Journal of Chromatography B (v.878, #15-16)

In this study, thermo-sensitive N-alkyl substituted polyacrylamide polymer PNNB was synthesized by using N-hydroxymethyl acrylamide(NHAM), N-isopropyl acrylamide (NIPA) and butyl acrylate (BA) as monomers, and its low critical solution temperature (LCST) was controlled to be 28 °C. The recovery of the thermo-sensitive polymer was over 98%. Butanol as a hydrophobic ligand was covalently attached onto polymer PNNB and butyl ligand density was 80 μmol g−1 polymer. The affinity polymer was used for purification of lipase from crude material. Optimized condition was pH 7.0, 35 °C adsorption temperature, 120 min adsorption time and 0.5 mg ml−1 initial concentration of lipase. The adsorption isotherm accords with a typical Langmuir isotherm. The maximum adsorption capacity (Q m) of the affinity polymer for lipase was 24.8 mg g−1polymer. The affinity copolymer could be recycled by temperature-inducing precipitation and there was only about 6% loss of adsorption capacity after five recyclings. Specific activity of lipase was improved from 14 IU mg−1 to 506 IU mg−1 protein, and its recovery achieved 82%. The affinity polymer is suitable for the purification of target proteins from the crude material with large volume and dilute solution.
Keywords: Affinity precipitation; Thermo-sensitive polymer; Hydrophobic ligand; Butyl glycidyl ether; Adsorption capacity; Lipase;

Study of the mechanism of interaction of antibody (IgG) on two mixed mode sorbents by S. Shiva Ranjini; Devla Bimal; A.P. Dhivya; M.A. Vijayalakshmi (1031-1037).
Purification of target proteins from a crude biological mixture containing proteins, peptides and other biomolecules is the chromatographic challenge. Mixed mode chromatography offers additional selectivities to improve the overall productivity of commercial bioprocesses with novel chromatographic sorbents being introduced to overcome the problem. HEA HyperCel™ (n-hexyl amine) and PPA HyperCel™ (phenyl propyl amine) are industry scalable mixed mode chromatography sorbents where both hydrophobic and electrostatic interactions are predominant. Our study focuses on understanding the underlying mechanism of interaction of protein with the sorbent. Parameters like buffer conditions, pH and temperature were tuned to study the adsorption and desorption conditions of the protein. Dynamic binding capacity of HEA HyperCel™ and PPA HyperCel™ sorbents was studied with human IgG as a model protein. Our study shows that, in HEA the interaction of IgG to the sorbent is predominantly hydrophobic as the binding is enhanced (50–60 mg/ml of sorbent) by presence of salt in buffer and increase in temperature. Binding capacity of PPA is 50–60 mg/ml of sorbent irrespective of temperature effect and/or the presence of salt. The chromatographic experiments show that the interaction could be hydrophobic or ionic or some charge transfer mechanism depending upon the buffer conditions.
Keywords: IgG purification; Mixed mode sorbents; HEA; PPA; Dynamic binding capacity;

Depletion of highly abundant proteins in blood plasma by hydrophobic interaction chromatography for proteomic analysis by Andrea Mahn; Alejandro Reyes; Mauricio Zamorano; Wildo Cifuentes; Maritza Ismail (1038-1044).
The proteomic analysis of plasma is extremely complex due to the presence of few highly abundant proteins. These proteins have to be depleted in order to detect low abundance proteins, which are likely to be of biomedical interest. In this work it was investigated the applicability of hydrophobic interaction chromatography (HIC) as a plasma fractionation method prior to two-dimensional gel electrophoresis (2DGE). The average hydrophobicity of the 56 main plasma proteins was calculated. Plasma proteins were classified as low, medium and highly hydrophobic through a cluster analysis. The highly abundant proteins showed a medium hydrophobicity, and therefore a HIC step was designed to deplete them from plasma. HIC performance was assessed by 2DGE, and it was compared to that obtained by a commercial immuno-affinity (IA) column for albumin depletion. Both methods showed similar reproducibility. HIC allowed partially depleting α-1-antitrypsin and albumin, and permitted to detect twice the number of spots than IA. Since albumin depletion by HIC was incomplete, it should be further optimized for its use as a complementary or alternative method to IA.
Keywords: Hydrophobic interaction chromatography; Two-dimensional gel electrophoresis; Plasma; Protein depletion;

Quantification of monofluoroacetate and monochloroacetate in human urine by isotope dilution liquid chromatography tandem mass spectrometry by Elizabeth I. Hamelin; Douglas B. Mawhinney; Ritchard Parry; Robert J. Kobelski (1045-1050).
The rodenticide monofluoroacetate (MFA) and monochloroacetate (MCA), a chemical intermediate from several chemical syntheses, have been identified as potential agents of chemical terrorism due to their high toxicity. In preparation for response to poisonings and mass exposures, we have developed a quantification method using isotopic dilution to determine MFA and MCA in urine from 50 to 5000 ng/mL. Both analytes were extracted from urine using solid-phase extraction; extraction recoveries were 62% (MFA) and 76% (MCA). The extracts were then separated with isocratic high-performance liquid chromatography and identified using electrospray ionization tandem mass spectrometry, with detection limits of 0.9 and 7.0 ng/mL for MFA and MCA, respectively. Selectivity was established for both analytes with unique chromatographic retention times which were correlated with isotopically labeled internal standards and the use of two mass spectral transitions for each compound. The intra-day variability was less than 5% for both analytes and the inter-day variability was 7% for MFA and 6% for MCA.
Keywords: Monofluoroacetate; Monochloroacetate; Tandem mass spectrometry; Compound; Urine excretion; LC/MS/MS; Solid-phase extraction; Chloroacetic acid;

Sedimentation field-flow fractionation separation of proliferative and differentiated subpopulations during Ca2+-induced differentiation in HaCaT cells by Ludovic Micallef; Serge Battu; Aline Pinon; Jeanne Cook-Moreau; Philippe J.P. Cardot; Christiane Delage; Alain Simon (1051-1058).
The spontaneously immortalized human keratinocyte cell line HaCaT is widely used as a human keratinocyte model. In a previous comparative study between normal human keratinocytes (NHKs) and HaCaT, we reported that Ca2+ concentrations greater than 1 mM induced differentiation in vitro in both cell types, notably characterized by increased expression of differentiation markers keratins 1 (K1), 10 (K10) and involucrin. Surprisingly, cells had a higher proliferative activity than those cultured with low Ca2+ levels. These results raised many questions; in particular concerning the emergence of HaCaT cells subpopulation which would have different differentiation states and/or proliferation rates throughout Ca2+-induced differentiation. To isolate these subpopulations, we used sedimentation field-flow fractionation (SdFFF). Results demonstrated that the most differentiated cells (HC-F1), characterized by the highest expression of keratinocyte differentiation markers, had the lowest proliferative activity. In contrast, less differentiated cells (HC-F2) maintained a higher proliferative activity. SdFFF is a tool to sort differentiated and/or proliferating cells from a total pool previously treated with a Ca2+ concentration inducing differentiation, and can be use to prepare biological models necessary for studying HaCaT cell proliferation after Ca2+-induced differentiation treatment.
Keywords: HaCaT cells; Calcium-induced differentiation; Proliferative activity; Sedimentation field-flow fractionation; Cell sorting;

Simple and selective method for the determination of various tyrosine kinase inhibitors used in the clinical setting by liquid chromatography tandem mass spectrometry by R. Honeywell; K. Yarzadah; E. Giovannetti; N. Losekoot; E.F. Smit; M. Walraven; J.S.W. Lind; C. Tibaldi; H.M. Verheul; G.J. Peters (1059-1068).
A fast, sensitive, universal and accurate method for the determination of four different tyrosine kinase inhibitors from biological material was developed using LC–MS/MS techniques. Utilizing a simple protein precipitation with acetonitrile a 20 μl sample volume of biological matrixes can be extracted at 4 °C with minimal effort. After centrifugation the sample extract is introduced directly onto the LC–MS/MS system without further clean-up and assayed across a linear range of 1–4000 ng/ml. Chromatography was performed using a Dionex Ultimate 3000 with a Phenomenex prodigy ODS3 (2.0 mm × 100 mm, 3 μm) column and eluted at 200 μl/min with a tertiary mobile phase consisting of 20 mM ammonium acetate:acetonitrile:methanol (2.5:6.7:8.3%). Injection volume varied from 0.1 μl to 1 μl depending on the concentration of the drug observed. Samples were observed to be stable for a maximum of 48 h after extraction when kept at 4 °C. Detection was performed using a turbo-spray ionization source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for Gefitinib (447.1  m/z; 127.9  m/z), Erlotinib (393.9  m/z; 278.2  m/z), Sunitinib (399.1  m/z; 283.1  m/z) and Sorafenib (465.0  m/z; 251.9  m/z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantification (LOQ) from cell culture medium were as follows: Gefitinib: 100.2 ± 3.8%, 11.2 nM; Erlotinib: 101.6 ± 3.7%, 12.7 nM; Sunitinib: 100.8 ± 4.3%, 12.6 nM; Sorafenib: 93.9 ± 3.0%, 10.8 nM, respectively. This was reproducible for plasma, whole blood, and serum. The method was observed to be linear between the LOQ and 4000 ng/ml for each analyte. Effectiveness of the method is illustrated with the analysis of samples from a cellular accumulation investigation and from determination of steady state concentrations in clinically treated patients.
Keywords: Gefitinib; Erlotinib; Sunitinib; Sorafenib; LC–MS/MS; Analysis;

Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography–tandem mass spectrometry by Vivekanandan Kannan; Deepak Gadamsetty; Madhankumar Rose; Stella Maria; Imran Mustafa; Anand Khedkar; Nitesh Dave; Muruganandam Arumugam; Harish Iyer (1069-1076).
A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 μm 50 mm × 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 μg/ml when 100 μl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C max) 0.40, 0.57, 1.95 μg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31 μg/ml on Day 28 for low, mid and high dose treated animals.
Keywords: Atosiban; Oxytocin receptor antagonist; LC–MS/MS; Solid phase extraction;

A rapid liquid chromatographic–tandem mass spectrometric (LC–MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC–MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 μg L−1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC–MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) were found to be below the chosen validation level of 1 μg L−1 for all compounds.
Keywords: LC–MS/MS; Bovine milk; Steroids; Bisphenol A; Analysis; Validation;

A quantitative assay for simultaneous measurement of individual human neutrophil peptide-1, -2 and -3 concentrations will aid in exploring the potential of these antimicrobial peptides as biomarkers for various diseases. Therefore, a liquid chromatography–tandem mass spectrometry method has been developed and validated to allow separate quantification of the three human neutrophil peptides in human plasma and serum. Plasma and serum samples (100 μl) were deproteinized by precipitation, followed by chromatographic separation on a Symmetry 300 C18 column (50 mm × 2.1 mm I.D., particle size 3.5 μm), using a water–methanol gradient containing 0.25% (v/v) formic acid and human alpha-defensin 5 as internal standard. Tandem mass spectrometric detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization. Despite low fragmentation efficiency of the antimicrobial peptides, multiple reaction monitoring was used for detection, though selecting the quaternary charged ions as both precursor and product. The method was linear for concentrations between 5 and 1000 ng/ml with a limit of detection around 3 ng/ml for all peptides. Intra- and inter-assay precisions were 14.8 and 19.1%, respectively, at the lowest measured endogenous concentration (6.4 ng/ml of HNP-1 in plasma), representing the lower limit of quantification of the assay. Recoveries of HNP-1, -2 and -3 from plasma and serum ranged between 85 and 128%. Analysis of serum samples from intensive care patients showed average concentrations of 362, 570 and 143 ng/ml for HNP-1, -2 and -3, respectively.
Keywords: Human neutrophil peptides; Quantification; LC–MS/MS;

In this paper, the amount of ascorbic acid (AA) in single rat peritoneal mast cell was determined by the method of capillary electrophoresis (CE) with electrochemical detection (ED) at a carbon fiber microdisk bundle electrode. The CE–ED system and the single-cell injection system were rearranged to make the operation more convenient and efficient. In the experiment, a self-made holder made of foam was used to keep the capillary from swing, which kept the stability of the baseline of the electropherogram. The single cell was lysed completely within 5 s using the 0.1% sodium dodecylsulfate (SDS) as the cell lysis solution together with the lysis voltage of 2 kV. The quantitation analysis was accomplished by the use of calibration curves, and the amount of AA in single rat peritoneal mast cell was from 2.4 to 7.1 fmol.
Keywords: Ascorbic acid; Capillary electrophoresis; Electrochemical detection; Rat peritoneal mast cell; Single-cell analysis;

Liquid chromatography–tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid by Maria van der Ham; Tom J. de Koning; Dirk Lefeber; André Fleer; Berthil H.C.M.T. Prinsen; Monique G.M. de Sain-van der Velden (1098-1102).
Analysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated.The method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2 → 87.0 (SA) and m/z 311.2 → 90.0 (13C3-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients’ samples with known differences in SA metabolites like meningitis (n  = 6), brain tumour (n  = 2), leukaemia (n  = 5), and Salla disease (n  = 1).Limit of detection (LOD) was 0.54 μM for FSA and 0.45 μM for TSA. Intra- and inter-assay variation for FSA (21.8 μM) were 4.8% (n  = 10) and 10.4% (n  = 40) respectively. Intra- and inter-assay variation for TSA (35.6 μM) were 9.7% (n  = 10) and 12.8% (n  = 40) respectively. Tested patients showed values of TSA above established reference value.The validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses.
Keywords: Sialic acid; Cerebrospinal fluid; High-performance liquid chromatography–tandem mass spectrometry;

Comparison of derivatization and chromatographic methods for GC–MS analysis of amino acid enantiomers in physiological samples by Magdalena C. Waldhier; Katja Dettmer; Michael A. Gruber; Peter J. Oefner (1103-1112).
GC–MS analysis of fluorinated and non-fluorinated chloroformate and anhydride derivatives of amino acid (AA) enantiomers on two different chiral columns was compared for the direct quantification of free l- and d-AAs in human serum and urine in a single analytical run. Best sensitivity was achieved with pentafluoropropionic anhydride/heptafluorobutanol derivatives separated on a Chirasil-l-Val column. However, the occurrence of racemization during derivatization precluded accurate quantification of AA enantiomers. Derivatization with methyl chloroformate/methanol and separation on an Rt-γDEXsa column did not exhibit racemization and yielded ten baseline separated racemates of proteinogenic AAs with resolution values greater than 2.4. However, protein and peptide hydrolysis occurred in serum and urine during the highly exothermal derivatization reaction under alkaline conditions. Removing serum proteins by precipitation before derivatization and performing the reaction at neutral pH enabled the determination of accurate free AA enantiomer concentrations. Accuracy of quantification was validated by an established nonchiral GC–MS method for AA analysis. Reliable quantification was achieved using stable-isotope labeled l-AAs as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) for the d-AAs were in the range of 3.2–446 nM and 0.031–1.95 μM, respectively. Relative standard deviations (N  = 6) for the measurement of AAs in urine and serum ranged from 0.49–11.10% to 0.70–3.87%, respectively. The method was applied to the analysis of urine from 19 patients with renal insufficiency. In comparison to healthy probands, D-ratios of Ala, Val, Pro, Thr, Asp, and Asn were significantly increased.
Keywords: Amino acid enantiomers; GC–MS; Urine; Serum; Pre-column derivatization; Methyl chloroformate; Racemization; Renal failure;

The metabolites in rats after administration of icariside II, icariin, epimedin C and extracts of four Epimedium species were investigated. Feces, bile, plasma and urine samples were detected comprehensively using HPLC-ESI-MS n method. The structures of metabolites were identified on the basis of their characteristic fragmentations in MS n experiments. Totally, 54 metabolites were identified in these biosamples. Specific hydrolysis of 7-O glucosides in gut lumen and glucuronic acid conjugation in liver were considered as the main physiologic processes of prenylflavonoids. Icariside II and anhydroicaritin were the major intermediate products in forming of mono- and di-glucuronic acid conjugations in vivo. In general, this study revealed the possible metabolite profiles of prenylflavonoids in rats, and might aid the clinical use of different Epimedium species.
Keywords: Epimediums; Prenylflavonoids; Metabolite profile; HPLC-ESI-MS n ;

A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein–hapten binding in the skin, is presented. Mass spectra of BSA–FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 mM. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined, 28 possible and 2 non-binding sites for FITC.
Keywords: Capillary electrophoresis; MALDI-TOF-MS; Contact allergy; Peptide-fluorescein isothiocyanate adducts; Bovine serum albumin;

A reagent, 1-(4-isopropyl) phenyl-3-methyl-5-pyrazolone (PPMP) has been synthesized and used for high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) determination of pre-column labeled carbohydrates. Monosaccharides have been quantitatively converted into mono-PPMP-labeled derivatives with 28% aqueous ammonia as a catalyst at 80 °C during 70 min. Mono-PPMP derivatives have been demonstrated to exhibit better chemical stability than bis-PMP ones. PPMP-labeled mixture of twelve monosaccharides (galactosamine, glucosamine, galacturonic acid, glucuronic acid, galactose, glucose/N-acetylgalactosamine, N-acetylglucosamine, xylose, arabinose, mannose, fucose, and rhamnose) has been well separated by a reverse-phase HPLC and detected by on-line ESI-MS method under optimized conditions. The data on characteristic fragment ions of the 13 PPMP-labeled monosaccharides with MS2 data have been collected. The suggested method exhibits good linearity (correlation coefficients > 0.9975) between the peak areas and the concentration of monosaccharides in a broad concentration range and good reproducibility (RSD < 3.19%). The developed method has been successfully applied to analyze the monosaccharide composition of natural Spirulina polysaccharide SPPB-1.
Keywords: Carbohydrate; 1-(4-Isopropyl) phenyl-3-methyl-5-pyrazolone (PPMP); Derivatization; High-performance liquid chromatography (HPLC); Electrospray ionization mass spectrometry (ESI-MS);

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.
Keywords: Lysophosphatidic acid (LPA); Lysophosphatidylcholine (LPC); LC/MS/MS; Rat brain tissue; Tissue section; Liquid–liquid extraction;

Analysis of cyclosporine A and its metabolites in rat urine and feces by liquid chromatography–tandem mass spectrometry by Zhi-gang Fang; Ben-gang You; Ya-gen Chen; Jian-kang Zhang; Yue-qing Liu; Xue-nong Zhang; Qiang Zhang (1153-1162).
A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of cyclosporine A (CyA) and the identification of its metabolites in rat urine and feces. The analytes were extracted from waste samples via liquid–liquid extraction. A Turboionspray source was used as a detector. It was operated in a positive ion mode with transitions of m/z 1225 →  m/z 1112 for CyA and in a selected multiple reactions monitoring (MRM) mode with transitions of m/z 1239 →  m/z 1099 for the internal standard (cyclosporine D, CyD). Linear calibration curves were obtained for CyA concentration ranges of 12.5–250 ng mL−1 in urine and 2.5–375 ng mg−1 in feces. The intra- and inter-day precision values (relative standard deviation) obtained were less than 8%, and the accuracy was within ±15% for each of the analytes. Extraction recoveries of CyA and CyD were both over 80%. The identification of the metabolites and elucidation of their structure were performed on the basis of their retention times and mass spectrometry fragmentation behaviors. A total of seven metabolites in rat feces were identified as dimethyl CyA, hydroxy CyA, and dihydroxy CyA after the oral administration of cyclosporine A-Eudragit® S100 nanoparticles (CyA-NP). Six of these metabolites were also detected in rat urine. A possible metabolic pathway was also proposed. The newly developed method was proven to be sensitive, simple, reproducible, and suitable for the rapid determination of CyA. It was successfully employed to study the excretion of CyA in rats and could be used to better understand the in vivo metabolism of CyA-NP, a potentially effective nanoparticle system.
Keywords: Cyclosporine A; Metabolites; LC–MS/MS;

Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay by Johannes M.W. van den Ouweland; Antonius M. Beijers; Pierre N.M. Demacker; Henny van Daal (1163-1168).
The plasma 25-OH vitamin D concentration is a reliable biomarker for vitamin D status but assay's variability makes adequate monitoring of vitamin D status difficult. We employed isotope-dilution liquid chromatography (LC) tandem-mass spectrometry (MS/MS) for the measurement of both 25-OH vitamin D3 and 25-OH vitamin D2 in human serum. Hexadeuterium labelled 25-OH vitamin D3 internal standard (IS) was added to calibrators (prepared in phosphate-buffered saline with 60 g/L albumin), controls or patient sera and 25-OH vitamin D metabolites were released from vitamin D binding protein by adding sodium hydroxide prior to protein precipitation by acetonitrile/methanol (9:1, v/v). Subsequent off-line solid-phase extraction was followed by chromatographic separation on a C-18 column using a water/methanol/ammonium acetate gradient. Detection was by Atmospheric Pressure Electrospray Ionisation (AP-EI) followed by selected reaction monitoring. We compared the LC-MS/MS assay to the DiaSorin radioimmunoassay (RIA) and a recently re-standardised version of an automated electrochemiluminescent immunoassay (ECLIA) from Roche Diagnostics. We also analysed external quality control samples from the International Vitamin D External Quality Assessment Scheme (DEQAS) for comparison with other participating laboratories using LC-MS. The method was linear from 5 to at least 550 nmol/L with intra- and interday CV's ≤6% for both 25-OH vitamin D3 and 25-OH vitamin D2. Recoveries ranged between 94.9 and 106.9% for 25-OH vitamin D3 and 82.7 and 100.3% for 25-OH vitamin D2. Our results for the DEQAS serum pools averaged −7.2% from the overall LC-MS method mean. The DiaSorin RIA agreed well with the LC-MS/MS method (r 2  = 0.90; average bias 1.61 nmol/L), the Roche ECLIA considerably disagreed (r 2  = 0.58; bias 10.13 nmol/L). This LC-MS/MS method is reliable and robust for the measurement of both 25-OH vitamin D3 and 25-OH vitamin D2 in human serum.
Keywords: 25-Hydroxyvitamin D; LC-MS/MS; Radioimmunoassay; Method comparison;

Simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma by LC–MS/MS—A high throughput approach to evaluate drug–drug interactions by Wei Zhang; Futian Han; Ping Guo; Harry Zhao; Zhongping (John) Lin; Mike-Qingtao Huang; Kirk Bertelsen; Naidong Weng (1169-1177).
Drug–drug interactions involving cytochrome P450 (CYP450s) are an important factor for evaluation of a new chemical entity (NCE) in drug development. To evaluate the potential inhibitory effects of a NCE on the pharmacokinetics of a cocktail of representative probes of CYP enzymes (midazolam for CYP3A4, tolbutamide for CYP2C9, omeprazole for CYP2C19 and dextromethorphan for CYP2D6) and the safety and tolerability of the NCE in the presence of probe substrates, a high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma using tolbutamide-d9, midazolam-d4, (±)-omeprazole-d3, and dextromethorphan-d3 as the internal standards (ISs). Human plasma samples of 50 μL were extracted by a simple protein-precipitation procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. Reversed-phase HPLC separation was achieved with a Hypersil GOLD AQ column (50 mm × 4.6 mm, 5 μm). MS/MS detection was set at mass transitions of 271 → 172 m/z for tolbutamide, 346 → 198 m/z for omeprazole, 326 → 291 m/z for midazolam, 272 → 171 m/z for dextromethorphan, 280 → 172 m/z for tolbutamide-d9 (IS), 349 → 198 m/z for (±)-omeprazole-d3 (IS), 330 → 295 m/z for midazolam-d4 (IS), and 275 → 171 m/z for dextromethorphan-d3 (IS) in positive mode. The high throughput LC–MS/MS method was validated for accuracy, precision, sensitivity, stability, recovery, matrix effects, and calibration range. Acceptable intra-run and inter-run assay precision (<10%) and accuracy (<10%) were achieved over a linear range of 50–50,000 ng/mL for tolbutamide, 1–1000 ng/mL for omeprazole, 0.1–100 ng/mL for midazolam and 0.05–50 ng/mL for dextromethorphan in human plasma. Method robustness was demonstrated by the 100% pass rate of 24 incurred sample analysis runs and all of the 50 clinical study samples used for incurred sample reproducibility (ISR) test having met the acceptance criterion (%Diff within 20%). The overall ISR results for all compounds showed that over 95% of the samples had a %Diff of less than 10%. The method is simple, rapid and rugged, and has been applied successfully to sample analysis in support of a drug–drug interaction study.
Keywords: Drug–drug interaction; P450; Tolbutamide; Omeprazole; Midazolam; Dextromethorphan; LC–MS/MS; Incurred sample reproducibility (ISR);

Simultaneous speciation of selenomethionine and 2-hydroxy-4-methylselenobutanoic acid by HPLC–ICP MS in biological samples by Véronique Vacchina; Marc Moutet; Jean-Claude Yadan; Frédéric de Baene; Bernard Kudla; Ryszard Lobinski (1178-1180).
An analytical method was developed for the simultaneous speciation of selenomethionine (SeMet) and 2-hydroxy-4-methylselenobutanoic acid (NutraSelen®), a new SeMet precursor. The compounds could be baseline resolved by ion-pairing reversed-phase HPLC using ICP MS detection. Detection limits of 1 ng mL−1 (Se content) could be reached. SELM-1 reference material was used to validate the SeMet measurement. Additionally, the quantification of NutraSelen® was validated by standard addition together with checking the Se mass balance. The procedure developed was then applied to the monitoring of the conversion of NutraSelen® into SeMet by yeast.
Keywords: Selenium; Selenomethionine; NutraSelen®; HPLC–ICP MS;

Determination of picamilon concentration in human plasma by liquid chromatography–tandem mass spectrometry by Wenqi Cui; Xiaoyan Chen; Yan Zhan; Zhenzhong Zhang; Yifan Zhang; Dafang Zhong (1181-1184).
A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of picamilon concentration in human plasma. Picamilon was extracted from human plasma by protein precipitation. High performance liquid chromatography separation was performed on a Venusil ASB C18 column with a mobile phase consisting of methanol −10 mM ammonium acetate–formic acid (55:45:01, v/v/v) at a flow rate of 0.65 ml/min. Acquisition of mass spectrometric data was performed in selected reaction monitoring mode, using the transitions of m/z 209.0 →  m/z (78.0 + 106.0) for picamilon and m/z 152.0 →  m/z (93.0 + 110.0) for paracetamol (internal standard). The method was linear in the concentration range of 1.00–5000 ng/ml for the analyte. The lower limit of quantification was 1.00 ng/ml. The intra- and inter-assay precision were below 13.5%, and the accuracy was between 99.6% and 101.6%. The method was successfully applied to characterize the pharmacokinetic profiles of picamilon in healthy volunteers. This validated LC–MS/MS method was selective and rapid, and is suitable for the pharmacokinetic study of picamilon in humans.
Keywords: Picamilon; LC–MS/MS; Pharmacokinetics; Human plasma;

Simultaneous determination of procaine, lidocaine, ropivacaine, tetracaine and bupivacaine in human plasma by high-performance liquid chromatography by Wei-wei Qin; Zheng Jiao; Ming-kang Zhong; Xiao-jin Shi; Jun Zhang; Zhong-dong Li; Xue-yan Cui (1185-1189).
A simple and sensitive high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been developed and validated for simultaneous quantification of five local anesthetics in human plasma: procaine, lidocaine, ropivacaine, tetracaine and bupivacaine. In an ice-water bath, 500 μL plasma sample, containing 100 μg/mL neostigmine methylsulfate as anticholinesterase, was spiked with carbamazepine as internal standard and alkalized by sodium hydroxide. Liquid–liquid extraction with ethyl ether was used for plasma sample preparation. The chromatographic separation was achieved on a Kromosil ODS C18 column with a mobile phase consisting of 30 mM potassium dihydrogen phosphate buffer (0.16% triethylamine, pH adjusted to 4.9 with phosphoric acid) and acetonitrile (63/37, v/v). The detection was performed simultaneously at wavelengths of 210 and 290 nm. The chromatographic analysis time was 13 min per sample. The calibration curves of all five analytes were linear between 0.05 and 5.0 μg/mL (r 2  ≥ 0.998). Precision ranged from 1.4% to 7.9% and accuracy was between 91.7% and 106.5%. The validated method is applicable for simultaneous determination of procaine, lidocaine, ropivacaine, tetracaine and bupivacaine for therapeutic drug monitoring and pharmacokinetic study.
Keywords: Procaine; Lidocaine; Ropivacaine; Tetracaine; Bupivacaine; High-performance liquid chromatography;

Determination of trace endocrine disruptors in ultrapure water for laboratory use by the yeast estrogen screen (YES) and chemical analysis (GC/MS) by Katy Sanfilippo; Barbara Pinto; Maria Perla Colombini; Ugo Bartolucci; Daniela Reali (1190-1194).
High purity water for endocrine disruptors (EDs) analysis in experimental tests is an indispensable requirement for the preparation of reagents and solutions employed in biological laboratories. Commercial ultrapure water may contain traces of organic compounds, which can interfere with in vitro bioassays carried out to detect the potential estrogen-like activity of pure compounds and complex mixtures. This paper shows that solid-phase extracts of different types of ultrapure water (UPW) purchased or produced in situ for laboratory analysis (mQ-UPW) may contain organic molecules able to antagonize the binding of E2 to the human estrogen receptor α in the yeast estrogen screen (YES) assay. GC/MS analysis detected the presence of bis(2-ethylhexyl) phthalate (DEHP) (0.033 ppm ± 0.006) in mQ-UPW extracts. The dose–response curve of DEHP in the YES assay showed a relevant antagonist effect of this phthalate. Agreement between content of DEHP chemically detected in UPW extract and the magnitude of biological effects induced was pointed out. It would be appropriate that chemical analyses were complemented by biological tests to establish concentration limits for chemical contaminants in UPW that do not induce biological effects detectable in vitro. The yeast assay used in this study has previously proved to be a sensitive tool in assessing the presence of agonistic/antagonistic chemicals at the ng/l level in complex mixtures and may be successfully used to identify trace amounts of estrogenic/antiestrogenic chemicals, which can represent critical issues influencing the experimental results in environmental testing laboratories.
Keywords: Ultrapure water; Antagonist activity; Endocrine disruptors; YES assay; GC/MS;

Aldosterone and cortisol are useful biomarkers of dehydration and stress, respectively. The aim of this study was to develop an HPLC–tandem mass spectrometric method for the simultaneous measurement of aldosterone and cortisol in human plasma that could be applied to the study of athletes undergoing exercise and rehydration. Samples were prepared and analysed using an on-line sample preparation/HPLC system coupled to a triple quadrupole tandem-mass spectrometer. Samples (200 μL) were pre-treated and extracted on Hysphere C18 HD cartridges (7 μm, Spark Holland). Chromatography was performed on a Sunfire C18 analytical column (50 mm × 3.0 mm, 3 μm, Waters) under isocratic conditions at a flow rate of 0.3 mL/min. The mobile phase consisted of 35% acetonitrile/water. Mass spectrometric detection was by selected reaction monitoring using negative electrospray ionization conditions. The assay had an analytical range of 25–500 pg/mL and 25–500 ng/mL for aldosterone and cortisol, respectively (r 2  > 0.992, n  = 22). Inter-day accuracy and imprecision for quality control samples was 99.4–106% and <16%, respectively (n  = 10). In a study of nine human subjects, both aldosterone and cortisol concentrations reflected the expected physiological responses to dehydration, rehydration and exercise when measured by this method. The reported method is suitable to facilitate the study of athletes undergoing dehydration and rehydration protocols.
Keywords: Aldosterone; Cortisol; Mass spectrometry; Exercise; Dehydration; Stress;

Validated liquid chromatography–tandem mass spectrometry method for quantitative determination of dauricine in human plasma and its application to pharmacokinetic study by Xiaoying Liu; Qian Liu; Dongmei Wang; Xueya Wang; Peng Zhang; Haiyan Xu; Hui Zhao; Huaiqing Zhao (1199-1203).
A highly sensitive and selective LC–MS/MS method was developed and validated for the determination of dauricine in human plasma, using protopine as internal standard (IS). The analyte and IS were extracted by liquid–liquid extraction and analyzed by LC–MS/MS. Chromatographic separation was performed on Agilent TC-C18 column with a mobile phase of methanol–water–glacial acetic acid (60:40:0.8, v/v/v) at a flow rate of 0.7 mL/min. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The method was linear over the concentration range of 1–200 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL in human plasma with acceptable precision and accuracy. The intra- and inter-day precision was less than 5.9% determined from quality control (QC) samples at concentrations of 2.0, 20.0 and 160 ng/mL, and the accuracy was within ±9.9%. This method was successfully applied for the evaluation of pharmacokinetics of dauricine after oral doses of 100, 300 and 600 mg phenolic alkaloids of menispermum dauricum tablet (PAMDT) to 12 Chinese healthy volunteers.
Keywords: LC–MS/MS; Pharmacokinetics; Dauricine;