Journal of Chromatography B (v.853, #1-2)

The analysis of exhaled air has several advantages since it is a noninvasive method applicable to a large number of toxic agents, in addition to being a simpler matrix than those of other biological samples such as urine and blood. However, it presents some challenges, such as the necessity of a more sensitive sampling procedure, since the chemical substances eliminated through exhaled air are unchanged in form, not being metabolized, and exhaled compounds are present at extremely low concentrations, i.e. in the nanomolar range. To improve the sensitivity and precision of measurement of the concentration of these substances in exhaled air, the sample usually has to be concentrated before assay by gas chromatography. To this end, the use of the solid-phase microextraction (SPME) technique has been proposed as an efficient sampling method. This paper presents a revision of breath analysis as a biomarker for occupational and environmental exposure to chemicals. The sampling methods and the potential use of SPME for determining chemical substances in exhaled air are discussed.
Keywords: Breath analysis; Gas chromatography; Solid-phase microextraction; Biomarker; Occupational exposure; Environmental exposure;

3-(4-Bromophenyl)-5-acetyloxymethyl-2,5-dihydrofuran-2-one (LNO-18-22) is a representative member of a novel group of potential antifungal drugs, derived from a natural 3,5-disubstituted butenolide, (-)incrustoporine, as a lead structure. This lipophilic compound is characterized by high in vitro antifungal activity and low acute toxicity. For the purpose of in vivo studies, a new bioanalytical high-performance liquid chromatographic method with UV photodiode-array and mass spectrometric detection (HPLC-PDA-MS), involving a direct injection of diluted mouse urine was developed and used in the evaluation of the metabolic profiling of this drug candidate. The separation of LNO-18-22 and its phase I metabolites was performed in 37 min on a 125 mm × 4 mm chromatographic column with Purospher RP-18e using an acetonitrile–water gradient elution. Scan mode of UV detection (195–380 nm) was employed for the identification of the parent compound and its biotransformation products in the biomatrix. Finally, the identity of LNO-18-22 and its metabolites was confirmed using HPLC-MS analyses of the eluate. These experiments demonstrated the power of a comprehensive analytical approach based on the combination of xenobiochemical methods and the results from tandem HPLC-PDA-MS (chromatographic behaviour, UV and MS spectra of native metabolites versus synthetic standards). The chemical structures of five phase I LNO-18-22 metabolites and one phase II metabolite were elucidated in the mouse urine, with two of these metabolites having very unexpected structures.
Keywords: Incrustoporine derivatives; 3-(4-Bromophenyl)-5-acetyloxymethyl-2,5-dihydrofuran-2-one (LNO-18-22); Metabolites in mouse urine; HPLC with UV PDA, APCI-MS and ESI-MS detection;

Sample preparation and bioinformatics in MALDI profiling of urinary proteins by Panagiotis Zerefos; Julien Prados; Sophia Kossida; Alexandros Kalousis; Antonia Vlahou (20-30).
One of the challenges of current proteomics research is identifying healthy and diseased mass spectrometric (MS) patterns within biological fluids. As a result, sample preparation methodologies, as well as the mathematical tools utilized for MS data analysis become pivotal. This study involves a thorough evaluation of the reproducibility and protein resolution that various urinary protein preparation methodologies provide in MALDI MS analysis. Several precipitation approaches, ultrafiltration, as well as direct dilution of urine in MALDI MS compatible buffers were applied in combination to a thorough bioinformatics analysis of the generated MS data. Our results indicate that ultrafiltration, as well as direct dilution of urine in TFA, can provide information rich and reproducible spectra for mass ranges up to 20 kDa. The importance of the presence of peak reproducibility filters when generating disease classification models is suggested.
Keywords: Urine; Proteomics; MALDI; Bioinformatics; Biomarker research;

Control of electroosmotic flow by a cation additive to enhance the separation of amino acids by micellar electrokinetic chromatography by Qishu Qu; Xiaoqing Tang; Debby Mangelings; Chengyin Wang; Gongjun Yang; Xiaoya Hu; Chao Yan (31-37).
The effect of a divalent cation (Mg2+) and 3 monovalent cations (Na+, Li+, and K+) as buffer additives on the electroosmotic flow (EOF) was investigated in order to improve the separation performance of p-acetamidobenzenesulfonyl fluoride (PAABS-F) derivatives of 20 standard amino acids by micellar electrokinetic chromatography (MEKC). The EOF can be decreased with increasing concentration of cations with the order of cations as Mg2+  > K+  > Na+  > Li+. However, it was found that the resolution cannot be improved easily using a buffer cation which is more capable of decreasing the EOF. Taking the migration time, resolution, and peak shape into account, Na+ is the best buffer additive, although the EOF decreased most using Mg2+. Using 20 mmol/L borate at pH 9.3 containing 140 mmol/L SDS and 20 mmol/L Na+ as electrolyte, 20 standard amino acids were successfully separated within 14 min.
Keywords: Micellar electrokinetic chromatography; Amino acids; Electroosmotic flow; Cation additive;

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.
Keywords: Plasmid DNA; Methacrylate monolith; Fast purification; Enzyme-free process;

Determination of eprosartan in human plasma and urine by LC/MS/MS by Xue-Ning Li; Hong-Rong Xu; Wei-Li Chen; Gang-Yi Liu; Nan-Nan Chu; Chen Yu (47-53).
A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mm × 2.0 mm, 5 μm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 μg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.
Keywords: Eprosartan; LC/MS/MS; Protein precipitation; Plasma; Urine;

Halitosis associated volatiles in breath of healthy subjects by Sandra van den Velde; Marc Quirynen; Paul Van hee; Daniel van Steenberghe (54-61).
Halitosis can have an intra- or extra-oral origin. In all cases, bad breath is caused by the presence of volatile organic compounds originating from the mouth or the expired air. They can be specific for certain diseases or infections.This study explored the presence and concentration of these volatile compounds normally associated with halitosis in the breath of healthy symptomless volunteers.Alveolar and mouth air of 40 healthy volunteers as well as environmental air were analyzed by gas chromatography–mass spectrometry (GC–MS) and by a commercially available GC device (OralChroma®).14 compounds, associated with halitosis could be detected. All of them except carbon disulfide, appeared to be (partly) produced endogenously and/or in the mouth. Acetone, 2-butanone, 2-pentanone and 1-propanol were common to all volunteers in both alveolar and mouth air and indole and dimethyl selenide in alveolar air.GC–MS seems a promising tool for differential diagnosis of halitosis, with the possibility to detect extra-oral causes, which often remain undetected unless characterized by a specific smell.
Keywords: Halitosis; VOC's; GC/MS; Alveolar air; Breath air;

Crosslinking copolymer microbeads with a diameter range of 100–150 μm were synthesized by suspension copolymerization of glycidyl methacrylate (GMA), acrylamide (AM) and N,N′-methylene bisacrylamide (MBA). Subsequently, polyethyleneimine (PEI) was grafted on the surfaces of the terpolymer microbeads GMA/AM/MBA via the ring-opening reaction of the epoxy groups, and the grafting microbeads PEI-GMA/AM/MBA were prepared. In this paper, the adsorption property of the grafting microbeads for bilirubin was mainly investigated, and the effects of various factors, such as pH value, ionic strength and grafting degree of PEI on the surface of grafting microbeads and the adsorption capacity of the grafting microbeads for bilirubin were examined. The batch adsorption experiment results show that by right of the action of grafted polyamine macromolecules PEI, the grafting microbeads PEI-GMA/AM/MBA have quite strong adsorption ability for bilirubin; the isotherm adsorption conforms to Freundlich equation. The pH value of the medium affects the adsorption capacity greatly, As in the nearly neutral solutions with pH 6, the grafting microbeads have the strongest adsorption ability for bilirubin, whereas in acidic and basic solutions their adsorption ability is weak. The ionic strength hardly affects the adsorption ability of the grafting microbeads. The grafting degree of PEI on the surfaces of the grafting microbeads also has a great effect on the adsorption capacity, and higher the grafting degree of PEI on the surface of the microbead PEI-GMA/AM/MBA, the stronger is the adsorption ability of the microbeads.
Keywords: Bilirubin; Polyethyleneimine; Grafting; Glycidyl methacrylate; Terpolymer microbead; Adsorption;

Imidafenacin (KRP-197/ONO-8025, IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a new antimuscarinic agent currently under application for the indication of treatment of overactive bladder in Japan. We developed and validated the sensitive and selective bioanalytical methods for the extremely low levels of IM and its metabolite, M-2 (Method 1), M-4 (Method 2) and M-9 (Method 3) in human plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS). In each method, plasma sample was extracted by solid phase extraction, separated on a semi-micro high performance liquid chromatography column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization or ionspray interface. Selected reaction monitoring mode was used for quantification. Each method was found to have acceptable accuracy, precision, stability, selectivity and linearity over the concentration range of 10–500 pg/mL for IM and M-2, 10–1000 pg/mL for M-4 and 50–5000 pg/mL for M-9. Using these analytical methods, concentration profiles of IM and its metabolites in human plasma were successfully determined even in the low pg/mL levels after oral administration of IM at the therapeutic dosage of 0.1 mg.
Keywords: Imidafenacin; KRP-197; ONO-8025; Metabolites; Human-plasma; LC–MS/MS;

Development and validation of a liquid chromatography–mass spectrometry assay for the determination of pyronaridine in human urine by Himanshu Naik; She-Yi Wei; Mark S. Schmidt; Daryl J. Murry; Lawrence Fleckenstein (80-87).
A reliable method has been developed for the determination of pyronaridine in human urine using amodiaquine as an internal standard. Liquid–liquid extraction was used for sample preparation. Analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. The extracted ion for pyronaridine was m/z 518.20 and for amodiaquine was m/z 356.10. Chromatography was carried out using a Gemini 5 μm C18 3.0 mm × 150 mm column using 2 mM perflurooctanoic acid and acetonitrile mixture as a mobile phase delivered at a flow rate of 0.5 mL/min. The mobile phase was delivered in gradient mode. The retention times of pyronaridine and amodiaquine were 9.1 and 8.1 min respectively, with a total run time of 14 min. The assay was linear over a range of 14.3–1425 ng/mL for pyronaridine (R 2  ≥ 0.992, weighted 1/Concentration). The analysis of quality control samples for pyronaridine at 28.5, 285, 684 and 1140 ng/mL demonstrated excellent precision with relative standard deviation of 5.1, 2.3, 3.9 and 9.2%, respectively (n  = 5). Recoveries at concentrations of 28.5, 285, 684 and 1140 ng/mL were all greater than 85%.This LC–MS method for the determination of pyronaridine in human urine has excellent specifications for sensitivity, reproducibility and accuracy and can reliably quantitate concentrations of pyronaridine in urine as low as 14.3 ng/mL. The method will be used to quantify pyronaridine in human urine for pharmacokinetic and drug safety studies.
Keywords: Pyronaridine; Antiparasitic; Liquid chromatography–mass spectroscopy;

Concurrent determination of ezetimibe and its phase-I and II metabolites by HPLC with UV detection: Quantitative application to various in vitro metabolic stability studies and for qualitative estimation in bile by Shaik Jafar Sadik Basha; Shaik Abdul Naveed; Nirbhay Kumar Tiwari; Dhanya Shashikumar; Syed Muzeeb; Thammera Ranjith Kumar; Nyavanandi Vijay Kumar; Nadipalli Prabhakar Rao; Nanduri Srinivas; Ramesh Mullangi; Nuggehally R. Srinivas (88-96).
Simultaneous separation and quantification of ezetimibe (EZM) and its phase-I metabolite i.e., ezetimibe ketone (EZM-K) and phase-II metabolite i.e., ezetimibe glucuronide (EZM-G) in various matrices was accomplished by gradient HPLC with UV detection. The assay procedure involved deproteinization of 500 μL of either incubation or bile sample containing analytes and internal standard (IS, theophylline) with 75 μL acetonitrile containing 25% perchloric acid. An aliquot of 100 μL supernatant was injected onto a C18 column. The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid:acetonitrile:methanol:water at a flow rate of 1.0 mL/min. The detection of analyte peaks were achieved by monitoring the eluate using an UV detector set at 250 nm. Nominal retention times of IS, EZM-G, ezetimibe ketone glucuronide (EZM-KG), EZM and EZM-K were 9.39, 24.23, 27.82, 29.04 and 30.56 min, respectively. Average extraction efficiencies of EZM, EZM-G and IS was >75–80% and for EZM-K was >50% from all the matrices tested. Limit of quantitation (LOQ) for EZM, EZM-K and EZM-G was 0.02 μg/mL. Due to the lack of availability of reference standard of EZM-KG, the recovery and LOQ aspects for this metabolite were not assessed. Overall, the method is suitable for simultaneous measurement of EZM, and its phase-I and phase-II metabolite (EZM-G) in in vitro and in vivo studies.
Keywords: Ezetimibe; Ezetimibe ketone; Ezetimibe glucuronide; Ezetimibe ketone glucuronide; HPLC; Metabolism;

Determination of Cloretazine™ (VNP40101M) and its active metabolite (VNP4090CE) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC–ESI-MS/MS) by Feng Bai; Patton Minkin; Charles H. Fraga; Melinda A. O'Shaughnessy; Sri Gururangan; Clinton F. Stewart (97-104).
A sensitive method for the determination of Cloretazine™ (VNP40101M) and its metabolite (VNP4090CE) with an internal standard (ISTD) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Acidified plasma samples (500 μL) were prepared using solid phase extraction (SPE) columns, and 25 μL of the reconstituted sample was injected onto an Ascentis C18 HPLC column (3 μm, 5 cm × 2.1 mm) with an isocratic mobile phase. Analytes were detected with an API-3000 LC–MS/MS System at unit (Q1) and low (Q3) resolution in negative multiple reaction monitoring mode: m/z 249.0 (precursor ion) to m/z 114.9 (product ion) for both Cloretazine™ (at 3.64 min) and VNP4090CE (at 2.91 min), and m/z 253.0 (precursor ion) to m/z 116.9 (product ion) for the ISTD. The mean recovery for Cloretazine™ (VNP40101M) and its metabolite (VNP4090CE) was greater than 87% with a lower limit of quantification of 1.0 ng/mL for Cloretazine™ (S/N = 9.7, CV ≤ 12%) and 0.5 ng/mL for VNP4090CE (S/N = 11.3, CV ≤ 9.7%). This method was validated over a linear range of 1.0–1000 ng/mL for Cloretazine™ and 0.5–100 ng/mL for VNP4090CE, and results from a five day validation study demonstrated good within-day and between-day precision and accuracy. This method has been used to measure plasma Cloretazine™ and its metabolite concentrations in a Phase I study in children with recurrent progressive or refractory primary brain tumors.
Keywords: Plasma; Cloretazine; Liquid chromatography; Electrospray tandem mass spectrometry (LC–ESI-MS/MS);

Use of magnetic poly(glycidyl methacrylate) monosize beads for the purification of lysozyme in batch system by Evrim Banu Altıntaş; Nalan Tüzmen; Nilgün Candan; Adil Denizli (105-113).
The hydrophobic affinity ligand l-tryptophan immobilized magnetic poly(glycidyl methacrylate) [m-poly(GMA)] beads in monosize form (1.6 μm in diameter) were used for the affinity purification of lysozyme from chicken egg white. The m-poly(GMA) beads were prepared by dispersion polymerization in the presence of Fe3O4 nano-powder. The epoxy groups of the m-poly(GMA) beads were converted into amino groups with 1,6 diaminohexane (i.e., spacer arm). l-tryptophan was then covalently immobilized on spacer arm attached m-poly(GMA) beads. Elemental analysis of immobilised l-tryptophan for nitrogen was estimated as 42.5 μmol/g polymer. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration and temperature). Maximum lysozyme adsorption amount of m-poly(GMA) and m-poly(GMA)-l-tryptophan beads were 1.78 and 259.6 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption–elution cycle, m-poly(GMA)-l-tryptophan beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. It was found to be successful in achieving purification of lysozyme in a high yield of 76% with a purification fold of 71 in a single step. The specific activity of the eluted lysozyme (62,580 U/mg) was higher than that obtained with a commercially available pure lysozyme (Sigma (60,000 U/mg).
Keywords: Magnetic monosize beads; Lysozyme purification; Poly(glycidyl methacrylate);

The influence of the chemical substitutions on the interfacial interactions of pyrimidines with the phospholipid-mimic immobilized-artificial-membrane (IAM) chromatographic stationary phase was evaluated. Monocyclic pyrimidine nucleic acid bases (nucleobases) were revealed behaving differently from their bicyclic purine counterparts substantially. The computed electrostatic potential surfaces for both the IAM phase and the interacting nucleobases are intuitive in deconvoluting the retention patterns of pyrimidines molecularly. A structure-retention model has also been derived using quantitative 3D-QSAR methodology pertinent to the IAM-retention of pyrimidines for the potential use in molecular design. IAM phase is found particularly suitable in assessing the retention of pyrimidines with bulky or elongated exocyclic substituents in the biological context than the alkyl-based chromatographic counterparts.
Keywords: Pyrimidine; IAM; Retention; CoMSIA; ESP; Modeling;

A HPLC method utilizing solid phase extraction was developed to analyze doripenem (formerly S-4661) in human and mouse serum. A reversed-phase column was used with a UV detector set at 295 nm. The mobile phase consisted of methanol and phosphate buffer at a flow rate of 1.5 ml/min. Meropenem was used as the internal standard. The standard curve was linear over a range of 0.5–40 μg/ml. The assay is simple, reproducible, and accurate and has been used successfully to analyze doripenem concentrations from a murine pharmacokinetic study.
Keywords: Doripenem; HPLC; Murine; Human;

Determination of dimenhydrinate in human plasma by liquid chromatography–electrospray tandem mass spectrometry: Application to a relative bioavailability study by V. Tavares; C.C. Macedo; L. Montanhez; F.A.P. Barros; E.C. Meurer; D.R. Campos; E.C Coelho; S.A. Calaffati; J. Pedrazzoli (127-132).
Here we present a sensitive and specific liquid chromatography–tandem mass spectrometric method for the quantification of dimenhydrinate (I) in human plasma. Sample preparation is conducted using citalopram (II) addition as an internal standard (IS), liquid–liquid extraction with basified plasma using a mixture hexane/acetate (1:1, v/v) as the extracting solvent, and the final extract reconstituted in the mobile phase. I and II (IS) were injected in a C8 column with the mobile phase composed of methanol:isopropanol:water:formic acid (78.00:19.92:2.00:0.08, v/v/v/v) and monitored using a positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 256.0 > 167.0 and m/z 325.0 > 109.0 for I and II, respectively. The limit of quantification (LOQ) was 0.4 ng/mL, the dynamic range being 0.4–200 ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of plasma samples taken up to 24 h after oral administration of 100 mg of dimenhydrinate in healthy volunteers demonstrated its applicability to bioavailability studies.
Keywords: Mass spectrometry; Bioavailability; Dimenidrynate;

Detection of altered N-glycan profiles in whole serum from rheumatoid arthritis patients by Hiroaki Nakagawa; Megumi Hato; Yasuhiro Takegawa; Kisaburo Deguchi; Hiroki Ito; Masahiko Takahata; Norimasa Iwasaki; Akio Minami; Shin-Ichiro Nishimura (133-137).
Altered N-glycosylation occurs in many diseases. In rheumatoid arthritis (RA), for example, reduction in galactose residues in IgG and an increase in fucose residues in α1-acid glycoprotein have been observed. To further analyse N-glycans in disease, we show N-glycan profiling from whole serum employing reversed phase high performance liquid chromatography/negative-ion mode by sonic spray ionization ion trap mass spectrometry with pyridylamination. Profiles from female 15 RA patients and 18 aged-matched healthy women were compared. The most significant change seen in RA was decreased levels of mono-galactosyl bi-antennary N-glycans, in agreement with the previous reports regarding IgG. We also show previously unreported differences between isomers and increased tri-antennary oligosaccharides. These results indicate that LC–MS analysis of whole serum N-glycans can identify N-glycan alterations in RA and that this is a promising method both for studies of RA mechanisms and diagnosis.
Keywords: N-glycan; Serum; Rheumatoid arthritis; LC–MS;

High throughput quantification of phytoestrogens in human urine and serum using liquid chromatography/tandem mass spectrometry (LC–MS/MS) by Philip B. Grace; Nayan S. Mistry; Megan H. Carter; Anthony J.C. Leathem; Philip Teale (138-146).
Phytoestrogens are currently the subject of intense study owing to their potential protective effects against a number of complex diseases. However, in order to investigate the interactions between phytoestrogens and disease state effectively, it is necessary to have analytical methods which are sensitive, reproducible, and require low sample volumes. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (equol and O-desmethylangolensin), three lignans (secoisolariciresinol, enterodiol, and enterolactone), and one flavanone (naringenin) in human urine and serum. A high throughput of samples has been achieved via the use of 96-well plate sample extraction and liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis incorporating column switching, thus making the assay suitable for use on large sample numbers, such as those found in epidemiological studies. The robustness of the assay was proven via the comparison of data generated on two different LC–MS/MS systems, with and without column switching.
Keywords: Phytoestrogens; Isoflavones; Lignans; LC–MS/MS; Urine; Serum; Column switching;

A sensitive high performance liquid chromatography–electrospray ionization-mass spectrometry (HPLC–ESI-MS) assay was developed to determine raltitrexed in human plasma. After addition of benazeprilat as the internal standard (IS), methanol was used to produce a protein-free extract. Chromatographic separation was achieved with a Zorbax SB-C18 column (Narrow-Bore 2.1 mm × 150 mm, 5-μm) using a mobile phase of acetonitrile–water containing 0.1% formic acid and 2% methanol (21.9:78.1, v/v). Raltitrexed was determined with electrospray ionization-mass spectrometry. HPLC–ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at [M  + H]+ m/z 459.1 for raltitrexed and [M  + H]+ m/z 397.1 for IS. Calibration curves were linear over the range of 2.0–3000 ng/ml. The lower limit of quantification was 2.0 ng/ml. The intra- and inter-batch variability values were less than 6.7% and 10.3%, respectively. The mean plasma extraction recovery of raltitrexed was in the range of 85.2–91.1%. The method was successfully applied to determine the plasma concentrations of raltitrexed in eight Chinese colorectal cancer patients.
Keywords: Raltitrexed; HPLC–ESI-MS; Pharmacokinetics;

An efficient and sensitive method for simultaneous determination of 102 pesticide residues in teas has been established and validated. The multi-residue analysis of the pesticides in teas involved extraction with acetone–ethyl acetate–hexane, clean-up using gel permeation chromatography (GPC) and solid-phase extraction (SPE), and subsequent identification and quantification of the selected pesticides by gas chromatography–mass spectrometry (GC–MS) under retention time locked (RTL) conditions. For most of the target analytes, the optimized pretreatment processes led to no significant interference on analysis from sample matrix, and the determination of 102 compounds was achieved in about 120 min. Pesticide residues could be determined in low sub-ppb range, from 0.01 μg/mL for hexachlorobenzene to 2.5 μg/mL for propargite, with average recoveries ranging from 59.7 to 120.9% (mean 88%) and relative standard deviations (RSDs) in the range 3.0–20.8% (mean 13.7%) for all analytes across three fortification tea levels. The limits of detection (LODs) were much lower than the maximum residue levels established by the European Union (EU) legislations.
Keywords: Pesticides; Multi-residue analysis; Tea; GPC; SPE; GC–MS; Validation;

A simple reversed phase high-performance liquid chromatographic method was established for the separation and quantification of a novel prodrug of zidovudine in rat plasma. Zidovudine was one of the original drugs used to treat HIV infection. Appropriate aliquots of rat plasma were spiked with cholesteryl carbonate zidovudine (AZTC) and treated with acetonitrile to precipitate plasma proteins. The supernatant after supercentrifugation was collected and an aliquot of 20 μL was injected directly into an HPLC system consisting of a Diamonsil™ C18 column and a diode array detector. The mobile phase consisted of methanol (85%, v/v) and diethyl ether (15%, v/v) at a flow rate of 1.0 mL/min. The extraction recoveries of AZTC at the three concentrations examined were all higher than 80%. The HPLC assay was linear over the concentration range 0.5–80 μg/mL. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration–time profile for AZTC after administration via the tail vein. The mean elimination half-life (t 1/2) was 292.4 min. This RP-HPLC method will be useful for the evaluation of the pharmacokinetics of AZTC in rats.
Keywords: Cholesteryl carbonate zidovudine; Pharmacokinetics; HPLC; Rat plasma;

Development and validation of an LC/MS/MS assay for mycophenolic acid in human peripheral blood mononuclear cells by Henri Bénech; Sophie Hascoët; Valérie Furlan; A. Pruvost; A. Durrbach (168-174).
The aim was to develop a LC/MS/MS method able to quantify mycophenolic acid (MPA) in the peripheral blood mononuclear cells (PBMCs) of transplanted patients. PBMCs were isolated from blood by a density gradient separation. The chromatographic separation was carried out on a Zorbax Stable Bond CN, 150 mm × 2.1 mm, and MS/MS detection was performed after positive electrospray ionisation of the protonated parent ion. The calibration range was from 0.25 to 100 ng/sample. Extraction from the cells and ionisation recoveries reached 73.5 and 37.9%, respectively. Inaccuracy was always <10% with CVs <15%. MPA was stable at room temperature in the autosampler over 48 h and at −20 °C over 1.5 months. Application to clinical samples taken from patients treated with mycophenolate mofetil indicated that the method is suitable for measuring intracellular MPA.
Keywords: Mycophenolic acid; PBMC; Intracellular; Tandem mass spectrometry; Clinical pharmacology; Patient;

Piceatannol, compared with the renowned resveratrol, is a better anticancer agent and a superior agent with other biological activities. However, as there are only few plants reported to contain minute quantity of piceatannol, the scarcity of sources greatly impedes the piceatannol-related researches. To explore new sources of piceatannol, we established a sample preparation approach for screening the piceatannol in plants using HPLC-UV-fluorescence detection. When the HPLC is coupled with UV and fluorescence detectors, the decrease of signals in interferences and increase of signal in piceatannol in the fluorescence chromatogram mark clearly the position of the piceatannol peak; ultimately, it allows identification without standards. In this study, we systematically evaluated the factors affecting the extraction efficiency of piceatannol in sample preparation. Of the sample preparation strategies studied, direct solvent extraction and liquid nitrogen treatment followed by solvent extraction gave satisfactory recoveries for both piceatannol and resveratrol. These approaches avoided time-consuming lyophilization procedure. In addition, all procedures must be done in the dark to avoid negative impact of irradiation from fluorescence light on the recoveries of piceatannol and resveratrol. With the present method, we re-examined the plants previously claimed to contain only resveratrol for their piceatannol contents. The species examined included Polygonum cuspidatum, Arachis hypogaea, Vitis thunbergii, and Ampelopsis brevipedunculaata. The results showed, for the first time, all these plants contain piceatannol. The finding implies that the resveratrol-containing plants may also contain piceatannol. The results demonstrate the feasibility of these sample preparation approaches and may further the understanding for the distribution of piceatannol in plants.
Keywords: Anticancer agent; Piceatannol; Resveratrol; Sample preparation; HPLC; Fluorescence detection;

Oral bioavailability of curcumin in rat and the herbal analysis from Curcuma longa by LC–MS/MS by Kuo-Yi Yang; Lei-Chwen Lin; Ting-Yu Tseng; Shau-Chun Wang; Tung-Hu Tsai (183-189).
This study presents a validated liquid chromatography technique coupled with tandem mass spectrometry (LC–MS/MS) to measure curcumin in rat plasma and provide curcuminoids analysis from the extract of Curcumin longa L. This method was applied to investigate the pharmacokinetics of curcumin in a freely moving rat. The analytes were separated by a reversed phase C18 column (150 × 4.6 mm I.D., particle size 5 μm) and eluted with acetonitrile-1 mM HCOOH mobile phase (70:30, v/v) with a flow rate of 0.8 ml/min in rat plasma and herbal extracts. Multiple reaction monitoring (MRM) was used to monitor the transition of the deprotonated molecule m/z of 367 [M − H] to the product ion 217 for curcumin, a m/z of 337–217 for demethoxycurcumin and a m/z of 265–224 for honokiol (internal standard) analysis. The limit of detection (LOD) and quantification (LOQ) of curcumin in the rat plasma were 1 and 5 ng/ml, respectively. The method was linear in the range of 5–1000 ng/ml with a coefficient of correlation greater than 0.996 in the rat plasma. After curcumin (500 mg/kg, p.o.) administration, the maximum concentration (C max) and the time to reach maximum concentration (T max) were 0.06 ± 0.01 μg/ml and 41.7 ± 5.4 min, respectively. The elimination half-life (t 1/2,β) were 28.1 ± 5.6 and 44.5 ± 7.5 min for curcumin (500 mg/kg, p.o.) and curcumin (10 mg/kg, i.v.), respectively. The oral bioavailability was about 1%.
Keywords: Bioavailability; Curcuma longa; Curcumin; Herbal analysis; Pharmacokinetics;

The goal of the project was the extraction of PCR-compatible genomic DNA representative of the entire microbial community from municipal biogas plant samples (mash, bioreactor content, process water, liquid fertilizer). For the initial isolation of representative DNA from the respective lysates, methods were used that employed adsorption, extraction, or precipitation to specifically enrich the DNA. Since no dedicated method for biogas plant samples was available, preference was given to kits/methods suited to samples that resembled either the bioreactor feed, e.g. foodstuffs, or those intended for environmental samples including wastewater. None of the methods succeeded in preparing DNA that was directly PCR-compatible. Instead the DNA was found to still contain considerable amounts of difficult-to-remove enzyme inhibitors (presumably humic acids) that hindered the PCR reaction. Based on the isolation method that gave the highest yield/purity for all sample types, subsequent purification was attempted by agarose gel electrophoresis followed by electroelution, spermine precipitation, or dialysis through nitrocellulose membrane. A combination of phenol/chloroform extraction followed by purification via dialysis constituted the most efficient sample treatment. When such DNA preparations were diluted 1:100 they did no longer inhibit PCR reactions, while they still contained sufficient genomic DNA to allow specific amplification of specific target sequences.
Keywords: Biogas plant; Genomic DNA; Humic acids; PCR;

HPLC method for determination of in vitro delivery through and into porcine skin of adefovir (PMEA) by Kateřina Vávrová; Kateřina Lorencová; Jana Klimentová; Jakub Novotný; Alexandr Hrabálek (198-203).
A simple HPLC/UV method for the determination of the transdermal permeation and dermal penetration of a broad-spectrum antiviral drug adefovir (PMEA) was developed. The separation was achieved on a C18 column with the mobile phase composed of 10 mM KH2PO4 and 2 mM Bu4NHSO4 at pH 6.0 and 7% acetonitrile. The method was validated with respect to selectivity, linearity (0.1–50 μg/ml), precision, accuracy, and stability. Transdermal permeation of 2% PMEA was studied in vitro using the Franz diffusion cell and porcine skin. The flux values were 1.8, 3.0, and 0.6 μg/cm2/h from aqueous donor samples at pH 3.4 and 7.4, and isopropyl myristate, respectively. The respective skin concentrations at 48 h were 294, 263, and 971 μg/g from these vehicles. These results will serve as a lead for further studies on transdermal and topical delivery of antivirals from the group of acyclic nucleoside phosphonates.
Keywords: Adefovir; Acyclic nucleoside phosphonate; Antiviral; Transdermal transport; Dermal absorption; HPLC/UV;

Monitoring phosphatase reactions of multiple phosphorylated substrates by reversed-phase HPLC by Kathleen Teichmann; Robert Winkler; Kornelia Hampel; Antje Trümpler; Frank D. Böhmer; Diana Imhof (204-213).
In an approach to gain insight into the sequence-dependent dephosphorylation of multiple phosphotyrosyl-containing peptides by the phosphatases SHP-1 and PTP1B, we applied a chromatographic technique for the analysis of the dephosphorylation products. Mono-, bi- and triphosphorylated reference peptides corresponding to positions 1999–2014 in the activation loop of the receptor tyrosine kinase Ros were first analyzed by reversed-phase HPLC and MALDI-TOF/TOF mass spectrometry. Then, the respective products from enzymatic treatment were investigated by HPLC and compared to the standard peptides. The results obtained in this study emphasize the advantage of monitoring phosphatase reactions for mono- and biphosphorylated peptides using the described procedure rather than spectrophotometric and fluorimetric methods that do not allow for a clear identification of the products formed.
Keywords: Phosphopeptide; Dephosphorylation; HPLC; MALDI-TOF/TOF;

A new hydrophilic interaction liquid chromatography method for the simultaneous determination of ascorbic acid (AA), erythorbic acid (EA), 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) and 2-O-β-d-glucopyranosyl-l-ascorbic acid (AA-2βG) was developed using a diol column with an isocratic solution of acetonitrile-66.7 mM ammonium acetate solution (85:15, v/v) at a detection wavelength of 260 nm. The calibration curves were found to be linear in the range of 1–50 μg/ml for AA and EA and in the range of 2.5–100 μg/ml for AA-2G and AA-2βG. Detection limits of AA, EA, AA-2G and AA-2βG were 0.3, 0.3, 0.03 and 0.03 μg/ml, respectively. This method was satisfactorily applied to the determination of AA, EA, AA-2G and AA-2βG in a fruit, a food and beverages. The results show that the procedure is simple and sensitive and that it can be employed for the simultaneous determination of AA and its related compounds in foods and beverages.
Keywords: Ascorbic acid 2-glucoside; AA-2G; AA-2βG; Ascorbic acid; Erythorbic acid; Hydrophilic interaction chromatography;

A high-performance liquid chromatography (HPLC) method was developed and validated for the determination of orientin in rabbit plasma using ultraviolet (UV) absorbance detection. Orientin is the active constituent of purified herbal extract (TRO PE) from the flower of Trollius chinensis Bunge. Protein precipitation was used as the sample preparation technique. A Diamonsil C18 column (150 mm × 4.6 mm, 5 μm) was equilibrated with a mobile phase composed of 0.1% acetic acid/methanol/acetonitrile (80/5/15, v/v/v). The calibration curve of orientin in rabbit plasma was linear in the concentration range of 0.530–53.0 μg/mL. This validated method was successfully applied to a pharmacokinetic study in rabbits after the intravenous administrations of orientin and TRO PE at three different doses.
Keywords: Orientin; Liquid chromatography with ultraviolet detection; Rabbit plasma; Pharmacokinetics;

A sensitive and reliable high performance liquid chromatography-tandem time-of-flight mass spectrometry method (HPLC/TOF MS) has been developed to determine three active bufadienolides from Liu-Shen-Wan (LSW) in dog's plasma. Enhanced selectivity and sensitivity in comparison with traditional HPLC/DAD method could be obtained through this method. Bufodienolides could be well separated and distinguished from its nominally isobaric endogenous components by HPLC/TOF MS, with the linear calibration range covering from 0.5 ng/mL to 100 ng/mL and Limit of Detection (LOD) being about 0.15 ng/mL. This method was also proved to be quite stable, with the intra-day precision and the inter-day precision results being lower than 6.39% and 7.44%, respectively. Meanwhile HPLC/TOF MS was successfully used in the pharmacokinetic study of LSW. For resibufogenin, the major pharmacokinetic parameters AUC 0−t , C max and t 1/2α were 160.72 ± 21.97 ng/mL min, 2.35 ± 0.71 ng/mL and 20.74 ± 5.89 min, respectively, and for bufalin the corresponding parameters were 55.55 ± 7.55 ng/mL min, 0.91 ± 0.15 ng/mL and 25.45 ± 13.28 min, respectively.
Keywords: Liu-Shen-Wan; Pharmacokinetics; Bufadienolides; HPLC/TOF MS;

Liquid–liquid extraction (LLE) is widely used as a simple and robust sample preparation technique in bioanalytical sample preparation. When extracting ionisable compounds, most bioanalysts adjust the pH of the sample to achieve fully unionized compounds. Usually, a generally accepted rule is applied to adjust the pH of the aqueous phase, known as the pK a  ± 2 rule, depending on the acid/base characteristics of the analyte. By taking a closer look at the general equations that describe the extraction behaviour of ionisable compounds, we extended this pH adjustment rule by taking the distribution ratio and the volume of both liquid phases into account. By choosing an extraction pH based on this extended rule, the selectivity of the extraction can be influenced without loss of recovery. As a measure of this selectivity, two equations were proposed to indicate the ability of the extraction system to discriminate between two compounds. Also, milder extraction pH can be used for pH labile analytes. To use this new rule quantitatively, a new calculation method for the determination of the distribution ratio was derived. These calculations were based on normalized recoveries making this method less susceptible to errors in absolute recovery determination. The proposed equations were supported by demonstrating that careful pH adjustment can lead to higher selectivity. The main conclusion was that a closer look at the extraction pH in bioanalytical methods extends the possibilities of obtaining a higher selectivity or the possibilities of extracting pH labile analytes at milder pH conditions without loss of recovery.
Keywords: Liquid–liquid extraction; pH adjustment; Bioanalysis; Modelling; Recovery; Selectivity;

Analysis of 8-hydroxy-2′-deoxyguanosine in urine using high-performance liquid chromatography–electrospray tandem mass spectrometry by Minna Harri; Hiroshi Kasai; Toshiaki Mori; Jarkko Tornaeus; Kirsti Savela; Kimmo Peltonen (242-246).
8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used biomarker of oxidative stress in research related to DNA, protein damage as well as lipid peroxidation. HPLC–MS/MS with electrospray ionization (ESI) and the use of isotopically labelled 8-OHdG as an internal standard allows a simple quantification of 8-OHdG in urine samples. HPLC separation utilized the peak cutting technique and a 1.5 mm × 120 mm analytical anion exchange column. Novel method entails only minimal sample handling including the addition of a buffer and an internal standard followed by centrifugation before the samples are ready for analysis. The levels of 8-OHdG in human urine samples (n  = 246) varied from 0.16 to 16.48 μg/L and the corresponding creatinine-normalized values were ranged from 0.49 to 14.27 μg of 8-OHdG/g creatinine. The correlation between the developed HPLC–MS/MS method and the existing HPLC-EC method was good with an R 2 value of 0.8707.
Keywords: DNA damage; Monitoring; Radicals;

In this study, a high efficiency immunomagnetic affinity matrix was developed to eliminate l-cysteine desulfhydrase (CD), which decomposes l-cysteine, in crude enzyme extracts from Pseudomonas sp. TS1138. After cloning and expression in Escherichia coli, recombinant CD was purified to raise polyclonal antibodies from mice. The anti-CD antibody was cross-linked to staphylococcal protein A-magnetic cellulose microspheres (MCMS) with dimethyl pimelimidate (DMP). The natural CD was eliminated from the crude enzyme extracts by treatment with the cross-linked antibody-protein A-MCMS, resulting in a high level of l-cysteine production. The conversion rate of dl-2-amino-Δ2-thiazoline-4-carboxylic acid (dl-ATC) to l-cysteine increased significantly from 61.9 to 96.2%. The cross-linked antibody-protein A-MCMS showed its durability after repetitive use, maintaining a constant binding capacity for CD during five cycles. This study may lead to a convenient and cost-efficient method to produce l-cysteine by enzymatic conversions.
Keywords: Pseudomonas sp. TS1138; l-Cysteine; l-Cysteine desulfhydrase; Immunomagnetic separation; Magnetic cellulose microspheres;

Development of a RP-HPLC method for screening potentially counterfeit anti-diabetic drugs by Jing Yao; Ya-Qin Shi; Zhuo-Rong Li; Shao-Hong Jin (254-259).
Pharmaceutical counterfeiting is becoming a serious problem in the world, especially in developing countries including China. Herein an isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for screening counterfeit medicines and adulterated dietary supplement products. The developed method could be employed to separate and determine simultaneously six anti-diabetic drugs (glipizide, gliclazide, glibenclamide, glimepiride, gliquidone, repaglinide) on an isocratic solvent system using an Alltima C18 column (5 μm, 150 mm × 4.6 mm) with an isocratic mobile phase of methanol–phosphate buffer (pH 3.0; 0.01 mol/L) (70:30, v/v), at a flow rate of 1.0 mL/min and at a wavelength of 230 nm. The proposed method was successfully applied to the analysis of medicinal and dietary supplement samples purchased from the local market in China.
Keywords: Anti-diabetic drugs; Counterfeit drugs; Substandard drugs; RP-HPLC; Screening analysis;

In the present work, a method was developed and optimized aiming to determinate the herbicides paraquat (PQ) and diquat (DQ) in human plasma and urine samples. An initial procedure of chemical reduction of the analytes by adding NaBH4 directly in the buffered samples (pH 8.0) was performed. This procedure was necessary to convert the quaternary ammonium substances into more volatile compounds for gas chromatographic analysis. The reduction compounds were extracted with C18 cartridges (solid-phase extraction). Ethyl paraquat (EPQ) was used as internal standard (IS). Gas chromatography–mass spectrometry (GC–MS) was used to identify and quantify the analytes in selected ion monitoring (SIM) mode. The limits of detection were 0.05 mg/l for both PQ and DQ. By using the weighted least squares linear regression ( 1 / x 1 2 for plasma and 1/y for urine), the accuracy of the analytical method was improved at the lower end of the calibration curve (from 0.1 to 50 mg/l; r  > 0.98). This method can be readily utilized as an important tool to confirm the suspicion of PQ and/or DQ poisoning and evaluate the extent of the intoxication.
Keywords: Paraquat; Diquat; Plasma and urine samples; SPE; GC–MS;

High-performance liquid chromatographic method with UV photodiode-array, fluorescence and mass spectrometric detection for simultaneous determination of galantamine and its phase I metabolites in biological samples by Jana Maláková; Milan Nobilis; Zbyněk Svoboda; Miroslav Lísa; Michal Holčapek; Jaroslav Květina; Jiří Klimeš; Vladimír Palička (265-274).
Galantamine, an alkaloid isolated from the bulbs and flowers of Caucasian snowdrop (Galanthus woronowii, Amaryllidaceae) and related species, is employed in human medicine for the treatment of various neuromuscular and neurodegenerative diseases. After the administration, the products of oxidative biotransformation (O-desmethyl-galantamine, N-desmethyl-galantamine, galantamine-N-oxide) and chiral conversion (epigalantamine) are formed in various concentrations from parent compound. For the identification and determination of galantamine and its phase I metabolites in blood plasma and tissues, a new bioanalytical method based on a reversed-phase high-performance liquid chromatography with UV photodiode-array, fluorescence and mass spectrometric detection was developed, validated and applied to pharmacokinetic and biotransformation studies. Sample preparation included a homogenization of the rat tissues (liver, brain, hypophysis) in a phosphate buffer 0.05 mol/L pH 7.4. Plasma samples and tissue homogenates were purified using a mixed-mode solid-phase extraction (Waters Oasis MCX cartridges). Galantamine, its above-mentioned metabolites and the internal standard codeine were separated on a Discovery HS F5 column (Supelco, 150 mm× 4.6 mm I.D., 5 μm) at flow rate of 1 mL/min using a linear gradient elution. UV photodiode-array and mass spectrometric detection were employed for the identification of individual galantamine metabolites in various biomatrices, the fluorescence detection (λ excit  = 280 nm/λ emiss  = 310 nm) was chosen for the quantification of galantamine and its metabolites. The developed method was applicable in liver tissue in the range from 0.50 to 63.47 nmol/g of galantamine, from 0.32 to 41.42 nmol/g of O-desmethyl-galantamine, from 0.54 to 69.40 nmol/g of N-desmethyl-galantamine and from 0.70 to 89.03 nmol/g of epigalantamine. Limit of detection was found to be 0.04 nmol/g for galantamine, 0.19 nmol/g for O-desmethyl-galantamine, and 0.07 nmol/g for N-desmethyl-galantamine and epigalantamine.
Keywords: High-performance liquid chromatography; Fluorescence detection; Mass spectrometry; Galantamine; Metabolites;

Determination of talinolol in human plasma by high performance liquid chromatography–electrospray ionization mass spectrometry: Application to pharmacokinetic study by Juan He; Bernd Terhaag; Li-Ying Yang; Bi-Kui Zhang; Fen-Li Su; Yun-Gui Zhu; Juan Song; Jing Tang; Xiao-Lei Liu; Wen-Xing Peng (275-280).
A rapid and sensitive method for determination and screening in human plasma of talinolol is described using propranolol as the internal standard. The analytes in plasma were extracted by liquid–liquid extraction using methyl t-butyl ether. After removed and dried the upper organic phase, the extracts were reconstituted with a fixed volume of buffer of ammonium acetate and acetonitrile (60:40, v/v). The extracts were analyzed by a HPLC coupled to electrospray ionization mass spectrometry (HPLC–MS/ESI). The HPLC separation of the analytes was performed on a Phenomenex C18 (250 mm × 4.6 mm, 5 μm, USA) column, with a flow rate of 0.85 mL/min. The complete elution was obtained within 5.5 min. The calibration curve was linear in the 1.0–400.0 ng/mL range for talinolol, with a coefficient of determination of 0.9996. The average extraction recovery was above 83%. The methodology recovery was between 101% and 102%. The limit of detection (LOD) was 0.3 ng/mL for talinolol. The intraday and inter-day coefficients of variation were less than 6%. This HPLC–MS/ESI procedure was used to assess the pharmacokinetics of talinolol. A single oral 50 mg dose of talinolol tablet was administered to 12 healthy Chinese volunteers, the main pharmacokinetic data are as follows: C max was 147.8 ± 63.8 ng/mL; t max was 2.0 ± 0.7 h; t 1/2 was 12.0 ± 2.6 h. The method is accurate, sensitive and simple for the pharmacokinetic study of talinolol.
Keywords: Talinolol; Pharmacokinetics; HPLC–MS/ESI;

A simple and sensitive liquid chromatography with amperometric detection was developed for the first time to monitor the protein-unbound acteoside in the rat blood and brain microdialysate by microdialysis technique. Microdialysis samples without further cleanup procedures were directly injected into the HPLC and separated using a reversed-phase C18 column (150 mm × 2 mm, i.d. 5 μm) maintained at ambient temperature and a mobile phase comprised of acetonitrile–50 mM monosodium phosphate (pH 2.8) (17:83, v/v) with a flow rate of 0.2 mL/min. Based on the experimental voltamogram, the applied potential was set at +0.9 V oxidative mode. The concentration–response relationship was linear (r 2  > 0.99) over a concentration range of 5–500 ng/mL; method precision and accuracy fell within predefined limits (less than 20%). The developed method was applied to assess the pharmacokinetics of acteoside, and the results suggested that acteoside was fitted better by the two-compartmental model following a single intravenous injection of acteoside. Acteoside was unable to be detected in the brain dialysate. The distribution and elimination half-lives of unbound acteoside in the blood were 5 and 28 min, respectively, which suggested the rapid distribution of acteoside.
Keywords: Acteoside; Herbal medicine; Microdialysis; Pharmacokinetics;

Cytochrome P450 enzymes catalyze oxidative metabolism of most pharmaceutical compounds. Consequently dextromethorphan, flurbiprofen, midazolam and other compounds are commonly used as probe substrates to evaluate cytochrome P450 function in humans. A “cocktail” approach employing simultaneous administration of two or more of the probe substrates has been used by various investigators in recent years. An analytical strategy to simultaneously extract and analyze dextromethorphan, flurbiprofen and midazolam and their major metabolites (dextrorphan, 4′-hydroxy-flurbiprofen and 1′-hydroxy-midazolam) by HPLC-MS/fluorescence was developed and is described here. The three probe substrates and their major metabolites were extracted simultaneously by means of a solid-phase (Bond Elut Certify ® cartridges) extraction procedure from 200 μl of pig plasma. The extraction efficiency was more than 79.5% for each of the six analytes. The extracted compounds were chromatographically separated on a Luna C8(II) column (50 mm L × 3 mm ID) in a single run of 20 min and analyzed by either fluorescence (flurbiprofen and 4′-hydroxy-flurbiprofen) or selective ion monitoring (dextromethorphan, dextrorphan, midazolam and 1′-hydroxy-midazolam) with positive electrospray ionization. The limit of quantification was 2.5 ng/ml for midazolam and 5 ng/ml for the other five analytes. The assay was precise and accurate (error: −9.1 to 12.1) with total CVs of 13.9% or better for each of the 6 analytes. This method was used to analyze concentrations of the three probes and their metabolites in plasma after intravenous administration to a healthy pig.
Keywords: Cytochrome P450; Dextromethorphan; Flurbiprofen; Midazolam; LC/MS; Phenotyping; Pharmacokinetics; Pig;

A sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and fully validated for the simultaneous quantification of vinflunine (VFL) and its pharmacologically active metabolite, 4-O-deacetyl vinflunine (DVFL). The two compounds, as well as vinblastine (used as internal standard), were deproteinised from blood and faeces, analysed on a cyano type column and detected on a Micromass Quattro II system in the positive ion mode after ionisation using an electrospray ion source. In blood, linearity was assessed up to 200 ng/ml for vinflunine and 100 ng/ml for 4-O-deacetyl vinflunine. The lower limit of quantification was validated at 250 pg/ml for both compounds. In other biological media, the linearity was assessed within the same range; the limit of quantification was adjusted according to the expected concentration levels of each compound. This method was first developed in order to identify the structures and to elucidate the metabolic pathway of vinflunine. Thanks to its high sensitivity and specificity, the method has enabled the quantification of vinflunine and 4-O-deacetyl vinflunine in blood at trace levels, and has contributed to the knowledge of vinflunine metabolism by monitoring up to 10 metabolites.
Keywords: Vinflunine; 4-O-Deacetyl vinflunine; LC–MS/MS; Human; Metabolites;

Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS by Lan Gao; William Chiou; Hua Tang; Xueheng Cheng; Heidi S. Camp; David J. Burns (303-313).
Malonyl-CoA is a key intermediate involved in lipid synthesis and lipid oxidation. Here, we report on a novel method for the quantification of malonyl-CoA and seven other short-chain acyl-CoAs in various rat and mouse tissues using ion-pairing reversed-phase HPLC/MS. This method is capable of measuring malonyl-CoA, free coenzyme A (CoASH), acetyl-CoA, β-hydroxyl-butyryl-CoA (HB-CoA), 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), propionyl-CoA, succinyl-CoA, and isobutyryl-CoA simultaneously with a dynamic linear range over two orders of magnitude in a 7.0 min HPLC gradient run. The lower limit of quantification (LLOQ) was 0.225 pmol for all acyl-CoAs studied, except for HMG-CoA which had a higher LLOQ of 0.90 pmol. The interference of HB-CoA on the quantification of malonyl-CoA in animal tissues was also explored for the first time.
Keywords: Coenzyme A (CoA); Short-chain acyl-CoA; Malonyl-CoA; HPLC; LC/MS; Mass spectrometry; β-Oxidation; Lipid metabolism; Acetyl-CoA; β-Hydroxybutyryl-CoA; Succinyl-CoA; 3-Hydroxy-3-methylglutaryl-CoA; n-Propionyl-CoA; Isobutyryl-CoA;

Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography by Céline B. Parsy; Caroline J. Chapman; Antony C. Barnes; John F. Robertson; Andrea Murray (314-319).
As part of a study to purify the internal domain of HER2 (ICD) from recombinant expression, through metal immobilised affinity chromatography (IMAC), we encountered a contaminant, SlyD, a 29 kDa native E. coli protein. SlyD is a recurrent contaminant, with a histidine rich domain enabling binding to IMAC columns and thus co-elution with the target protein. Research has been carried out on this protein and its purification, however, no work mentions how to treat it as a true contaminant or describe procedures to isolate it from target proteins. In this report, we described a two-step chromatographic method for the purification of ICD, including IMAC as a capture step and size exclusion chromatography (SEC) as a polishing step. IMAC allowed us to purify ICD from bacterial crude with SlyD co-eluting. SEC then allowed us to resolve ICD from SlyD and achieve a purity greater than 95% for ICD. However, this method has been developed to accommodate any protein whose molecular weight is different enough from SlyD to be separated by SEC.
Keywords: Recombinant protein; SlyD; Size exclusion chromatography; Immobilized metal affinity chromatography;

High throughput LC–MS/MS method for simultaneous quantification of lamivudine, stavudine and nevirapine in human plasma by Hiren N. Mistri; Arvind G. Jangid; Ashutosh Pudage; Noel Gomes; Mallika Sanyal; Pranav Shrivastav (320-332).
A selective and high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated to separate, detect and simultaneously quantify lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma using metaxalone as internal standard (IS). After solid phase extraction (SPE), the analytes and the IS were chromatographed on a Symmetry C18 (150 mm × 3.9 mm i.d., 5 μm particle size) column using 5 μL injection volume with a run time of 4.5 min. An isocratic mobile phase consisting of 0.5% glacial acetic acid in water:acetonitrile (20:80, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The method was validated over the range of 25–3000 ng/mL for 3TC, 20–2000 ng/mL for d4T and 50–5000 ng/mL for NVP. The absolute recoveries for analytes (≥86%) and IS (98.12%) achieved from spiked plasma samples were consistent and reproducible. Inter-batch and intra-batch precision (%CV) across four validation runs (LLOQ, LQC, MQC and HQC) was less than 10. The accuracy determined at these levels was within ±8% in terms of relative error. The method was successfully applied to a pivotal bioequivalence study of [60 (3TC) + 12 (d4T) + 100 (NVP)] mg dispersible tablets in 60 healthy human subjects under fasting condition.
Keywords: Lamivudine (3TC); Stavudine (d4T); Nevirapine (NVP); LC–MS/MS; Multiple reaction monitoring (MRM); Pivotal bioequivalence;

A rugged, sensitive and efficient liquid chromatography–tandem mass spectrometry method was developed and validated for the quantitative analysis of firocoxib in urine from 5 to 3000 ng/mL and in plasma from 1 to 3000 ng/mL. The method requires 200 μL of either plasma or urine and includes sample preparation in 96-well solid phase extraction (SPE) plates using a BIOMEK® 2000 Laboratory Automated Workstation. Chromatographic separation of firocoxib from matrix interferences was achieved using isocratic reversed phase chromatography on a PHENOMENEX LUNA® Phenyl-Hexyl column. The mobile phase was 45% acetonitrile and 55% of a 2 mM ammonium formate buffer. The method was accurate (88–107%) and precise (CV < 12.2%) within and between sets. Extraction efficiencies (recovery) >93% were achieved and ionization efficiencies (due to matrix effects) were >72%. Extensive stability and ruggedness testing was also performed; therefore, the method can be used for pharmacokinetic studies as well as drug monitoring and screening. The data presented here is the first LC–MS/MS method for the quantitation of firocoxib in plasma (LLOQ of 1 ng/mL), a 25-fold improvement in sensitivity over the HPLC-UV method and the first quantitative method for firocoxib in urine (LLOQ of 5 ng/mL). Additionally the sample preparation process has been automated to improve efficiency.
Keywords: LC–MS/MS; NSAID; Urine; Plasma; Automation; Firocoxib; Horse; Dog;

The evaluation of molecularly imprinted polymers (MIPs) as selective sorbents for the solid-phase extraction of sildenafil and its principal metabolite, desmethylsildenafil, was investigated. Two MIPs were synthesised using structural analogues of sildenafil as templates, and a comparison of the performance of the two MIP sorbents in organic and aqueous media was performed. Additionally, the feasibility of applying molecularly imprinted solid-phase extraction (MISPE) to the clean-up of plasma samples containing sildenafil and desmethylsildenafil was investigated. A preliminary, quantitative MISPE for the determination of both compounds in plasma was also performed. The results showed that the MIPs used for the selective extraction of sildenafil gave better compound recovery when aqueous samples were used in comparison to organic-based samples. A preliminary, quantitative MISPE of sildenafil and desmethylsildenafil indicated that the imprinted materials could be used successfully as SPE sorbents for sample pre-treatment for the determination of sildenafil, and related compounds, in plasma.
Keywords: Molecularly imprinted polymers; Sildenafil; Solid-phase extraction;

One of the main stages of human recombinant insulin (HRI) production is the hormone purification by means of reversed phase high-performance liquid chromatography (RP HPLC). The optimization of this stage determines the increase of the total manufacturing yield. Therefore, the cost of the sorbent used in HPLC influences the cost of the manufacturing product, i.e. HRI substance. However, resolution between HRI and its admixtures decreases with time. The reason for this is the sorbent contamination with strongly adsorbed impurities (SAI) which are accumulated during elution. In the following research several methods for sorbent protection are studied. The opinion that SAI are mainly high-molecular weight compounds was examined using gel filtration. Different sorbent types were tested for the use in guard column. The results obtained were applied and improved at preparative level.
Keywords: Human recombinant insulin; Strongly adsorbed impurities; Reversed phase high-performance liquid chromatography; A21-desamidoinsulin; High-molecular weight compounds; Gel filtration; Guard column;

A simple and selective analytical method for the quantitative determination of cyanuric acid, the degradation product of sodium dichloroisocyanurate (NaDCC), in human urine is reported herein. The sample preparation involved the use of diatomaceous earth extraction columns. Quantification was achieved by liquid chromatography mass spectrometry using negative ion electrospray with a cyano (CN) column. Between day relative standard deviation less than 10% (n  = 6) was obtained at the 5 mg L−1 level. The assay was linear over the investigated range 0–20 mg L−1 and the limit of detection (LOD) was confirmed to be 0.1 mg L−1. The method was applied to monitoring levels of cyanuric acid in healthcare workers using disinfectants products containing NaDCC.
Keywords: Cyanuric acid; Liquid chromatography; Mass spectrometry; Biological monitoring; Urine;

A rapid, sensitive and specific method to determination of ambroxol in human plasma using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC–MS/ESI) was described. Ambroxol and the internal standard (I.S.), fentanyl, were extracted from plasma by N-hexane-diethyl ether (1:1, v/v) after alkalinized with ammonia water. A centrifuged upper layer was then evaporated and reconstituted with 100 μl mobile phase. Chromatographic separation was performed on a BDS HYPERSIL C18 column (250 mm × 4.6 mm, 5.0 μm, Thermo electron corporation, USA) with the mobile phase consisting of 30 mM ammonium acetate (0.4% formic acid)–acetonitrile (64:36, v/v) at a flow-rate of 1.2 mL min−1. The total run time was 5.8 min for each sample. Detection and quantitation was performed by the mass spectrometer using selected ion monitoring at m/z 261.9, 263.8 and 265.9 for ambroxol and m/z 337.3 for fentanyl. The calibration curve was linear within the concentration range of 1.0–100.0 ng mL−1 (r  = 0.9996). The limit of quantification was 1.0 ng mL−1. The extraction recovery was above 83.3%. The methodology recovery was higher than 93.8%. The intra- and inter-day precisions were less than 6.0%. The method is accurate, sensitive and simple for the study of the pharmacokinetics and metabolism of ambroxol.
Keywords: Ambroxol; HPLC–MS/ESI;