Journal of Chromatography B (v.818, #2)

Amosulalol is an antihypertensive drug with selective postsynaptic alpha 1 and non-selective beta blocking effects. A simple solid-phase extraction and high-performance liquid chromatographic (HPLC) method has been developed and validated for the quantitative determination of amosulalol in human plasma. A reversed phase C18 column was used for the separation of amosulalol and ethyl paraben (internal standard) with a mobile phase composed of 0.025 M phosphate buffer (pH 6.0)·acetonitrile (73:27, v/v) at a flow rate of 1.5 mL/min. The ultraviolet detector was operated at the 272 nm wavelength. Intra- and inter-day precision and accuracy were acceptable for all quality control samples including the lower limit of quantification of 30 ng/mL. Recovery of amosulalol from human plasma was >95.6%. Amosulalol was stable in human plasma under various storage conditions. This method was used successfully for a pharmacokinetic study in plasma after oral administration of a single 20 mg dose of amosulalol hydrochloride to 16 healthy volunteers.
Keywords: Amosulalol;

Non-enzymatic hinge region fragmentation of antibodies in solution by Armando J. Cordoba; Bao-Jen Shyong; Deirdre Breen; Reed J. Harris (115-121).
Liquid formulations of monoclonal antibodies (MAbs) typically undergo fragmentation near the papain cleavage site in the hinge region, resulting in Fab and Fab + Fc forms. The purpose of this study was to investigate whether this fragmentation is due to proteases. Four closely-related MAbs were exchanged into a pH 5.2 acetate buffer with NaCl and stored at −20 °C, 5 °C, 30 °C, or 40 °C for 1 month. Fragmentation generated size-exclusion chromatography (SEC) peak fractions that were analyzed by electrospray mass spectrometry to identify the cleavage sites. The effects of protein inhibitors or host cell proteins on fragmentation were also studied. The extent of fragmentation was equivalent for all four antibodies, occurring in the heavy chain hinge region Ser–Cys–Asp–Lys–Thr–His–Thr sequence. The fragment due to cleavage of the Asp–Lys bond showed two forms that differ by 18 Da. A synthetic peptide with the hinge region sequence terminating with Asp did not show fragmentation or the loss of 18 Da after incubation. Protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. Fragmentation appears to be a kinetic process that is not caused by low levels of host cell proteases.
Keywords: Hinge region fragmentation; Protease inhibitors; Monoclonal antibodies; Size-exclusion chromatography; Stability;

Copper–zinc superoxide dismutase (Cu,Zn SOD) has been extracted, purified and characterized from Radix lethospermi seed (RLS), a kind of Chinese traditional medicine. Before extraction, the lipid was removed by super critical fluid extraction (SCF). Partial protein fractionation in the crude extract was affected by using 50–75% (NH4)2SO2. Subsequently, superoxide dismutase was fractionated by column chromatographies on DEAE-52, Sephadex G-200 and DEAE-52 again. Pure Cu,Zn SOD had a specific activity of 4843 U/mg protein and was purified 267.2-fold, with a yield of 23.55%. The purified enzyme has a molecular weight of about 30,500 ± 100 and is composed of two non-covalently joined equal subunits. Purity was confirmed by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), HPLC and mass spectroscopy. Amino acid content has been investigated. The enzyme was found to remain stable in the pH range 6.0–9.0 at 25 °C and up to 45 °C at pH 7.8 for a 30 min incubation period. RLS Cu,Zn SOD appeared to have significant thermal stability lower than other Cu,Zn SODs, as revealed by irreversible heat inactivation at 60 °C. The enzyme was not inhibited by DTT, NaN3 and β-mercaptoethanol, but was inhibited by cyanide and hydrogen peroxide. Finally, in the presence of 2 mM ethylendiamine tetra acetic acid (EDTA) and sodium dodecyl sulphate (SDS), the enzyme showed approximately 18 and 34% activity loss.
Keywords: Superoxide dismutase; Radix lethospermi seed; Purification; pH stability; Thermal stability; Amino acid analysis;

Quantitative analysis of chondroitin sulfate in raw materials, ophthalmic solutions, soft capsules and liquid preparations by Joon-Soo Sim; Gyungjin Jun; Toshihiko Toida; So Yean Cho; Don Woong Choi; Seung-Yeup Chang; Robert J. Linhardt; Yeong Shik Kim (133-139).
We performed the quantitative analysis of chondroitin sulfate (CS) obtained from raw materials and various pharmaceutical preparations. To quantify CS content in raw materials and in an ophthalmic solution, each test sample and the authentic CS were first digested by chondroitinase ABC. The CS disaccharides produced were analyzed by strong anion-exchange high-performance liquid chromatography (SAX-HPLC) and CS content was quantified by calculating the total peak areas of the disaccharides derived from a CS calibration curve. In the case of soft capsules, CS was first extracted with hexane followed by phenol–chloroform to remove oil and protein ingredients. The extracted CS samples were depolymerized by chondroitinase ABC and CS content was determined. Quantitative analysis of the disaccharides derived from raw materials and an ophthalmic solution showed the CS contents (%) were 39.5 ± 0.1 to 105.6 ± 0.1 and 103.3 ± 1.2, respectively. In case of CS analysis in soft capsules and liquid preparations, the overall recovery (%) of the spiked CS was 96.79 ± 0.53–103.54 ± 1.78 and 97.10 ± 1.82 to 103.17 ± 2.34, respectively. In conclusion, the quantitative analysis of the disaccharides produced by enzymatic digestion can be used in the direct quantitation of CS containing pharmaceutical formulations.
Keywords: Chondroitin sulfate; Chondroitinase ABC; Formulation material; Ophthalmic solutions; Soft capsules; Liquid preparations;

Metal ion-selective membrane prepared by surface molecular imprinting by Kosuke Araki; Tatsuo Maruyama; Noriho Kamiya; Masahiro Goto (141-145).
Surface molecular imprinting was applied to the preparation of an ion-selective polymeric membrane for the first time. The use of acrylonitrile-butadiene rubber and a porous solid support in the polymer matrix resulted in improved flexibility and mechanical strength of the imprinted membrane. The asymmetric porous structure of the membrane was observed by scanning electron microscopy. The selectivity of the zinc(II)-imprinted membrane was evaluated by competitive adsorption and permeation studies. The imprinted membrane showed higher adsorption affinity and permeation selectivity towards the imprinted zinc ion than the non-imprinted counterpart. On the basis of the results obtained, the permeation mechanism of the metal ions was considered to be hopping of metal ions on the binding sites in the membranes.
Keywords: Metal ion; Membrane; Surface template polymerization; Separation; Ion exchange;

A novel solid-phase microextraction (SPME) method was developed for isolation of dextromethorphan (DM) and its main metabolite dextrorphan (DP) from human plasma followed by GC-MS determination. Three different polymers, poly(dimethylsiloxane) (PDMS), poly(ethylenepropyleneglycol) monobutyl ether (Ucon) and polyethylene glycol (PEG) were synthesized as coated fibers using sol–gel methodologies. DP was converted to its acetyl-derivative prior to extraction and subsequent determination. The porosity of coated fibers was examined by SEM technique. Effects of different parameters such as fiber coating type, extraction mode, agitation method, sample volume, extraction time, and desorption condition, were investigated and optimized. The method is rapid, simple, easy and inexpensive and offers high sensitivity and reproducibility. The limits of detection are 0.010 and 0.015 ng/ml for DM and DP, respectively. The precisions for both analytes are below 5% (n  = 5). The correlation coefficient was satisfactory (r 2  > 0.99) for both DM and DP. Linear ranges were obtained from 0.03 ng/ml to 2 μg/ml for DM and from 0.05 ng/ml to 2 μg/ml for DP.
Keywords: Sol–gel technology; Solid-phase microextraction; Gas chromatography-mass spectrometry; Dextromethorphan; Dextrophan; Plasma samples;

Drug development of cucurbitacins requires derivatives that have lower cytotoxicity. Therefore, the effect of structural modification on in vitro cytotoxicity has been investigated. Lipophilicity or chromatographic hydrophobicity index (CHI) was chosen as molecular property. CHI was determined by RP-HPLC in both aqueous acetonitrile and aqueous methanol. Compounds CHI range was wide and better defined in acetonitrile (CHIACN  = 46–88 and 38–102) than in methanol (CHIMeOH  = 56–78). Higher resolution was achieved in acetonitrile, and higher precision on the shorter C18 column. Cucurbitacins cytotoxicity (IC50) was measured on the hepatocyte-derived HepG2 cells. Strong relationship between CHI and logarithmic IC50 was found. As a result, cytotoxicity increased linearly with increasing hydrophobicity (r  ≥ 0.90). Other lipophilicity parameters, such as log  P and C  log  P were also estimated. Cytotoxicity correlated well with log  P (r  = 0.95) and slightly with C  log  P (r  = 0.74). The log  P and C  log  P data showed good correlation with CHI (r  > 0.92). Overall, alkylation of C1 hydroxyl, unsaturation of C1―C2 bond, and acetylation of C25 hydroxyl increased both lipophilicity and cytotoxicity. This assay should prove useful for monitoring cucurbitacin homologues or other drug candidates for their cytotoxicity.
Keywords: Cucurbitacin; Chromatographic hydrophobicity index; CHI; HepG2; IC50; Cytotoxicity; Structure-activity relationship; SAR; log  P; C  log  P; RP-HPLC;

High performance liquid chromatographic–mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies by Hai-Tang Xie; Guang-Ji Wang; Jian-Guo Sun; Ian Tucker; Xiao-Chen Zhao; Yuan-Yuan Xie; Hao Li; Xi-ling Jiang; Rui Wang; Mei-Juang Xu; Wei Wang (167-173).
To support pharmacokinetic studies of ginsenosides, a novel method to quantitatively analyze ginsenoside Rg3 (Rg3), its prosapogenin ginsenoside Rh2 (Rh2) and aglycone 20(S)-protopanaxadiol (ppd) in rat plasma was developed and validated. The method was based on gradient separation of ginsenosides present in rat plasma using high performance liquid chromatography (HPLC), followed by detection with electrospray ionization(ESI) mass spectrometry (MS) in negative ion mode with the mobile phase additive, ammonium chloride (500 μM). Differentiation of ginsenosides was achieved through simultaneous detection of the [M+Cl] adduct of ginsenoside Rg3 and [M+Cl] adducts of its deglycosylated metabolites Rh2 and ppd, and other ions after solid phase extraction (SPE). The /specific ions monitored were m/z 819.50 for Rg3, m/z 657.35 for Rh2, m/z 495.40 for ppd and m/z 799.55 for the internal standard (digitoxin). The mean recoveries for Rg3, Rh2 and ppd were 77.85, 82.65 and 98.33%, respectively using 0.1 ml plasma for extraction. The lower limits of quantification were 10.0, 2.0 and 8.0 ng/ml (equivalent to 0.1, 0.02 and 0.08 ng in each 10 μl injection onto the HPLC column) for Rg3, Rh2 and ppd, respectively. The method has been demonstrated to be highly sensitive and accurate for the determination of Rg3 and its metabolites in rat plasma.
Keywords: Ginsenoside Rg3; Rh2; 20(S)-Protopanaxadiol (ppd); Pharmacokinetics;

The most common commercially available silylating reagents, N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), N,O-bis-(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS) and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) were evaluated to achieve optimal derivatization conditions for analyzing various benzodiazepines based on gas chromatography–electron impact ionization–mass spectrometry (GC–EI–MS). Sensitivity, repeatability, retention times and stability of the derivatives, as well as specificity of mass fragmentation, were studied in detail. Also other parameters affecting the derivatization chemistry of benzodiazepines are discussed. tert-Butyldimethylsilyl (TBDMS) derivatives proved to be more stable, reproducible and sensitive than corresponding trimethylsilyl (TMS) derivatives for the GC–EI–MS analysis of benzodiazepines. Based on the TBDMS derivatives, a rapid, reliable, sensitive and quantitative GC–MS method was developed for the determination of 14 benzodiazepines and two hydroxy metabolites, as well as two non-benzodiazepine hypnotic agents, zolpidem and zaleplon, using 500 μl of whole blood. The method was completely validated in terms of accuracy, intra- and interday precision, limit of detection (LOD), limit of quantitation (LOQ), linearity, selectivity and extraction efficiency; these were all within the required limits, except for the accuracy of nitrazepam at a medium concentration level.
Keywords: GC–MS; Silylation; Benzodiazepines; Zolpidem; Zaleplon;

An assay comprising two simple, selective and isocratic HPLC methods with UV detection was developed and validated for measuring warfarin enantiomers and all five warfarin monohydroxylated metabolites in patient blood plasma. Following liquid/liquid extraction from 1 ml of blood plasma a baseline separation of analytes was achieved on chiral (α1 acid glycoprotein – AGP) and achiral (C18) column. Both methods were consistent (R.S.D. < 6.9% for warfarin enantiomers and < 8.9% for monohydroxylated metabolites) and linear (r  > 0.998). The limits of detection were 25 ng/ml for warfarin enantiomers, 25 ng/ml for 4′-, 10-, 6- and 7-hydroxywarfarin, 35 ng/ml for 8-hydroxywarfarin and 50 ng/ml for racemic warfarin. In a clinical study in 204 patients, it was confirmed that the assay is appropriate for evaluation of influences of genetic polymorphisms, demographic factors and concomitant drug treatment on warfarin metabolism.
Keywords: Warfarin enantiomers; Warfarin monohydroxylated metabolites; Human plasma;

Simple and sensitive high-performance liquid chromatography (HPLC) assays were developed and validated for the quantitation of the investigational anticancer drug 9-nitrocamptothecin (9-NC) as the lactone form and as the total of the lactone(I) and carboxylate(II) forms in human plasma. For the assay of lactone form (9NC-lac), the analytical method involved a protein precipitation step with adding a mixture of cold acetonitril–chloroform (5:1 (v/v), −20 °C) to plasma sample that stabilized the pH-dependent conversion of I to II. After evaporation under gentle stream of nitrogen gas (40 °C) the dry extract was dissolved in mobile phase (pH 5.5). For determination of the total of the lactone and carboxylate forms of the drug (9NC-tot), plasma samples were deproteinated with cold acetonitril (−20 °C) acidified with perchloric acid (5%), which resulted in the conversion of the carboxylate into the lactone form. After centrifugation the upper solvent was evaporated (nitrogen, 40 °C) and the dry extract was dissolved in mobile phase (pH 3.5). All separations were performed on a RP-C8 column, using a mixture of acetonitril–water as eluent (pH 3.5 for total form and pH 5.5 for lactone form) and UV detection. The presented assay was linear over a concentration range of 25–1500 ng/ml with lower limit of quantitation of 25 ng/ml for both 9NC-tot and 9NC-lac. Within-run and between-run precision was always less than 7.5% in the concentration range of interest. The reported assay method showed good characteristics of linearity, sensitivity, selectivity and precision allowing applying in pharmacokinetic studies.
Keywords: 9-Nitrocamptothecin; HPLC; Lactone; Total; Plasma;

A selective and highly sensitive isocratic high performance liquid chromatographic (HPLC) method is described for simultaneous determination of lactone and carboxylate species of topotecan, in plasma. The method utilizes a protein precipitation step with cold methanol (−20 °C) for sample preparation followed by separation on a Novapack C18 column using ammonium acetate buffer, acetonitrile and triethylamine (84:16:1.5, v/v) containing tetrabutyl ammonium hydrogen sulfate (TBAHS) (2 mM) with a pH of 5 as the mobile phase. The eluted peaks were detected by a fluorescence detector was set at an excitation wavelength of 380 nm and an emission wavelength of 527 nm. The method was validated in the range of lactone and carboxylate forms of topotecan concentrations from 0.05 to 75 ng/ml. Intra- and inter-day precision expressed by the relative standard deviation was less than 8.50% and inaccuracy did not exceed 10% for lactone and carboxylate forms of topotecan. The limit of quantitation was 0.05 ng/ml using 0.50 ml plasma. Stability studies in plasma and plasma extract indicated that topotecan is stable for at least 2 weeks at −70 °C.
Keywords: Topotecan; HPLC; Plasma; Lactone; Carboxylate;

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study by Sonu Sundd Singh; Manish Jain; Kuldeep Sharma; Bhavin Shah; Meghna Vyas; Purav Thakkar; Ruchy Shah; Shriprakash Singh; Brajbhushan Lohray (213-220).
A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm × 4.6 mm, 5 μm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31–3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17–9.50%. Serum samples containing itopride were stable for 180.0 days at −70 ±5 °C and for 24.0 h at ambient temperature (25 ± 5 °C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.
Keywords: Itopride; Human serum; Validation; Bioequivalence;

Determination of Salvinorin A in body fluids by high performance liquid chromatography–atmospheric pressure chemical ionization by Mark S. Schmidt; Thomas E. Prisinzano; Kevin Tidgewell; Wayne Harding; Eduardo R. Butelman; Mary J. Kreek; Daryl J. Murry (221-225).
Salvinorin A was quantitated in human and rhesus monkey plasma, rhesus monkey cerebrospinal fluid, and human urine by negative ion LC–MS/APCI. The method for Salvinorin A has been fully validated, the LLOQ using FDA guidelines is 2 ng/mL for 0.5 mL plasma samples. The linear range was from 2 to 1000 ng/mL. Several derivatives in the Salvinorin family can also be analyzed by this method; d3-Salvinorin A was prepared and used as internal standard. The metabolite Salvinorin B can be semi quantitatively determined. The method has been used to establish that Salvinorin B is the principal metabolite of Salvinorin A ex vivo and to establish the analytical method to study in vivo samples.
Keywords: Salvinorin; LC–MS; APCT; Analysis; Urine; Plasma; Body fluids; Metabolism; Salvinorin A;

Glutathione (GSH) and glutathione disulfide (GSSG) are important thiols, which provide defence against oxidative stress by scavenging free radicals or causing the reduction of hydrogen peroxide. The ratio GSH/GSSG is often used as a sensitive index of oxidative stress in vivo. In this paper, a direct electrochemical method using an electrode modified with functionalized carbon nanotubes as electrochemical detector (ED) for liquid chromatography (LC) was described. The electrochemical behaviors of GSH and GSSG on this modified electrode were investigated by cyclic voltammetry and it was found that the functionalized carbon nanotubes exhibited efficiently electrocatalysis on the current responses of GSH and GSSG. In LC-ED, both of the analytes showed good and stable current responses. The detection limit of GSH was 0.2 pmol on column and that of GSSG was 1.2 pmol on column, which were low enough for the analysis of real small samples. The method was sensitive enough to detect difference in concentration of GSH and GSSG in hepatocytes from animals with and without introduction of oxidation stress by glucose or hydrogenperoxide.
Keywords: Glutathione; Glutathione disulfide; Hepatocytes; Functionalized carbon nanotubes;

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the simultaneous determination of buprenorphine and buprenorphine propionate in human and animal blood. Buprenorphine propionate was also proven to be a prodrug of buprenorphine. It was comprised of only a one-step extraction procedure with ethyl acetate and normal-phase chromatography on a Betasil Silica column. The recoveries of buprenorphine and buprenorphine propionate were above 84%. Calibration graphs were linear for buprenorphine over the concentration range 2–1500 ng/ml and for buprenorphine propionate over the concentration range 20–1500 ng/ml with a coefficient of variation, both within- and between-day, or less than 10% at any level. The limits of quantitation of buprenorphine and buprenorphine propionate in human or animal blood were 2.0 and 20 ng/ml, respectively, based on a single-to-noise ratio of 3. The method has been successfully applied to pharmacokinetic studies of buprenorphine and buprenorphine propionate in rabbits. The results demonstrated that buprenorphine propionate was rapidly and totally converted to its parent drug, buprenorphine, following intravenous administration. Buprenorphine propionate is a prodrug of buprenorphine.
Keywords: Buprenorphine; Propionate; HPLC; Pharmacokinetic;

Concerns in the development of an assay for determination of a highly conjugated adsorption-prone compound in human urine by Yang Xu; Lihong Du; Mark J. Rose; Irong Fu; Eric J. Woolf; Donald G. Musson (241-248).
Concerns in pre-analytical handling of urine samples are discussed using a new KDR kinase inhibitor, 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (compound A), as an example of a case where high light sensitivity and low analyte recovery (high affinity for container surface) were found. The absence of these problems in plasma samples may be a result of the plasma protein content. Low recovery of the analyte from urine can be remedied by either changing the container or by using additives, such as bovine serum albumin (BSA) or non-ionic surfactant Tween-20. In the case of compound A, changing containers (polypropylene versus glass vial) or addition of BSA did bring analyte recovery up to 80%. However, the addition of 0.2% Tween-20 into urine quality controls (QCs) gave more than 95% analyte recovery, indicating effective reduction of analyte loss to the surface of containers. The urine assay using mixed-mode SPE and LC–MS/MS was not affected significantly by introducing Tween-20 into the samples. The mean SPE extraction recovery was 68.4% and matrix suppression of ionization on MS was less than 8% at all analyte concentrations. The linear range of the calibration curve was 0.5–400 ng/mL on PE Sciex API 3000 LC–MS/MS system. The assay intraday accuracy and precision were 92.1–104.8% and <4.2% (%CV), respectively. Urine QC samples, containing 0.2% Tween-20, gave excellent recovery after three cycles of freeze and thaw. Since analyte loss to its urine container surface is not unique to compound A (M. Schwartz, W. Kline, B. Matuszewski, Anal. Chim. Acta 352 (1997) 299–307; A.L. Fisher, E. DePuy, T. Shih, R. Stearns, Y. Lee, K. Gottesdiener, S. Flattery, M. De Smet, B. Keymeulen, D.G. Musson, J. Pharm. Biomed. Anal. 26 (2001) 739–752), we suggest an evaluation of the potential problem in the early stages of urine assay development to ensure reliable quantitation of analytes. The addition of Tween-20 can serve as a useful analytical tool to other analytes with similar situations.
Keywords: Urine assay; Low QC recovery from urine; Tween-20; Solid-phase extraction and LC–MS/MS;

DE-310 is a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, DX-8951, conjugated to a water-soluble polymer by means of a peptide spacer. New assay methods have been developed to determine the polymer-bonded DX-8951 conjugate, free DX-8951, and Glycyl-DX-8951 in human plasma. Solid-phase extraction was used to extract free DX-8951 and Glycyl-DX-8951 from plasma, and LC/MS/MS (Method I) was used to determine the amount of each analyte. Protein precipitation was used to extract Conjugated DX-8951, which was then digested with thermolysin. HPLC (Method II) was used to determine the productive compound (Phenylalanyl-Glycyl-DX-8951). The lower limit of quantitation of DX-8951 was 50 pg/ml, of Glycyl-DX-8951 was 80 pg/ml, and of Conjugated DX-8951 was 100 ng/ml (as DX-8951 equivalent). Both methods showed satisfactory sensitivity, precision, and accuracy.
Keywords: HPLC; LC/MS/MS; DE-310; Camptothecin;

A high performance liquid chromatography (HPLC) method for the estimation of pravastatin in human plasma and urine samples has been developed. The preparation of the samples was performed by automated solid phase extraction using clonazepam as internal standard. The compounds were separated by isocratic reversed-phase HPLC (C18) and detected at 239 nm. The method was linear up to concentrations of 200 ng/ml in plasma and 2000 ng/ml in urine. The intra-assay variability for pravastatin in plasma ranged from 0.9% to 3.5% and from 2.5% to 5.3% in urine. The inter-assay variability ranged from 9.1% to 10.2% in plasma and from 3.9% to 7.5% in urine. The validated limits of quantification were 1.9 ng/ml for plasma and 125 ng/ml for urine estimation. These method characteristics allowed the determination of the pharmacokinetic parameters of pravastatin after administration of therapeutic doses.
Keywords: Pravastatin; OATP-C; Pharmacokinetic;

Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase by R. Moaddel; R. Yamaguchi; P.C. Ho; S. Patel; C.-P. Hsu; V. Subrahmanyam; I.W. Wainer (263-268).
Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(−)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [3H]-methyl phenyl pyridinium ([3H]-MPP+) as the marker ligand and MPP+, verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The K d values calculated from the chromatographic studies correlated with previously reported K i values (r 2  = 0.9987; p  < 0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP+ and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil.
Keywords: hOCT1; Drug transporters; Affinity chromatography; MDCK cells;

Using the CNS of Lymnaea stagnalis a method is described for the rapid analysis of neurotransmitters and their metabolites using high performance liquid chromatography coupled with electrochemical detection. Tissue samples were homogenised in ice-cold 0.1 M perchloric acid and centrifuged. Using a C18 microbore column the mobile phase was maintained at a flow rate of 100 μl/min and consisted of sodium citrate buffer (pH 3.2)–acetonitrile (82.5:17.5, v/v) with 2 mM decane-sulfonic acid sodium salt. The potential was set at +750 mV versus Ag|AgCl reference electrode at a sensitivity of 50 nA full scale deflection. The detection limit for serotonin was 11.86 ng ml−1 for a 5 μl injection. Preparation of tissue samples in mobile phase reduced the response to dopamine and serotonin compared with perchloric acid. In addition it was found that the storage of tissue samples at −20 °C caused losses of dopamine and serotonin. As a result of optimising the sample preparation and mobile phase the total time of analysis was substantially reduced resulting in a sample preparation and assay time of 15–20 min.
Keywords: Microbore HPLC; Serotonin; Catecholamines; Lymnaea stagnalis;

Benzene is a human carcinogen and its metabolite, urinary trans,trans-muconic acid (ttMA), is a biomarker for risk assessment. However, most of the existing methods were not sensitive enough for monitoring of low level exposure. This paper describes a HPLC-UV method for ttMA determination with enhanced selectivity and sensitivity. A 30 mg Oasis®MAX cartridge was used to clean-up 50 μl of urine sample and gradient elution was performed on a Zorbax SB-C18 column (30 °C). ttMA was detected at wavelength 263 nm using a UV diode array detector (DAD). The two mobile phases used were (A) 150 mM ortho-phosphoric acid containing of 9% (v/v) methanol; and (B) 125 mM ortho-phosphoric acid containing 30% (v/v) acetonitrile. The method was validated with 61 urine samples collected from non-occupationally benzene exposed individuals and 14 quality control specimens from an international quality assessment scheme. The urinary ttMA concentrations (mean ± S.D. μg/g creatinine) were 90 ± 34 for smokers (n  =26), 49 ± 39 for non-smokers (n  = 21) and 23 ± 18 for non-smoking hospital staff (n  = 14). A correlation coefficient, r  = 0.99 was found with 14 external quality specimens for ttMA ranged from 0.4 to 6.8 mg/l. The recovery and reproducibility were generally over 90% and the detection limit was 5 μg/l.
Keywords: Biomarker; Interference of sorbic acid; Low level benzene exposure; Cigarette smoking;

Solid-phase microextraction and chiral HPLC analysis of ibuprofen in urine by Anderson Rodrigo Moraes de Oliveira; Evandro José Cesarino; Pierina Sueli Bonato (285-291).
A simple and rapid solid-phase microextraction method was developed for the enantioselective analysis of ibuprofen in urine. The sampling was made with a polydimethylsiloxane-divinylbenzene coated fiber immersed in the liquid sample. After desorptioning from the fiber, ibuprofen enantiomers were analyzed by HPLC using a Chiralpak AD-RH column and UV detection. The mobile phase was made of methanol–pH 3.0 phosphoric acid solution (75:25, v/v), at a flow rate of 0.45 mL/min. The mean recoveries of SPME were 19.8 and 19.1% for (−)-R-ibuprofen and (+)-(S)-ibuprofen, respectively. The method was linear at the range of 0.25–25 μg/mL. Within-day and between-day assay precision and accuracy were below 15% for both ibuprofen enantiomers at concentrations of 0.75, 7.5 and 20 μg/mL. The method was tested with urine quality control samples and human urine fractions after administration of 200 mg rac-ibuprofen.
Keywords: Solid-phase microextraction; Ibuprofen enantiomers; Enantiomeric separation; Urine;

Application of the standard addition approach for the quantification of urinary benzene by Pascale Basilicata; Nadia Miraglia; Maria Pieri; Antonio Acampora; Leonardo Soleo; Nicola Sannolo (293-299).
Urinary benzene is used as biomarker of exposure to evaluate the uptake of this solvent both in non-occupationally exposed population and in benzene-exposed workers. The quantitative determination of benzene in urine is carried out in a three steps procedure: urine collection, sample analysis by head space/solid phase microextraction/gas chromatography/mass spectrometry and analyte quantification. The adopted quantification method influences the initial step, hence the whole procedure. Two quantification approaches were compared as regards precision and accuracy: the calibration curves and the standard addition method. Even if calibration curves obtained by using urine samples from different subjects were always linear, their slopes and intercepts showed noteworthy variations, attributable to the influence of the biological matrix on benzene recovery. The standard addition method showed to be more suitable for compensating matrix effects, and a three-point standard addition protocol was used to quantify benzene in urine samples of 11 benzene-exposed workers (smokers and non-smokers). Urine from occupationally exposed workers was collected before and after work-shift. Besides urinary benzene, the applicability of the method was verified by measuring the urinary concentration of the S-phenylmercapturic acid, a specific benzene metabolite, generally adopted as biomarker in biological monitoring procedures. A similar trend of concentration levels of both analytes measured in urine samples collected before work-shift with respect to the after work-shift ones was found, showing the actual applicability of the standard addition method for biological monitoring purposes.
Keywords: Urinary benzene; Standard addition approach; Occupational exposure;

New methods employing capillary liquid chromatography in combination with time-of-flight mass spectrometry (μLC-TOF/MS) were developed for the rapid determination of salcatonin in human urine and plasma. The present approaches utilize 13C6-leucine (19)-labeled salcatonin as internal standard, small matrix volumes and simple sample preparation procedures. They allow TOF/MS to be used as a highly selective detector for providing accurate quantitation of salcatonin. Data acquisition was performed in enhanced mode optimizing the signal for the triply charged species of salcatonin and its internal standard. We demonstrate that the determination of salcatonin is straightforward and reliable and can be performed with excellent linearity (R 2  > 0.999), precision and accuracy over the concentration ranges of 2.9–290 pmol/mL in human urine, and 7.3–730 pmol/mL in human plasma.
Keywords: Salcatonin; Capillary chromatography; Time-of-flight mass spectrometry; Quantitation; Isotope dilution; Plasma; Urine;

The presence of succinylacetone in urine or blood or amniotic fluid is pathognomonic of an inherited metabolic disorder, named tyrosinemia type I. We developed a capillary electrophoretic method for the fast analysis of succinylacetone in urine samples. The separation was performed at reversed polarity mode using either a cationic surfactant as the buffer additive, or a capillary coated with a positively charged polyelectrolyte. Under these conditions, urine samples were directly injected to the capillary without any pretreatment step. The utility of the method was demonstrated by the identification of succinyacetone in urine from patients with hereditary tyrosinemia type I. For all patients, diagnostic peaks at the expected migration times were detected. The developed method is rapid, simple, inexpensive, and suitable for the determination of succinylacetone in clinical urine samples.
Keywords: Hereditary tyrosinemia type I; Succinylacetone; Capillary electrophoresis;

Author Index (313-315).

Keyword Index (317-323).