liquid chromatography by Renata Gadzała-Kopciuch; Júlia Ričanyová; Bogusław Buszewski (1177-1184).
Naturally occurring steroids such as progesterone, testosterone and 17β-estradiol were analyzed in this study. These bio-identical molecules paradoxically can be either beneficial or harmful. Unfortunately as growth promoters can be toxic and cancerogenic at elevated levels. Due to difficulty in monitoring at trace quantities of these hormones in biological matrices specific adsorption materials molecularly imprinted polymers (MIPs) were used for preconcentration and clean up in sample preparation step. A non-covalent imprinting approach was used for bulk polymerization of progesterone, testosterone and 17β-estradiol imprinted polymers. Synthesis of MIPs was achieved by thermal, UV and γ irradiation initiated polymerization whereby were used methacrylic acid (MAA), 4-vinylpyridine (4-VP) as functional monomers, ethylene glycol dimethacrylate (EDMA), trimethylolpropane trimethacrylate (TRIM) as cross-linking agents and acetonitrile, isooctane–toluene (1:99, v/v) and chloroform as porogen solvents. It was also used as initiator 2,2′-azobis(2-methylpropionitrile) (AIBN) or benzyl methyl ether (BME). The MIPs were applied as selective sorbents in solid-phase extraction (SPE). Molecularly imprinted solid-phase extraction (MISPE) considered as hyphenated technique were applied in extraction step before HPLC-DAD analysis of steroids from human urine.
Keywords: Steroids; MIP; MISPE; HPLC-DAD; Urine samples;

Simultaneous determination of four tobacco-specific N-nitrosamines (TSNA) in human urine by Dominique Kavvadias; Gerhard Scherer; Michael Urban; Francis Cheung; Graham Errington; Jim Shepperd; Mike McEwan (1185-1192).
Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). TSNA are suggested to play an important role in tobacco smoke carcinogenesis. We have developed and validated an LC–MS/MS method for the determination of total (free and conjugated) TSNA in human urine. The limits of detection (LOD) were 2.0, 0.8, 1.1 and 0.7 pg/ml for NNAL, NNN, NAB and NAT, respectively. Smokers were found to have significantly higher levels of TSNA in their urine than nonsmokers. In conclusion, the newly developed method is suitable for assessing the tobacco use-related exposure to NNK, NNN, NAB and NAT.
Keywords: Tobacco-specific N-nitrosamines (TSNA); 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK); 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL); N-nitrosonornicotine (NNN); N-nitrosoanabasine (NAB); N-nitrosoanatabine (NAT); Biological monitoring; Tobacco smoking; Human urine;

Proteomic Helicobacter pylori biomarkers discriminating between duodenal ulcer and gastric cancer by Ghalia Khoder; Yoshio Yamaoka; Jean-Louis Fauchère; Christophe Burucoa; Christo Atanassov (1193-1199).
Protein patterns of 129 Helicobacter pylori strains isolated from Korean and Colombian patients suffering from duodenal ulcer or gastric cancer were analyzed by the high-throughput methodology SELDI-TOF-MS. Eighteen statistically significant candidate biomarkers discriminating between the two clinical outcomes were selected by using the Mann–Whitney test. Three biomarker proteins were purified and identified as a neutrophil-activating protein NapA (HU HPAG1_0821), a RNA-binding protein (HPAG1_0813), and a DNA-binding histone-like protein HU, respectively (jhp0228). These novel biomarkers can be used for development of diagnostic assays predicting the evolution to gastric cancer in H. pylori-infected patients.
Keywords: Helicobacter pylori; SELDI-TOF-MS; Biomarker; Gastric cancer; Duodenal ulcer;

Removal of autoantibodies by 4-mercaptoethylpyridine-based adsorbent by Jun Ren; Lingyun Jia; Li Xu; Xue Lin; Zhiqian Pi; Jian Xie (1200-1204).
Extracorporeal immunoadsorption (ECI) therapy using Staphylococcal Protein A columns has proven effective for removing autoantibodies and circulating immune complexes from patients selectively, providing a promising treatment for autoimmune diseases. However, due to the drawbacks of Protein A in terms of cost and stability, the widespread use of Protein A based ECI is limited. In this study, we investigated the feasibility of 4-mercaptoethylpyridine (MEP, MW 139 Da), a simple and inexpensive synthetic compound, as an alternative to Protein A for autoantibody removal therapy. MEP-based adsorbents were prepared by coupling MEP to Sepharose CL-6B. We found that ligand density was an adjustable parameter for the synthesis of adsorbents aiming at different pathogenic factors, depending on the class of antibody. MEP-Sepharose with a ligand density of 98.8 μmol/ml could remove 80% of the anti-double-stranded DNA antibodies from human serum, whereas a ligand density of 64.5 μmol/ml was enough to remove 96% of the rheumatoid factor (RF) in the serum. Moreover, MEP-based adsorbents showed a lower degree of individual differences compared to Protein A-Sepharose. RF removal of 90% was achieved for all 12 serum samples from different individuals. Among the 14 serum samples derived from systemic lupus erythematosus patients, 11 samples had markedly reduced antinuclear antibody titers. In addition, non-specific adsorption of plasma components to MEP-Sepharose was limited, and the binding capacity of the absorbent for IgG was still about 20 mg/ml of gel after 10 cycles. The results indicated that MEP-based adsorbent could offer a new type of adsorber for the treatment of autoimmune diseases.
Keywords: Adsorbent; Autoantibody; Autoimmune disease; Blood purification;

A high performance liquid chromatographic method for determination of moxifloxacin in human plasma was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C18 column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.125 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.
Keywords: Moxifloxacin; Plasma; HPLC;

An isocratic RP-HPLC method for the determination of retinol and α-tocopherol in serum, with fluorescence and UV/VIS detection, respectively, was developed and validated according to international guidelines. Detection (retinol 0.015 mg/L, α-tocopherol 0.29 mg/L) and quantification (retinol 0.05 mg/L, α-tocopherol 0.95 mg/L) limits were determined. Repeatability was <3.3% and <2.9% and intermediate precision was <4.6% and <3.2%, for retinol and α-tocopherol, respectively. Certified reference materials were utilised to assess bias and guarantee traceability to SI units. Expanded uncertainties (retinol 8.9%; α-tocopherol 7.9%), estimated according to the EURACHEM/CITAC guide from method validation data, satisfied fit-for-purpose requirements based on biological variability.
Keywords: Serum; Retinol; α-Tocopherol; HPLC; Validation; Uncertainty of measurement; Biological variability; Quality specifications;

Displacement effect during HPLC preparative purification of human insulin by D. Gusarov; V. Nekipelova; V. Gusarova; V. Lasman; D. Bairamashvili (1216-1220).
HPLC plays a key role in the preparative purification of human insulin. A21-desamidoinsulin is one of the impurities that possesses the chromatographic behavior similar to that of insulin and hence separation from this by-product is rather difficult at the process scale. During the optimization of insulin reversed-phase HPLC purification, when a column was sufficiently overloaded, the effect of displacement of A21-desamidoinsulin molecules from active groups of sorbent by insulin ones was observed. It was suggested that monocarboxylic acid and organic modifier in mobile phase are responsible for the esterification during which the formed ester promotes the displacement effect. This effect was studied in order to optimize the purification of human insulin at the process scale.
Keywords: Preparative chromatography; Displacement; Human insulin; A21-desamidoinsulin;