Journal of Chromatography B (v.833, #2)

A sensitive, specific and reproducible fluorescence high performance liquid chromatography (HPLC) assay has been developed for the separate or simultaneous measurement of AQ-13 (a candidate 4-aminoquinoline antimalarial), chloroquine (CQ), and their metabolites in whole blood. After liquid–solid extraction using commercially available extraction cartridges, these two aminoquinolines (AQs) and their metabolites were separated on C18 (Xterra RP18) columns using a mobile phase containing 60% borate buffer (20 mM, pH 9.0) and 40% acetonitrile with isocratic elution at a flow-rate of 1.0 ml/min. The assay uses a biologically inactive 8-chloro-4-aminoquinoline (AQ-18) as its internal standard (IS). There is a linear relationship between the concentrations of these AQs and the peak area ratio (ratio between the peak area of the AQ or metabolite and the peak area of the IS) on the chromatogram. Linear calibration curves with correlation coefficients ≥0.997 (r 2  ≥ 0.995, p  < 0.001) were obtained for AQ-13, CQ and their N-dealkylated metabolites. Reproducibility of the assay was excellent with coefficients of variation (CVs) ≤3.8% for AQ-13 and its metabolites, and ≤2.5% for CQ and its metabolites. The sensitivity of the assay is 5 nM using 1.0 ml of blood and a 20 μl injection volume, and can be increased by using 5.0 ml of blood with an injection volume of 40 μl.
Keywords: Chloroquine; Aminoquinoline; Fluorescence HPLC; AQ-13; Malaria;

The capillary electrophoresis (CE) system with optical fiber light-emitting diode (optical fiber LED) induced fluorescence detector was developed for the analysis of the excitatory amino acids (EAAs) tagged with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of EAAs was carried out in an uncoated fused-silica capillary (50 cm × 75 μm i.d.) with a buffer of 10 mM borate at pH 9.3 and an applied voltage of 20 kV. High sensitivity was obtained by the use of optical fiber LED induced fluorescence detector with a violet LED as the excitation light source. The limits of detection (S/N = 3) for glutamic acid (Glu) and aspartic acid (Asp) were 2.1 × 10−8 and 2.3 × 10−8  M, respectively. The detection approach was successfully applied to the analysis of Glu and Asp in biological fluids including human serum, rabbit serum and human cerebrospinal fluid (CSF) with satisfactory results.
Keywords: Capillary electrophoresis; Optical fiber; Light-emitting diode; Excitatory amino acids; Biological fluids;

Aqueous two-phase systems extraction of α-toxin from Clostridium perfringens type A by Maria Taciana Holanda Cavalcanti; Tatiana Souza Porto; Benicio de Barros Neto; José Luiz Lima-Filho; Ana Lúcia Figueiredo Porto; Adalberto Pessoa (135-140).
Two sequential half-fraction designs were applied to studying the α-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an α-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.
Keywords: Aqueous two-phase system; PEG/phosphate; α-Toxin; Fractional design; Purification;

Simultaneous quantitation of 7-methyl- and O6-methylguanine adducts in DNA by liquid chromatography–positive electrospray tandem mass spectrometry by Fagen Zhang; Michael J. Bartels; Lynn H. Pottenger; B. Bhaskar Gollapudi; Melissa R. Schisler (141-148).
A methodology has been developed and validated for the simultaneous quantitation of O6-methyl- and 7-methylguanine in DNA isolated from in vitro exposure to the model alkylating agents: N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS). After exposure, DNA was isolated and directly hydrolyzed under acid conditions to hydrolytes containing DNA bases (modified and unmodified). The hydrolytes were used for direct O6- and 7-methylguanine quantitation using a rapid and selective liquid chromatography–electrospray tandem mass spectrometry (LC/ESI-MS/MS). The lower limits of quantitation for O6-methyl- and 7-methylguanine were 75.8 and 151.5 fmol, respectively. Linearity of the calibration curve was greater than 0.999 from 75.8 to 151,600.0 fmol for O6-methylguanine and 0.999 from 151.5 to 303,200.0 fmol for 7-methylguanine. The intra-day assay precision relative standard deviation (R.S.D.) values for O6-methylguanine for quality control (QC) samples were ≤9.2% with accuracy values ranging from 90.8 to 118%, and for 7-methylguanine the R.S.D. values for QC samples were ≤11%, with accuracy values ranging from 92.9 to 119%. The inter-day assay precision (R.S.D.) values for O6-methylguanine QC samples were ≤7.9% with accuracy values ranging from 94.5 to 116%, and for 7-methylguanine QC samples were ≤7.1% with accuracy values ranging from 95.2 to 110.2%. This method was used for simultaneous determination of the levels of 7-methyl- and O6-methylguanine in DNA acidic hydrolytes present in a series of incubations from salmon testis DNA treated with either MNU or MMS.
Keywords: Quantitation; 7-Methyl- and O6-methylguanine adducts; Liquid chromatography–tandem mass spectrometry;

Identification of bilirubin reduction products formed by Clostridium perfringens isolated from human neonatal fecal flora by Libor Vítek; Filip Majer; Lucie Muchová; Jaroslav Zelenka; Alena Jirásková; Pavel Branný; Jiří Malina; Karel Ubik (149-157).
Urobilinoids belong to the heterogenous group of degradation products of bilirubin formed in the gastrointestinal tract by intestinal microflora. Among them urobilinogen and stercobilinogen with their respective oxidation products, urobilin and stercobilin, are the most important compounds. The aim of present study was to analyze the products of bacterial reduction of bilirubin in more detail. The strain of Clostridium perfringens isolated from neonatal stools, capable of reducing bilirubin, was used in the study. Bacteria were incubated under anaerobic conditions with various native as well as synthetic bile pigments, including radiolabeled unconjugated bilirubin (UCB). Their reduction products were extracted from media and separated following thin layer chromatography. Pigments isolated were analyzed by spectrophotometry, spectrofluorometry and mass spectrometry. In a special set of experiments, bilirubin diglucuronide was incubated with either bacterial lysate or partially purified bilirubin reductase and β-glucuronidase to reveal whether bilirubin glucuronides may be directly reduced onto conjugated urobilinoids. A broad substrate activity was detected in the investigated strain of C. perfringens and a series of bilirubin reduction products was identified. These products were separated in the form of their respective chromogens and further oxidized. Based on their physical–chemical properties, as well as mass spectra, end-catabolic bilirubin products were identified to belong to urobilinogen species. The reduction process, catalyzed enzymatically by the studied bacterial strain, does not proceed to stercobilinogen. Bilirubin diglucuronide is not reduced onto urobilinoid conjugates, glucuronide hydrolysis must precede double bond reduction and thus UCB is reduced much faster.
Keywords: Bilirubin; Bacterial reduction; Intestinal microflora; Urobilinoids; Bile pigments;

Interaction study between double-stranded DNA and berberine using capillary zone electrophoresis by Juan-Fang Wu; Ling-Xin Chen; Guo-An Luo; Yong-Suo Liu; Yi-Ming Wang; Zhi-Hong Jiang (158-164).
Two non-self-complementary 17-mer double-stranded DNA (dsDNA) with four different central base pairs were designed to systematically investigate the binding affinity and sequence specificity of berberine with dsDNA by capillary zone electrophoresis (CZE). The data analysis with the Kenndler model proved only low affinity between dsDNA and berberine and suggested some weak binding preference of berberine for AATT-containing to GGCC-containing dsDNA. The binding constant, K a, between berberine and dsDNAAB was about (1.0 ± 0.7) × 103  M−1. In addition, the separation of single-stranded DNA (ssDNA) from dsDNA under simple electrophoretic conditions enabled CZE to be a potentially alternative tool to check the extent of DNA annealing, which is usually done by the time-consuming and labor-intensive slab electrophoresis.
Keywords: Berberine; DNA; Capillary zone electrophoresis; Interaction; DNA annealing;

High throughput metabolic stability screen for lead optimization in drug discovery by Xinchun S. Tong; Suoyu Xu; Song Zheng; James V. Pivnichny; Jesus Martin; Claude Dufresne (165-173).
A high throughput approach for the determination of in vitro metabolic stability and metabolic profiles of drug candidates has been developed. This approach comprises the combination of a Biomek FX liquid handling system with 96-channel pipetting capability and a custom-designed 96-well format on-line incubator with efficient thermal conductivity. This combination facilitates automated reagent preparation, sample incubation, and sample purification for microsome stability studies. The overall process is both fast and accurate and meets the challenges of high throughput screening for drug discovery. A custom designed, user-friendly computer program has been incorporated for large-scale data processing and report generation. Several applications are discussed that implement this strategy for rapid selection of compounds in early drug discovery.
Keywords: High throughput; Metabolic stability; Automation;

We investigated the complex formation between Cu(II) and human serum albumin (HSA) through a biuret reaction by use of capillary electrophoretic system incorporating an ultra-violet absorption (UV) and chemiluminescence (CL) dual detector. Cu(II)–tartrate complex and Cu(II)–human serum albumin complex were detected by UV detection (282 nm) with on-capillary, followed by CL detection (luminol–hydrogen peroxide CL reaction) with end-capillary. We examined the effects of the reaction time and temperature on the UV and CL responses. On the basis of the obtained results we considered the Cu(II)–human serum albumin complex formation processes and its catalytic activity for the CL reaction. The system easily, rapidly, and simultaneously produced useful information concerning the complex formation of Cu(II) and human serum albumin due to the presence of the both detectors.
Keywords: Capillary electrophoresis; Chemiluminescence; Biuret reaction; Dual detector; Cu(II)–human serum protein complex;

A rapid LC–MS method for determination of plasma anion profiles of acidotic patients by William McKinnon; Gwyn A. Lord; Lui G. Forni; Jean-Marie R. Peron; Philip J. Hilton (179-185).
In metabolic acidosis, the concentrations of anions associated with intermediary metabolism are increased and can make a significant contribution to the observed acidosis. Here we describe a method for the rapid determination of the plasma ultrafiltrate profile of these anions using liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The ultrafiltrate from patients with acidosis resulting from various causes were examined and the results compared to control values. Using the LC/ESI-MS method described, a unique plasma ultrafiltrate anion profile was obtained for each of the groups studied that provides rapid diagnosis of the type of underlying acidosis.
Keywords: Acidosis; Patients; Plasma; Ultrafiltrate; Krebs cycle; Anion gap; Intermediatry metabolism; Liquid chromatography; Mass spectrometry;

A simple and reliable capillary electrophoresis (CE) method with UV–vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 μg/ml was shown to be linear, and the limit of detection was 0.15 μg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.
Keywords: Capillary electrophoresis; Enantiomer separation; Vigabatrin; Dehydroabietylisothiocyante;

An isocratic liquid chromatographic method with pulsed electrochemical detection is described for the determination of neomycin in the presence of its impurities. The mobile phase is composed of an aqueous solution containing 35 g/l of sodium sulphate, 1 g/l of sodium 1-octanesulfonate, 14 ml/l of tetrahydrofuran (THF) and 50 ml/l of 0.2 M phosphate buffer pH 3.0. Sodium hydroxide was added post column to enhance the detection. An investigation of different reversed-phase columns indicated that the Discovery (C18 5 μm, 250 mm × 4.6 mm I.D.) column was the most suitable. The proposed method shows high efficiency, allowing the separation of the main component neomycin B from neomycin C and 15 other impurities. A central composite design was used to assess the robustness of the method. The method showed good selectivity, repeatability, linearity and sensitivity. This method was applied to analyse commercial samples.
Keywords: Pulsed electrochemical detection; Neomycin sulphate; Purity testing;

A simple and rapid HPLC method for quantitation of interferon-α2b in dosage forms and delivery systems by Abdolhossein Zarrin; Mahshid Foroozesh; Mehrdad Hamidi; Soleyman Mohammadi-Samani (199-203).
Development of reliable assay methods for quantitation of interferons in dosage forms has encountered serious limitations because of the physicochemical nature of these proteins as well as the sensitivity/selectivity issues. A rapid, available, and easy-to-use reversed-phase HPLC method has been developed for quantitative analysis of interferon-α2b in pharmaceuticals. The reversed- phase method was based on a gradient system using a wide-pore C4 column and produced linear response in drug concentration range of 0.25–5 MIU (r  = 0.9997). The average within-run and between-run variables of the method were 4.19 and 9.40%, respectively, with corresponding average accuracies of 99.48 ± 4.11 and 102.83 ± 9.51%. The limits of detection (LOD) and quantitation (LOQ) of the method were 0.125 and 0.25 MIU/ml, respectively. The practical applicability of the method was proven throughout a post-marketing quality control program on interferon-α2b products (PD-feron®) produced and marketed in Iran.
Keywords: Interferon-α2b; HPLC; Gradient HPLC; protein assay;

Quantification of Gluten Exorphin A5 in cerebrospinal fluid by liquid chromatography–mass spectrometry by Giuseppe Fanciulli; Emanuela Azara; Troy D. Wood; Alessandra Dettori; Giuseppe Delitala; Mauro Marchetti (204-209).
In the present work, for the first time, a method for the quantification of the alimentary opioid peptide Gluten Exorphin A5 (GE-A5; Gly-Tyr-Tyr-Pro-Thr) in cerebrospinal fluid (CSF) was developed. Aliquots (5 μL) of CSF were injected into a liquid chromatography–mass spectrometry (LC–MS) instrument equipped with a reversed-phase C18 column at a flow-rate of 0.4 mL/min. The mobile phase consisted of Eluent A water with 0.6% acetic acid as an ion-pairing reagent and Eluent B acetonitrile/methanol (75:25, v/v). The LC–MS system was programmed to divert column flow to waste for 4 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. No significant interfering peaks were detected at the retention times of GE-A5 in CSF blanks. The lower limit of detection and the lower limit of quantitation values for GE-A5 in CSF were established at 0.60 and 1.50 ng/mL, respectively. The intra- and inter-day precision values were <5% relative standard deviation. The intra- and inter-day accuracy were 99.6–102.8% and 100.0–101.9%, respectively. The reported assay employs extremely small volumes of CSF, thus allowing the analysis of GE-A5 from both small and large animal models.
Keywords: Gluten Exorphin A5; Cerebrospinal fluid; Liquid chromatography–mass spectrometry;

Simultaneous quantification of methamphetamine, cocaine, codeine, and metabolites in skin by positive chemical ionization gas chromatography–mass spectrometry by Wonkyung Yang; Allan J. Barnes; Mary G. Ripple; David R. Fowler; Edward J. Cone; Eric T. Moolchan; Heesun Chung; Marilyn A. Huestis (210-218).
A positive chemical ionization gas chromatography–mass spectrometric method was validated to simultaneously quantify drugs and metabolites in skin collected after controlled administration of methamphetamine, cocaine, and codeine. Calibration curves (2.5–100 ng/skin biopsy) for methamphetamine, amphetamine, cocaine, norcocaine, benzoylecgonine, cocaethylene, norcocaethylene, anhydroecgonine methyl ester, morphine, codeine, and 6-acetylmorphine (5–100 ng/skin biopsy for ecgonine methyl ester and ecgonine ethyl ester) exhibited correlation coefficients >0.999 and concentrations ±20% of target. Intra- and inter-run precisions were <10%. This procedure should be useful for postmortem analysis; data are included on drug concentrations in skin after controlled drug administration.
Keywords: Skin; Tissue; Biopsy; Methamphetamine; Cocaine; Codeine;

Glycerophosphocholines (GPCho's) are known to cause liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) matrix ionization effects during the analysis of biological samples (i.e. blood, plasma). We have developed a convenient new method, which we refer to as “in-source multiple reaction monitoring” (IS-MRM), for detecting GPCho's during LC–MS/MS method development. The approach uses high energy in-source collisionally induced dissociation (CID) to yield trimethylammonium-ethyl phosphate ions (m/z 184), which are formed from mono- and disubstituted GPCho's. The resulting ion is selected by the first quadrupole (Q1), passed through the collision cell (Q2) in the presence of collision gas at low energy to minimize fragmentation, and m/z 184 selected by the third quadrupole. This approach can be combined with standard multiple reaction monitoring (MRM) transitions with little compromise in sensitivity during method development and sample analysis. Hence, this approach was used to probe ionization matrix effects in plasma samples. The resulting information was employed to develop LC–MS/MS analyses for drugs and their metabolites with cycle times less than 5 min.
Keywords: LC–MS/MS; Plasma; Phospholipids; Lecithin; Glycerophosphocholines; Matrix suppression; Drug discovery; Pharmaceutical analysis; Electrospray mass spectrometry; Matrix effects;

Rutin deca(H-) sulfate sodium (RDS) is one of the most important drug candidates, which possesses very good activity as inhibitor of the complement system of warm-blooded animals and human immunodeficiency virus (HIV). In order to understand RDS metabolism and disposition, an ion-pairing coupled with solid-phase extraction technique (IP-SPE) was developed to extract RDS from rat plasma sample. Tetrabutyl ammonium bromide (TBAB) buffer (0.2 M, pH 8.0) was used as the ion-pairing extraction reagent and LC-18 was used as SPE sorbent. In addition, an ion-pairing HPLC method was established for the specific determination of RDS. A reversed phase C8 column was used for the separation of RDS and nitrendipine (internal standard). The mobile phase was composed of 10 mM phosphate buffer solution containing 25 mM TBAB–acetonitrile (52:48, v/v, pH 7.5). The calibration curve was linear from 0.3 to 30 nmol/mL. The analytical recovery from rat plasma was found to be 97.9 ± 4.1% (n  = 15). LOD and LOQ for RDS in plasma were calculated to be 0.12 nmol/mL and 0.30 ± 0.024 nmol/mL (R.S.D. = 8.2%, n  = 5), respectively. The intra- and inter-day precision was less than 9.2%. The assay was applied to a preliminary pharmacokinetic study in three male rats after those received a single intravenous bolus via caudal vein of 12 μmol/kg RDS.
Keywords: Rutin deca(H-) sulfate sodium; Ion-pairing solid-phase extraction; Ion-pairing chromatography; Plasma;

Preparation and characterization of magnetic microspheres for the purification of interferon α-2b by Yu Cao; Gang Bai; Jiaqi Chen; Wang Tian; Shenqi Wang; Wenbo Yang (236-244).
Magnetic agarose microspheres (MAMS), magnetic cellulose microspheres (MCMS), and magnetic poly(vinyl alcohol) microspheres (MPVAMS) were prepared by various different preparation methods. MCMS coupled with anti-IFN α-2b monoclonal antibodies (mAb) were selected for the purification of interferon α-2b (IFN α-2b) after performance characterization among microspheres. Parameters of immunomagnetic separation (IMS), including binding mAb, elution behavior, and sample pretreatment conditions, were optimized to improve the purification efficiency of the separation of IFN α-2b by MCMS. Size-exclusion HPLC (HPSEC) showed that the IFN α-2b was purified from crude cell lysate had an overall purity of 92.9%, while immunological and biological assays showed an activity recovery of 88.5% and specific antiviral activity of 2.7 × 108  IU/mg. Identity and molecular mass of purified IFN α-2b were confirmed by western blot and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. This study illustrated the favorable separation media which combined desired properties for the development of magnetic separation of biological materials.
Keywords: Agarose microspheres; Cellulose microspheres; PVA microspheres; Immunomagnetic separation; Interferon α-2b;

Altrenogest (17α-allyl-17β-hydroxyestra-4,9,11-trien-3-one) is a steroid used for the control of estrus in horses. This drug can potentially be abused in racehorses as the occurrence of estrus can alter their performance. This work describes an analytical method based on liquid chromatography–tandem mass spectrometry for the detection of altrenogest in horse urine down to a concentration of 13 pg/mL (0.042 nM). Furthermore, the qualitative aspect of metabolism of altrenogest in the horse has been studied. The main transformations that were found for this species were conjugation with glucuronic acid and sulfate. These phase II metabolites were identified by molecular mass and by comparison of their collision-induced dissociation product ion spectra with that of the synthetic aglycone at positive and negative potential, respectively. No phase I metabolites were discovered. In order to increase the ionisation in positive electrospray, a derivatisation procedure forming a basic oxime was tested. This process significantly increased the detection sensitivity for altrenogest glucuronide in horse urine.
Keywords: Altrenogest; Horse urine; LC–MS/MS; Derivatisation; Metabolism; Glucuronide; Sulfate; Conjugate; Doping control;

Determination of AKF-PD in whole blood of rat by HPLC-UV by Wei Cao; Lingyun He; Wenfang Liu; Zhizhuang Huang; Zeneng Cheng (257-259).
An HPLC-UV method was developed and validated for the determination of AKF-PD in whole blood of rat. Phenacetin was chosen as the internal standard, and the separation was achieved on a C18 column with methanol and 0.02 M phosphate buffer (pH 3.2) as mobile phase. The obtained calibration graphs were linear (r  = 0.9999, n  = 9) in the range of 0.203–52.0 μg ml−1. The low limit of quantitation was 0.203 μg ml−1. This method can be used to study the pharmacokinetics of AKF-PD in rat.
Keywords: AKF-PD; HPLC; Pharmacokinetics;

A method has been developed to determine the four water-soluble components—Danshensu (I), protocatechuic acid (II), protocatechuic aldehyde (III) and salvianolic acid B (IV) in Chinese medicine plant Salvia miltiorrhiza Bunge using high-performance liquid chromatography with a coulometric electrode array detection (HPLC–CEAD) system. Heat reflux extraction was used to pretreat the sample. This analysis was carried on a column of Hypersil C18 (250 mm × 4.6 mm, 5 μm) with a mobile phase of sodium acetate (pH 2.5, 50 mM) and acetonitrile in gradient mode. An ESA electrochemical detector monitored the four compounds. Potentials of four electrodes in series were set at 100, 150, 200 and 250 mV, respectively. Optimization of the pH of mobile phase and the proportion of acetonitrile were also performed. Calibration curve showed good linearity with correlation coefficients (r) more than 0.9937. Average recoveries of the four compounds were more than 92% and relative standard deviations were less than 6.6%. This method appeared to be stable, sensitive and reproducible for determination of the four water-soluble compounds in Chinese medicine plant S. miltiorrhiza Bunge.
Keywords: HPLC; Electrochemical detector; Chinese medicine plant; Salvia miltiorrhiza Bunge;

Author Index (265-267).

Keyword Index (268-272).