Journal of Chromatography B (v.844, #1)

The aim of the present study is to develop an automated blood sampling (ABS) method coupled to a liquid chromatography–tandem mass spectroscopy (LC–MS/MS) method to evaluate the oral bioavailability of plumbagin in a conscious freely moving rat. Plumbagin, an herbal ingredient, was isolated from Plumbago zeylanica L. The separation was performed using a reversed phase C18 (150mm × 4.6 mm I.D.; 5 μm) column and was eluted with the mobile phase of water–acetonitrile (40:60, v/v) at a flow-rate of 0.8 ml/min. Multiple reaction monitoring (MRM) was used to monitor the transition of the deprotonated molecule m/z 187 [M―H] to the product ion m/z 159 [M―H―CO] for the plumbagin analysis. The calibration curve was linear over the concentration range of 10–2000 ng/ml with a coefficient estimation of 0.995. The intra- and inter-day variations (% relative standard deviation; RSD and % bias) of the assay for rat plasma samples were less than 17%. The limit of detection and the limit of quantification were 5 and 10 ng/ml, respectively. Recovery of plumbagin from the rat plasma was about 80%. This LC–MS/MS method has been validated to study the pharmacokinetics of plumbagin in rats. The oral bioavailability (AUCPO/DosePO)/(AUCIV/DoseIV) of plumbagin was about 38.7 ± 5%.
Keywords: Bioavailability; Herbal medicine; Pharmacokinetics; Plumbagin; Plumbago zeylanica L;

Hydrophobic interaction adsorption of whey proteins: Effect of temperature and salt concentration and thermodynamic analysis by Renata C.F. Bonomo; Luis A. Minim; Jane S.R. Coimbra; Rafael C.I. Fontan; Luis H. Mendes da Silva; Valéria P.R. Minim (6-14).
The adsorptive behavior of bovine serum albumin (BSA) and β-lactoglobulin (β-lg) on hydrophobic adsorbent was studied at four temperatures and different salt concentrations. The Langmuir model was fitted by experimental equilibrium data showing that an increase in temperature and salt concentration results in an increase on the capacity factor of both proteins. A thermodynamic analysis coupled with isotherm measurements showed that salt concentration and temperature affected the enthalpic and entropic behavior of the adsorption process of both proteins, mainly to the β-lg. The fast variation in the Z value for temperature over than 303.1 K suggest a great conformational change occurring in the β-lg structure, which almost duplicated the maximum adsorption capacity of this protein.
Keywords: Bovine serum albumin; β-Lactoglobulin; Thermodynamic parameters; Isotherm; Hydrophobic interaction;

Validation of an HPLC method for analysis of DB-67 and its water soluble prodrug in mouse plasma by Jamie Horn; Sherri L. Jordan; Lin Song; Michael J. Roberts; Bradley D. Anderson; Markos Leggas (15-22).
A method for the quantitation of DB-67 ((20S)-10-hydroxy-7-tert-butyldimethylsilylcamptothecin) lactone and carboxylate in mouse plasma has been developed, validated, and applied in pharmacokinetic studies. The analytes were separated by reversed-phase chromatography with fluorescence detection. Validation demonstrated the selectivity and specificity for the carboxylate and lactone, with linearity between 1–300 ng/mL and 2.5–300 ng/mL for the carboxylate and lactone, respectively (accuracy 90–110% of theory and coefficient of variation ≤5.7%). Carboxylate to lactone conversion was <4% using this method. The assay was found to be suitable for the analysis of DB-67 lactone and carboxylate in pharmacokinetic studies following intravenous administration of DB-67 or its δ-aminobutyric acid ester derivative.
Keywords: DB-67; Camptothecin analogs; Method validation;

The opposite effect of temperature on polyethylene glycol-based aqueous biphasic systems versus aqueous biphasic extraction chromatographic resins by Scott T. Griffin; Meghna Dilip; Scott K. Spear; Jonathan G. Huddleston; Robin D. Rogers (23-31).
Variation in operational temperatures has revealed differences in the partitioning behavior of probe solutes between the phases in aqueous biphasic systems (ABS) and the related aqueous biphasic extraction chromatographic resin (ABEC). This difference has been studied using the hydrophobic anion, 99TcO4 , as a probe and (NH4)2SO4 as the kosmotropic salt. Distribution of the hydrophobic anion 99TcO4 to the PEG-rich phase in a MePEG-5000/(NH4)2SO4 ABS increases with increasing temperature, but decreases are observed in batch uptakes of this anion to ABEC resins from (NH4)2SO4 solutions. Phase diagrams were constructed at five different temperatures from 10 to 50 °C using cloud point titration for the ABS and a correlation between the phase divergence, measured in terms of tie line length (TLL), and the temperature of the partitioning system was verified. Thermodynamic parameters (ΔH°, ΔS°, ΔG°, Δ C p ° ) as a function of temperature were calculated for the various systems studied and the results imply thermodynamic differences between partitioning in ABS versus ABEC.
Keywords: Aqueous two phase system; Aqueous biphasic extraction chromatography; Temperature effect; Thermodynamics;

Isotachophoresis preconcentration integrated microfluidic chip for highly sensitive genotyping of the hepatitis B virus by Dayu Liu; Ming Shi; Huaiqing Huang; Zhicheng Long; Xiaomian Zhou; Jianhua Qin; Bingcheng Lin (32-38).
The genotyping of hepatitis B virus (HBV) has become recently a valuable tool not only for epidemiological reasons but also for the clinical practice. Conventional methods for HBV genotyping typically include amplification of the target DNA sequences with a two-round nested PCR followed by separation of the amplified fragments by gel electrophoresis. A microfluidic chip that couples isotachophoresis (ITP) preconcentration and zone electrophoresis (ZE) separation may provide great advantages for sensitive, rapid and cost-effective clinical analysis. In this study, an HBV genotyping method with only one amplification round was developed by the application of the ITP-ZE chip. All the analysis steps of the ITP-ZE separation including sample injection, stacking and separation were performed continuously, controlled by sequential high-voltage switching. A 2.1 cm sample plug was preconcentrated between discontinuous buffers in ITP process, followed by ZE separation. Sensitivity enhancement was obtained through the increase of sample loading volume. The average LOD value of the ITP-ZE microfluidic chip was determined to be 0.0021 pg/μL. In a large-scale HBV genotyping test, single round PCR products were analyzed by ITP-ZE microfluidic chip, and the results were compared with that of the conventional method. Among the 200 cases studied, the classification rate obtained with microfluidic chip was 93%, which was 6% higher than that obtained with the conventional method. Method with ITP-ZE chip analysis provides HBV genotyping information in reduced PCR amplification time with higher detection rate when compared with conventional method. This method holds great potential for extrapolation to the abundance of similar molecular biology-based techniques in clinical diagnosis.
Keywords: Microfluidic chip; Sample preconcentration; Isotachophoresis; Hepatitis virus B; Genotyping;

A simplified two-stage method for B-phycoerythrin (BPE) recovery from Porphyridium cruentum was developed. The proposed method involved cell disruption by sonication and primary recovery by aqueous two-phase partition. The evaluation of two different methods of cell disruption and the effect of increasing concentration of cell homogenate from P. cruentum culture upon aqueous two-phase systems (ATPS) performance was carried out to avoid the use of precipitation stages. Cell disruption by sonication proved to be superior over manual maceration since a five time increase in the concentration of B-phycoerythrin release was achieved. An increase in the concentration of crude extract from disrupted P. cruentum cells loaded to the ATPS (from 10 to 40%, w/w) proved to be suitable to increase the product purity and benefited the processing of highly concentrated disrupted extract. Kinetics studies of phase separation performed suggested the use of batch settlers with height/diameter (H/D) ratio less than one to reduce the necessary time for the phases to separate. The proposed ATPS stage comprising of 29% (w/w) polyethylene glycol (PEG) 1000 g/mol, 9% (w/w) potassium phosphate, tie-line length (TLL) of 45% (w/w), volume ratio (V R) of 4.5, pH 7.0 and 40% (w/w) crude extract loaded in a batch settler with H/D ratio of 0.5 proved to be efficient for the recovery of 90% of B-phycoerythrin at the top PEG-rich phase. The purity of B-phycoerythrin increased up to 4.0 times after the two-stage method. The results reported here demonstrate the potential implementation of a strategy to B-phycoerythrin recovery with a purity of 3.2 (estimated by the absorbance relation of 545–280 nm) from P. cruentum.
Keywords: B-phycoerythrin; Recovery; Aqueous two-phase systems;

A quantitative assay method by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the simultaneous determination of azelnidipine and its two metabolites, M-1 (aromatized form) and M-2 (hydroxylated form), in human plasma was developed and validated. Plasma samples, each of 1.0 mL, were extracted by a single step liquid–liquid extraction using a mixture of ethyl acetate and hexane (1:1, v/v), and analyzed by the LC/ESI-MS/MS method. Three analytes were separated by isocratic elution on a C18 column, and ionized using a positive ion electrospray ionization source. The ion transitions were monitored in selected reaction monitoring (SRM) mode. The chromatographic run time was 11 min per injection, with retention time of 3.6, 10.2 and 6.8 min for azelnidipine, M-1 and M-2, respectively. The calibration curves for azelnidipine, M-1 and M-2 well fitted to equations by a weighted (1/X 2) quadratic regression over the range of 0.5–40.0 ng/mL (r 2  > 0.9979). The intra- and inter-assay precisions (coefficient of variation: C.V.), calculated from quality control (QC) samples, were less than 8.7 and 8.4%, 3.8 and 4.7%, and 11.9 and 13.9%, respectively, for azelnidipine, M-1 and M-2. The accuracy was within ±9% for azelnidipine, within ±7% for M-1 and within ±16% for M-2. The overall recoveries for azelnidipine, M-1 and M-2 were 68.8–78.6%, 54.3–62.9% and 80.4–89.7%, respectively. All analytes evaluated demonstrated acceptable short-term, long-term, auto-sampler and stock solution stabilities. Furthermore, the method developed was successfully applied to pharmacokinetic studies on azelnidipine, M-1 and M-2 after an oral dose of 16 mg CALBLOCK® tablets (2 mg × 8 mg tablets) to healthy volunteers.
Keywords: Azelnidipine; Two metabolites;

Parabens (alkyl esters of p-hydroxybenzoic acid) are widely used as antimicrobial preservatives in cosmetic products, pharmaceuticals, and food processing. However, weak estrogenicity of some parabens has been revealed from several studies. Human exposure to parabens may be assessed by measuring the conjugated or free species of these compounds or their metabolites in urine. We have developed a method using on-line solid phase extraction-high performance liquid chromatography–isotope dilution tandem mass spectrometry with peak focusing to measure the urinary concentrations of methyl, ethyl, propyl, n- and iso- butyl, and benzyl parabens. This method has good reproducibility and accuracy with detection limits for all analytes below 0.2 ng/mL in 100 μL of urine, and permits quick and accurate analysis of a large number of samples in epidemiologic studies for assessing the prevalence of human exposure to parabens. Using this method, we detected methyl, ethyl, and propyl parabens, mostly as conjugated species, in 22 urine samples collected from anonymous adults.
Keywords: Paraben; Exposure; Human; Biomonitoring; Urine; Biomarker;

Quantification of O 6-methyl and O 6-ethyl deoxyguanosine adducts in C57BL/6N/Tk +/− mice using LC/MS/MS by Mona I. Churchwell; Frederick A. Beland; Daniel R. Doerge (60-66).
The carcinogenicity of many alkylating agents is derived from their ability to form persistent DNA adducts that induce mutations. This paper presents and validates methodology, based on LC with tandem mass spectrometry, for the separate or concurrent quantification by isotope dilution of O 6-methyl-2′-deoxyguanosine (O 6Me-dG) and O 6-ethyl-2′-deoxyguanosine (O 6Et-dG) DNA adducts. The limits of quantification were estimated to be ≤0.2 adducts/108 nucleotides for either adduct. This sensitivity permitted evaluation of adduct levels in livers from separate groups of untreated adult C57BL/6N/Tk +/− and C57BL/6N X Sv129 mice (undetectable to 5.5 ± 6.7 O 6Me-dG/108 nucleotides; undetectable to 0.04 O 6Et-dG/108 nucleotides). Treatment of adult C57BL/6N/Tk +/− mice with equimolar doses (342 μmol/kg body weight) of N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea produced adduct levels in liver of 1700 ± 80 O 6Me-dG/108 nucleotides and 260 ± 60 O 6Et-dG/108 nucleotides, respectively, when assessed 4 h after dosing. These methods should be useful for evaluations of DNA adducts in relation to cellular processes that modify carcinogenic and toxicological responses in experimental animals and humans.
Keywords: O 6-Methyl-2′-deoxyguanosine; O 6-Ethyl-2′-deoxyguanosine; Mass spectrometry; DNA adducts;

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometrical (LC/ESI–MS/MS) method was developed for the identification and quantification of two polyisoprenylated benzophenones xanthochymol and isoxanthochymol in the extracts of the fruit rinds, stem bark, seed pericarps and leaves of Garcinia indica and in the fruit rinds of Garcinia cambogia. The separation of xanthochymol and isoxanthochymol was achieved on a RP-18 column using the solvent system consisting of a mixture of acetonitrile–water (9:1) and methanol–acetic acid (99.5:0.5) as a mobile phase at a flow rate of 0.4 ml/min. A multiple reaction monitoring (MRM) method was developed for quantification of xanthochymol and isoxanthochymol in the above extracts of Garcinia species. On the basis of signal to noise ratio of 3, the limits of detection in MRM mode for xanthochymol and isoxanthochymol were 1.0 ng/ml and 0.5 ng/ml, respectively. The method was validated in terms of linearity, accuracy and precision for 6 days. The method developed was found to be useful for identification and quantification of xanthochymol and isoxanthochymol in the extracts of the fruit rinds, stem bark, seed pericarps and leaves of G. indica and in the fruit rinds of G. cambogia.
Keywords: Xanthochymol; Isoxanthochymol; Liquid chromatography; Tandem mass spectrometry; Multiple reaction monitoring; Garcinia extracts;

The antifungal polyene antibiotics nystatin was tested in a clinical trial to describe pharmacokinetics and safety after repeated administration of Nystatin “Lederle” sterile powder in healthy volunteers. To monitor the nystatin concentration–time profile in plasma we developed a sensitive method in the range of 1–100 ng/ml based on liquid chromatography coupled with tandem mass spectrometry. The target substance was separated from the biological matrix on C18 solid-phase extraction cartridges with methanol. The Chromatography was performed isocratically using a reversed phase Caltrex Resorcinearene column. The mobile phase consisted of 5 mM ammonium formate buffer and acetonitrile (40:60, v/v). The mass spectrometer works with electrospray ionization in its positive selected ion monitoring (SIM) mode using the respective MH+ ions, m/z 926.6 for nystatin and m/z 924.4 for amphotericin B as internal standard. The method validation was performed according to the demands and international criteria for validation of bioanalytical methods and was successfully applied to the quantification of nystatin in human plasma in the pharmacokinetic trial.
Keywords: Nystatin; Inhaled aerosol; Drug assay; LC–MS;

A sensitive LC–MS/MS method with a simple solid-phase extraction for the determination of acteoside in rat plasma and tissue homogenates was established for the investigation of bioavailability and brain distribution in freely-moving rats. Acteoside in Cistanche deserticola and Boschniakia rossica was also determined. Acteoside and internal standard were separated on a RP-select B column (125 mm × 4.6 mm i.d., particle size 5 μm). The mobile phase consisted of 35% methanol and 65% acetic acid–water (1:100, v/v) at a flow-rate of 1 mL/min. Acteoside and the internal standard were monitored using the multiple-reaction monitoring (MRM) mode at m/z transitions of 623 → 161 and 609 → 301, respectively. The acteoside content was 38.4 ± 2.4 mg/kg (n  = 3) for B. rossica, which is obviously lower than 21134.2 ± 805.5 mg/kg (n  = 3) of C. deserticola. The protein binding in rat plasma was 75.5 ± 1.8%. The brain distribution result indicated that acteoside was evenly distributed in brain tissues (brain stem, cerebellum, the rest of the brain, cortex, hippocampus and striatum) which was about 0.45–0.68% of that in plasma (4.5 ± 0.5 μg/mL) after 15 min of acteoside administration (10 mg/kg, i.v.). After acteoside was given (3 mg/kg, i.v.; 100 mg/kg, p.o.), the oral bioavailability (AUCp.o./dosep.o.)/(AUCi.v./dosei.v.) was only 0.12%.
Keywords: Acteoside; Automated blood sampling; Bioavailability; LC–MS/MS; Pharmacokinetics;

The recent commercial availability of small particle packed columns (<2 μm) and associated instrumentation capable of withstanding the high pressures of such columns, has lead to an increase in the application of so called ultra-performance liquid chromatography (UPLC). It has recently been shown that the improved efficiency, resolution and peak capacity of these columns, when coupled to mass spectrometry, provides particular benefit for the identification of drug metabolites in complex biological samples. In this work, the ability of TopCount, a microplate scintillation counter, to act as a suitable radiodetection system for ultra-performance liquid chromatography methods is tested. TopCount, has innumerable benefits over more traditional on-line radioactivity flow detection methods, when dealing with the narrow peak widths and small peak volumes associated with the enhanced efficiency of sub-2 μm columns. The system is tested for robustness and sensitivity, and then used to undertake successful metabolite profiling of actual samples, and the data compared to traditional HPLC with on-line radioactivity flow detector.
Keywords: Ultra-performance liquid chromatography; Metabolites; Radiochemical detection; TopCount;

Optimization and validation of the direct HPLC method for the determination of moxifloxacin in plasma by Aleksandra Laban-Djurdjević; Milena Jelikić-Stankov; Predrag Djurdjević (104-111).
Moxifloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo-[3,4-b]pyridin-6-yl]-4-oxo-3-quinolinecarboxylic acid hydrochloride) is new, fourth generation fluoroquinolone with broaden spectrum of antibacterial activity. In the present work simple and rapid RP-HPLC method for the direct determination of moxifloxacin in human plasma is described. Separation of moxifloxacin from plasma components was achieved on Supelco LC-Hisep shielded hydrophobic phase column. The mobile phase consisted of acetonitrile and 0.25 mol/dm3 Na3PO4 (pH 3) in a volume percent ratio (5:95, v/v) and was delivered at a rate of 1 mL/min. Fluorescence detection was employed with excitation at 290 nm and emission at 500 nm. Ofloxacin was used as internal standard and sodium dodecylsulfate solution was used as a displacing agent. Sample preparation was simplified and involved only addition of displacing agent and internal standard and dilution with water. The separation conditions were optimized by the response surface method in two factor space, i.e. the dependence of the retention time on volume percent of acetonitrile and on pH of aqueous phase was optimized. The method was fully validated and validation parameters were: linearity range 3–1300 μg/L; correlation coefficient, 0.99986; mean recovery, 92.5%; limit of quantification, 3.0 μg/L and limit of detection, 1.0 μg/L. Method was applied for the determination of moxifloxacin in human plasma after single or repeated oral doses of 400 mg Avelox® tablets. The proposed method proved to be rapid and accurate and can be successfully used in pharmacokinetic studies and routine clinical practice.
Keywords: Moxifloxacin; RP-HPLC; Determination; Plasma;

The potential importance of the methylation cycle during embryonic development necessitates the establishment of methodology to detect alterations in the relative abundance of s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) in an embryonic experimental system. We have developed a precise and sensitive method for measurement of SAM and SAH based on liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in single neurulation-stage mouse embryos. Use of a penta-fluorinated high-performance liquid chromatography (HPLC) stationary phase gave enhanced sensitivity due to optimal ionisation in organic mobile phase and increased retention time compared to standard reversed-phase separation. Calibration curves suitable for the analysis of neurulation-stage mouse embryos (SAM 0.02–25.0 μM, SAH 0.01–10.0 μM) were linear (r 2  > 0.997) with limits of detection for SAM and SAH of 10 and 2.5 nmol/L, respectively.
Keywords: s-Adenosylmethionine; s-Adenosylhomocysteine; Pentafluorophenylpropyl; Tandem mass spectrometry; Mouse embryo;

Development and validation of a sensitive HPLC–ESI-MS/MS method for the direct determination of glucosamine in human plasma by Aldo Roda; Laura Sabatini; Anna Barbieri; Massimo Guardigli; Marcello Locatelli; Francesco Saverio Violante; Lucio C. Rovati; Stefano Persiani (119-126).
A sensitive and specific HPLC–ESI-MS/MS method for the direct determination of glucosamine in human plasma has been developed and validated. Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid using a polymer-based amino high-performance liquid chromatography (HPLC) column and a water/acetonitrile mobile phase elution gradient, with d-[1-13C]glucosamine as the internal standard. Detection was performed by mass spectrometry, using an electrospray source and employing multiple reaction monitoring to separately monitor glucosamine and the internal standard. The limit of quantification of the method was 10 ng/ml of glucosamine and the calibration curve showed a good linearity up to 1000 ng/ml. The precision (R.S.D.) and the accuracy (bias) of the method at the limit of quantification were 13.8 and 4.0%, respectively, and the mean recovery of glucosamine at three concentration levels was 101.6 ± 5.7%. The method was applied for the determination of glucosamine concentrations in human plasma samples collected from untreated healthy volunteers and, in a separate bioavailability study, to evaluate plasma glucosamine pharmacokinetics profiles after oral administration of crystalline glucosamine sulfate.
Keywords: Bioavailability; Glucosamine; HPLC; Mass spectrometry; Osteoarthritis;

Confirmation of diminazene diaceturate in bovine plasma using electrospray liquid chromatography–mass spectrometry by Sherri B. Turnipseed; Susan B. Clark; Wendy C. Andersen; Christine M. Karbiwnyk; Keith E. Miller; Jeffrey A. Hurlbut (127-133).
Diminazene diaceturate is used as a trypanocide for cattle in tropical regions. This paper describes a LC–MS n method to confirm the presence of diminazene in bovine plasma. Bound diminazene in plasma samples was freed with dilute phosphoric acid, then concentrated on a bonded C18 SPE cartridge. The LC–MS n method utilized electrospray ionization coupled with an ion trap mass spectrometer. Ions observed in MS2 and MS3 product ion spectra, as well as those from the MS1 spectrum, were monitored. The method was validated with plasma samples fortified with diminazene diaceturate (4–100 ng/mL). Diminazene was confirmed in samples fortified with diminazene diaceturate at levels of 6.4 ng/mL or higher.
Keywords: Diminazene diaceturate; Bovine plasma; Mass spectrometry;

Artemia assays and protein phosphatase assays are commonly used for the screening of microcystins (MCs) in algal samples instead of the standard mouse toxicity assay. However, it has been shown that their results are often biased because of the matrix effects. To eliminate the possible interferences in the algal matrices, a new solid-phase extraction (SPE) method using silica gel as a sorbent was developed and evaluated. Results show that this SPE method could not only reduce the toxicity of the Microcystis samples towards brine shrimp by 50–80% but also eliminate 90–100% of the endogenous phosphatase activity from Spirulina and Chlorella samples, thus improving the determination of microcystins in algal samples using either of the two bioanalytical methods. The application of this SPE method as an off-line cleanup for high-performance liquid chromatography (HPLC) with UV detection is also described in this study. After SPE, the HPLC chromatograms of Microcystis samples have clear baselines that have no interferences with the analyte peaks.
Keywords: Solid-phase extraction (SPE); Microcystis; Microcystin (MC); Artemia assay; Protein phosphatase assay; HPLC; Mouse Bioassay; Algal dietary supplement; Cyanobacteria;

Molecularly imprinted solid-phase extraction for rapid screening of mycophenolic acid in human plasma by Junfa Yin; Sumin Wang; Guanqun Yang; Gengliang Yang; Yi Chen (142-147).
A molecularly imprinted solid-phase extraction coupled with high performance liquid chromatography (MISPE–HPLC) method was developed for rapid screening of mycophenolic acid (MPA) in human plasma. MPA imprinted polymers (MPA-MIP) were synthesized and then tested for their performance both in organic and in aqueous solution. MPA was selectively trapped and preconcentrated on the MPA-MIP sorbent using different loading and washing conditions. The good selectivity of MPA-MIP enabled further clean-up of the interfering components in human plasma. For the proposed MISPE–HPLC method, the linearity between responses (peak area) and concentration was found over the range of 1–100 μg/ml with a linear regression coefficient (R 2) of 0.9989. The limit of detection (LOD) and theoretical limit of quantification (LOQ) for MPA in plasma were 0.10 and 0.32 μg/ml, respectively. The precisions were 7.3, 3.5 and 4.7% RSD for intra-day assay and 9.2, 4.1 and 5.5% RSD for inter-day reproducibility, respectively, at three concentration levels of MPA in spiked plasma (1, 10 and 100 μg/ml). Both recoveries for the extraction (more than 74%) and for the analytical method (more than 87%) were acceptable for screening MPA in plasma samples. Twelve-hour pharmacokinetic profile of MPA for a renal transplant recipient receiving chronic oral dosing of 500 mg mycophenolate mofetil (MMF) was investigated. Results indicated that this method could be applied for therapeutic drug monitoring of mycophenolic acid in patient plasma.
Keywords: Mycophenolic acid; Molecularly imprinted polymer; Molecularly imprinted solid-phase extraction;

A simple and rapid determination of biapenem in plasma by high-performance liquid chromatography by Kayo Ikeda; Kazuro Ikawa; Aki Ikeda; Yoshimi Nishikawa; Norifumi Morikawa (148-152).
A simple, rapid and precise HPLC method using ultrafiltration to remove plasma protein was developed to determine biapenem concentrations in human plasma. Plasma was separated by centrifugation at 4 °C from blood collected in heparinized vacuum tubes, and biapenem was stabilized by immediate mixing the plasma with 1 M 3-morpholinopropanesulfonic acid (MOPS) buffer (pH 7.0) (1:1). Biapenem was detected by ultraviolet absorbance at 300 nm with no interfering plasma peak. The calibration curve of biapenem in human plasma was linear from 0.04 to 50 μg/mL. The limit of detection was 0.01 μg/mL, which was more than 40-fold lower than that of conventional plasma protein precipitation using ammonium sulfate. The assay has been clinically applied to pharmacokinetic studies in patients.
Keywords: Biapenem; Ultrafiltration;

A quantitative method for the determination of total tiopronin (TP) in human plasma was developed by liquid chromatography with electrospray ionisation (ESI) mass spectrometric detection. After reduction with tris (2-carboxy-ethyl) phosphine (TCEP) and derivatization with methyl acrylate (MA) for the thiol group of TP, plasma samples were processed successively by deproteinization and solid phase extraction. N-acetyl-l-cysteine (NAC) was selected as internal standard undergoing the same treatment as TP. The method was validated that it could meet the need of biological analysis. The lower limit of quantitation (LLOQ) of TP in plasma was 0.02 μg/mL. Finally, the method was successfully applied to a pharmacokinetic study in 20 healthy Chinese male volunteers after an oral dose of 200 mg TP tablets.
Keywords: Tiopronin; LC–ESI–MS; Methyl acrylate; Tris (2-carboxy-ethyl) phosphine; Plasma; Pharmacokinetics;

Characterization of proteinase activation dynamics by capillary electrophoresis conjugating with fluorescent protein-based probe by Shixia Zhou; Juqiang Lin; Wei Du; Zhihong Zhang; Qingming Luo; Bi-Feng Liu; Yiqun Dai (158-162).
In this paper, a novel strategy was reported to characterize dynamics of proteinase activation based on capillary electrophoresis (CE), using caspase-2 as the model system. A fusion protein conjugating an amino acid sequence VDVAD with two fluorescent proteins enhanced cyan fluorescence protein (ECFP) and red fluorescence protein (DsRed), ECFP–VDVAD–DsRed, was specially designed and expressed in HeLa cells as the substrate of proteinase caspase-2. In this substrate, the sequence VDVAD could be specifically recognized and cleaved by caspase-2 as soon as its activation was initiated with treatment of a certain dose of cisplatin to HeLa cells, which led to a break of the substrate into two fragments. Analyses of the cell lysates using CE in a time course of the apoptosis illustrated the dynamics of caspase-2 activation. It showed that the employment of fusion fluorescent protein greatly facilitated both CE separation and identification of the analytes. This result from cell colony by CE was compared with that from single cell achieved by optical imaging.
Keywords: Capillary electrophoresis; Fluorescent protein; Protein activation; Dynamics; Caspase-2;

This paper describes a novel membrane chromatographic immunoassay technique suitable for rapid screening of antibodies in serum samples. This technique could potentially be utilized for antibody screening in situations where screening for exposure to one of several possible antigens is required. A synthetic microporous membrane is first selectively loaded with antibodies from the test serum sample by hydrophobic interaction. The in situ affinity membrane thus formed is sequentially pulsed with the antigens corresponding to the antibodies being screened. From the antigen chromatogram peak profile thus obtained, inferences about the antibodies present in the serum sample can then easily be made. This technique in addition to being rapid and direct is conceptually simple, and does not use any expensive media or reagents. It would potentially be useful for rapid diagnosis of infections as well as for rapid assessment of conditions such as envenomation or exposure to toxic substances.
Keywords: Immunoassay; Membrane; Antibody; Chromatography; Screening; Antigen; Hydrophobic interaction;

A simple method using liquid chromatography–linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1–3 ng/mL), recovery (89–101%), intraday precision (2.0–6.8%), interday precision (3.4–9.6%) and accuracy (101–103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 °C during a time-storage of a week lead to the conclusion that addition of sodium azide (10 mg/mL) is required for preservation of the analytes.
Keywords: Testosterone; Epitestosterone; Glucuronide; Sulfate; Liquid chromatography–mass spectrometry; Doping control;