Journal of Chromatography B (v.850, #1-2)
Editorial Board (iii).
Simultaneous enantiomer determination of 20 (R)- and 20 (S)-ginsenoside-Rg2 in rat plasma after intravenous administration using HPLC method by Fang-Jin Gui; Xiu-Wei Yang; Long-Yun Li; Jian-Ming Tian (1-6).
20 (R,S)-Ginsenoside-Rg2, an anti-shock agent, is prescribed as a racemate. To analyze simultaneously the enantiomers of 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2 in plasma, a simple and reproducible high-performance liquid chromatographic (HPLC) method has been developed. The enantiomeric separation and determination were successfully achieved using a Diamonsil™ ODS C18 reversed-phase column (5 μm, 250 mm × 4.6 mm) with an RP18 (5 μm) guard column and a mobile phase of MeOH-aq. 4% H3PO4 (65:35, v/v, pH 5.1) with UV detection at 203 nm. Both enantiomers, 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2, were well separated at 14.5 min and 13.6 min, respectively. The linear ranges of the standard curves were 2.0–250 μg/ml. The intra- and inter-day precision (R.S.D.) were ≤1.59% and ≤0.54% and the mean extraction recoveries were 95.8% and 96.5% for 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2 in rat plasma, respectively. The limits of detection and quantification were 2.0 μg/ml and 7.8 μg/ml (S/N = 3:1) for 20 (R)-ginsenoside-Rg2, and 2.0 μg/ml and 3.9 μg/ml (S/N = 3:1) for 20 (S)-ginsenoside-Rg2, respectively. This validated method was applicable to pharmacokinetic studies in rat plasma after intravenous administration of 20 (R,S)-ginsenoside-Rg2. A pharmacokinetic study in rat plasma indicated that the enantiomers were rapidly absorbed and eliminated. These assay results are necessary for the evaluation of the metabolism of ginsenoside-Rg2 in vivo.
Keywords: 20 (R,S)-Ginsenoside-Rg2; 20 (R)-Ginsenoside-Rg2; 20 (S)-Ginsenoside-Rg2; Enantiomeric separation; Pharmacokinetics;
Application of HPLC to study the kinetics of a branched bi-enzyme system consisting of hypoxanthine-guanine phosphoribosyltransferase and xanthine oxidase—an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine in ALL cell line by Sukirti Kalra; Manash K. Paul; Hemalatha Balaram; Anup Kumar Mukhopadhyay (7-14).
The thiopurine antimetabolite 6-mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). 6MP is mainly catabolized by both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine oxidase (XOD) to form thioinosinic monophosphate (TIMP) (therapeutically active metabolite) and 6-thiouric acid (6TUA) (inactive metabolite), respectively. The activity of both the enzymes varies among ALL patients governing the active and the inactive metabolite profile within the immature lymphocytes. Therefore, an attempt was made to study the kinetic nature of the branched bi-enzyme system acting on 6MP and to quantitate TIMP and 6TUA formed when the two enzymes are present in equal and variable ratios. The quantification of the branched kinetics using spectrophotometric method presents problem due to the closely apposed λ max of the substrates and products. Hence, employing an HPLC method, the quantification of the products was done with the progress of time. The limit of quantification (LOQ) of substrate was found to be 10 nM and for products as 50 nM. The limit of detection (LOD) was found to be 1 nM for the substrate and the products. The method exhibited linearity in the range of 0.01–100 μM for 6MP and 0.05–100 μM for both 6TUA and TIMP. The amount of TIMP formed was higher than that of 6TUA in the bi-enzyme system when both the enzymes were present in equivalent enzymatic ratio. It was further found that enzymatic ratios play an important role in determining the amounts of TIMP and 6TUA. This method was further validated using actively growing T-ALL cell line (Jurkat) to study the branched kinetics, wherein it was observed that treatment of 50 μM 6MP led to the generation of 12 μM TIMP and 0.8 μM 6TUA in 6 h at 37 °C.
Keywords: Reverse-phase HPLC; Branched bi-enzyme system; Acute lymphoblastic leukemia; 6-Mercaptopurine; 6-Thiouric acid; Thioinosine monophosphate;
Liquid chromatography–electrospray tandem mass spectrometric method for quantification of monensin in plasma and edible tissues of chicken used in pharmacokinetic studies: Applying a total error approach by Estelle Chéneau; Jérôme Henri; Yvette Pirotais; Jean-Pierre Abjean; Brigitte Roudaut; Pascal Sanders; Michel Laurentie (15-23).
A liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for use in pharmacokinetic studies in order to determine the concentrations of monensin in plasma and edible tissues of chicken. Two sample preparations were performed, one for determining monensin concentrations in plasma using acetonitrile for protein precipitation and another one for determining monensin concentrations in muscle, liver, and fat using methanol–water followed by a clean up on a solid-phase extraction cartridge. Sample extracts were injected into the LC–MS/MS system, and a gradient elution was performed on a C18 column. Narasin was used as internal standard. The LC–MS/MS method was validated using an approach based on accuracy profiles, and applicability of the method was demonstrated for the determination of monensin in chicken plasma, muscle, liver, and fat in a pharmacokinetic study.
Keywords: Coccidiostats; Monensin; Polyether ionophores; Chicken; Plasma; Muscle; Liver; Fat; Validation; Pharmacokinetics; Total error;
Molecularly imprinted polymer microspheres for solid-phase extraction of chloramphenicol residues in foods by Xizhi Shi; Aibo Wu; Sulian Zheng; Rongxiu Li; Dabing Zhang (24-30).
Preparation of molecularly imprinted polymer microspheres (MIPMs) for chloramphenicol (CAP) by aqueous suspension polymerization is reported for the first time in this study. The resulting MIPMs had the ability to specifically adsorb CAP, and the molecularly imprinted solid phase extraction (MISPE) based on the MIPMs was shown to be applicable for clean-up and preconcentration of trace CAP in milk and shrimp samples with high recoveries of 92.7% and 84.9%, respectively. Combined with MISPE, the conventional HPLC-UV analysis sensitivity for CAP in foods could be significantly increased.
Keywords: Molecularly imprinted polymer microspheres; Chloramphenicol; Solid-phase extraction; Suspension polymerization; Foods;
Validation of high-performance liquid chromatography assay for quantification of formoterol in urine samples after inhalation using UV detection technique by D.K. Nadarassan; H. Chrystyn; B.J. Clark; K.H. Assi (31-37).
A novel high-performance liquid chromatography (HPLC) assay for the estimation of formoterol in urine samples was developed and validated. A solid phase extraction (SPE) using Oasis HLB was optimised to isolate formoterol from a urine matrix followed by HPLC with UV detection. This extraction procedure concentrated the final analyte forty times so that UV detection can be used to determine even a low concentration of formoterol in urine samples. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The samples were injected onto a C18 Spherisorb® (250 mm × 4.6 mm × 5 μm) analytical column maintained at 30 °C. The mobile phase consisted of 5 mM of potassium dihydrogen orthophosphate buffer (adjusted to pH 3 with ortho phosphoric acid):acetonitrile (ACN) (70:30, v/v), and the formoterol peak was detected at wavelength 214 nm. The extraction recovery of formoterol from the urine sample was >95%. The calibration curve was linear (r 2 = 0.99) over formoterol concentrations ranging from 1.5 to 25 ng/mL (n = 6). The method had an accuracy of >92% and intra and inter-day precision CV% of <3.9% and <2.2%, respectively, at three different concentrations low, medium and high (10, 15, 20 ng/mL). The limit of quantification (LOQ) for formoterol was found to be 1.50 ng/mL. The accuracy and precision at the LOQ level were 95% and %CV <3.7% (n = 10), respectively. The method reported is simple, reliable, precise, and accurate and has the capacity to be used for determination of formoterol in urine samples.
Keywords: Formoterol; HPLC; Assay; Validation;
Combining poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and on-line pre-concentration-capillary electrophoresis for analysis of ephedrine and pseudoephedrine in human plasma and urine by Fang Wei; Min Zhang; Yu-Qi Feng (38-44).
A method based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) and field-enhanced sample injection (FESI) pre-concentration technique was proposed for sensitive capillary electrophoresis-ultraviolet (CE-UV) analysis of ephedrine (E) and pseudoephedrine (PE) in human plasma and urine. The PMME device consisted of a regular plastic syringe (1 mL), a poly (MAA-EGDMA) monolithic capillary (2 cm × 530 μm I.D.) and a plastic pinhead connecting the former two components seamlessly. The extraction was achieved by driving the sample solution through the monolithic capillary tube using a syringe pump, for the desorption step, an aliquot of organic solvent, which normally provided an excellent medium to ensure direct compatibility for FESI in CE, was injected via the monolithic capillary and collected into a vial for subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1 M phosphate electrolyte (pH 2.5) and 10% acetonitrile (v/v). The combination of both pre-concentration procedures allowed the detection limits of the analytes down to 5.3 ng/mL and 8.0 ng/mL in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range 50–5000 ng/mL in plasma and urine sample. Plasma and urine samples from volunteers receiving pseudoephedrine have also been successfully analysed.
Keywords: Poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction; Capillary electrophoresis; On-line concentration; Ephedrine; Pseudoephedrine;
Simultaneous determination of abacavir and zidovudine from rat tissues using HPLC with ultraviolet detection by Summer R. Lewis; Catherine A. White; Michael G. Bartlett (45-52).
A simple high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of abacavir and zidovudine (AZT) in rat plasma, amniotic fluid, fetal, and placental tissues. Extraction of abacavir, AZT, and the internal standard, azidouridine (AZDU) in amniotic fluid was carried out by protein precipitation. Extraction from plasma, fetal and placental homogenates was achieved by using a salting out technique. Chromatographic separation was performed using a C8 column (150 mm × 4.6 mm, 5 μm). The mobile phase consisted of 12% acetonitrile in 25 mM sodium phosphate buffer (adjusted to pH 7 with sodium hydroxide) for the fetus, placenta, plasma and amniotic fluid samples at a flow rate of 0.8 mL/min. The method was validated over the range from 0.05 to 50 μg/mL for both abacavir and AZT in the four biological matrices. The absolute recovery of abacavir ranged from 79 to 94%, while AZT recoveries ranged from 79 to 90% in the different biological matrices. The internal standard recovery ranged from 90 to 92%. Acceptable intra- and inter-day assay precision (<10% R.S.D.) and accuracy (<10% error) were observed over 0.05–50 μg/mL for all four matrices.
Keywords: Abacavir; Zidovudine; Validation; Bioanalytical; HPLC;
One-step immunochromatographic separation and ELISA quantification of glycyrrhizin from traditional Chinese medicines by Jinsen Xu; Hiroyuki Tanaka; Yukihiro Shoyama (53-58).
The bioactive constituent, glycyrrhizin or glycyrrhizic acid (GA), was purified from two traditional Chinese medicines (TCM), Shaoyao gancao tang and Dahuang gancao tang, and from crude extracts from licorice roots by means of immunoaffinity chromatography using anti-GA monoclonal antibody (MAb) and was quantified with an enzyme-linked immunosorbent assay (ELISA). Laboratory preparations included the synthesis of conjugate GA-human serum albumin (GA-HSA), the production of anti-GA-MAb, the optimization of the immunoaffinity column packed with the anti-GA-MAb coupled to hydrazide gel and the determination of the GA content in TCM and crude drugs from five different sources by ELISA and high performance liquid chromatography (HPLC). The experimental results reveal that the anti-GA-MAb coupled to Affi-Gel Hz gel results in a coupling efficiency of 95.2%, and the immunoaffinity chromatography gives a mean recovery of 97.6% of GA with a capacity of 33.5 ± 2.40 μg/mL of immunoaffinity gel under the given conditions. The GA content of the crude extracts (ranging 74.8–114.6 μg/mg) from different sources by the ELISA method is much greater than that of the TCM (16.4–25.1 μg/mg) which is, in good agreement with the results of the HPLC method. Our report provides a rapid, reliable and sensitive approach for one-step separation and quantification of GA.
Keywords: One-step separation; Glycyrrhizin; Immunoaffinity chromatography; Monoclonal antibody; ELISA;
Determination of captopril in human plasma, using solid phase extraction and high-performance liquid chromatography, coupled to mass spectrometry: Application to bioequivalence study by Kênnia R. Rezende; Iram M. Mundim; Leonardo S. Teixeira; Weidson C. Souza; Douglas R. Ramos; Cláudio R.F. Cardoso; Inácia C. Souza; Mário Z. Gratão; Karini B. Bellório (59-67).
A specific high performance liquid chromatography–mass spectrometric (LC–MS/MS) assay was developed for the determination of captopryl in plasma. The retention time was 1.45 and 1.37 min for captopril and enalapril, respectively. The overall mean recovery, using SPE extraction with OASIS® HLB cartridges, was found to be 107.2 ± 9.5 and 100.04 ± 2%, respectively. Calibration curves were linear in the concentration range of 10.00–2000.00 ng/ml, and the lower limit of quantification (LLOQ) was 10.00 ng/ml. The LLOQ was sensitive enough for detecting terminal phase concentrations of the drug. Inter-batch precision of the method ranged from 0.88 to 1.95%. Intra-batch accuracy ranged from 97.15 to 105.77%, while intra-batch precision ranged from 2.49 to 5.66% at concentrations of 30.00, 760.00 and 1500.00 ng/ml. The developed method was applied to study bioequivalence of captopril in a group of 25 human subjects at a single oral dose of a 50 mg tablet.
Keywords: Captopril; LC–MS; SPE; Bioequivalence; Pharmacokinetics; Stability validation;
High performance liquid chromatography–electrospray ionization mass spectrometric determination of balofloxacin in human plasma and its pharmacokinetics by Zuwei Bian; Yuan Tian; Zunjian Zhang; Fengguo Xu; Jinheng Li; Xiaomei Cao (68-73).
A selective and sensitive high performance liquid chromatography–electrospray ionisation–mass spectrometry method has been developed for the determination of balofloxacin (BLFX) in human plasma. The sample preparation was a liquid–liquid extraction, and chromatographic separation was achieved with an Agilent ZORBAX 300SB C18 2.1 mm × 150 mm column using a mobile phase comprised of methanol–water (10 mM CH3COONH4, pH 3.0) = 40:60 (v/v). Standard curves were linear (r = 0.9992) over the concentration range of 0.03–3 μg/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation (R.S.D.). The limit of detection (LOD) was 0.02 μg/ml. The validated HPLC–electrospray ionization (ESI)–MS method has been used successfully to study balofloxacin pharmacokinetics in healthy volunteers.
Keywords: Balofloxacin; HPLC–ESI–MS; Human plasma; Pharmacokinetics;
Development of an in vitro incubation procedure for screening of CYP2D6 intrinsic clearance by Sarah E.G. Porter; Richard B. Keithley; Sarah C. Rutan (74-82).
The in vitro intrinsic clearances (CLint) for the metabolism of p-methoxymethamphetamine (PMMA) and fluoxetine by the CYP2D6 enzyme were calculated using a steady-state (SS) approach and a new general enzyme (GE) method, which measures the formation of product and the depletion of substrate as a function of time. For PMMA, the SS experiment resulted in a CLint of 2.7 ± 0.2 μL pmol 2D6−1 min−1 and the GE experiment resulted in a CLint of 3.0 ± 0.6 μL pmol 2D6−1 min−1. For fluoxetine, the SS experiment resulted in a CLint of 0.33 ± 0.17 μL pmol 2D6−1 min−1 and the GE experiment resulted in a CLint of 0.188 ± 0.013 μL pmol 2D6−1 min−1. We used two kinetic modeling techniques that can accommodate atypical kinetic models. We also show that the addition of fluoxetine results in a 10-fold decrease in the observed intrinsic clearance of PMMA, confirming that fluoxetine is a potent inhibitor of the liver enzyme CYP2D6.
Keywords: Drug metabolism; p-Methoxymethamphetamine; Fluoxetine; CYP2D6; Inhibition; Enzyme kinetics;
Quantification of organic eluates from polymerized resin-based dental restorative materials by use of GC/MS by Vibeke Barman Michelsen; Grete Moe; Rita Skålevik; Einar Jensen; Henning Lygre (83-91).
Residual monomers, additives and degradation products from resin-based dental restorative materials eluted into the oral cavity may influence the biocompatibility of these materials. Emphasis has been placed on studies addressing cytotoxic, genotoxic and estrogenic potential of these substances. A prerequisite for analyzing the potential of exposure to eluted compounds from dental materials is reliable quantification methods, both real time and accelerated measurements. The purpose of the present study was to quantify nine eluates; 2-hydroxyethyl methacrylate (HEMA), hydroquinone monomethyl ether (MEHQ), camphorquinone (CQ), butylated hydroxytoluene (BHT), ethyl 4-(dimethylamino)benzoate (DMABEE), triethylene glycoldimethacrylate (TEGDMA), trimethylolpropane trimethacrylate (TMPTMA), oxybenzone (HMBP) and drometrizole (TIN P) leaching from specimens of four commonly used resin-based dental materials in ethanol and an aqueous solution. All analyses were performed by use of GC/MS, each component was quantified separately and the results presented in μg mm−2. This study has shown that elution from various materials differs significantly, not only in the types of eluates, but also regarding amounts of total and of single components. A high amount of HMBP, a UV stabilizer with potential estrogenic activity, was detected from one material in both solutions.
Keywords: Quantification; Monomers; Gas chromatography/mass spectrometry; Resin-based dental composites; Eluates;
Pharmacokinetics and metabolism of 3,4-dichlorophenyl-propenoyl-sec.-butylamine in rats by high performance liquid chromatography–ion trap mass spectrometry by Shu-Mei Wang; Gui-Fang Dou; Qiang Li; Ting Liu; Zhi-Yun Meng; Ya-Qing Lou; Guo-Liang Zhang (92-100).
The pharmacokinetics (PK) and metabolism of 3,4-dichlorophenyl-propenoyl-sec.-butylamine (3,4-DCPB), a novel antiepileptic drug, were investigated after its oral administration to rats (100 mg/kg) by HPLC. The absorption and elimination of 3,4-DCPB were rapid. 3,4-DCPB was found to undergo extensive metabolism as the major route of elimination. Structures of the metabolites present in rat plasma were identified with liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS/MS). It was concluded that 3,4-DCPB was involved in the multiple metabolic pathways (hydrolysis, dealkylation and oxidation) and the hydrolysis product, 3,4-dichloro-cinnamic acid (M1) appeared to be the major metabolite.
Keywords: 3,4-Dichlorophenyl-propenoyl-sec.-butylamine; Liquid chromatography–electrospray ionization-mass spectrometry; Pharmacokinetics; Metabolite;
Development of a liquid chromatography/tandem mass spectrometry assay for the determination of bestatin in rat plasma and its application to a pharmacokinetic study by Chen Wang; Guorong Fan; Mei Lin; Yi Chen; Weiquan Zhao; Yutian Wu (101-108).
Bestatin is a low molecular weight aminopeptidase inhibitor originally isolated from culture filtrates of Streptomyces olivoreticuli. We have developed a sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the quantitative determination of bestatin in rat plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 50 μL plasma samples by solid phase extraction (SPE). Reverse-phase HPLC separation was accomplished on a Lichrospher C18 column (4.6 mm × 50 mm, 5 μm) with a mobile phase composed of methanol–water–formic acid (70:30:0.5, v/v/v) at a flow rate of 0.8 mL/min. The method had a chromatographic total run time of 3 min. A Varian 1200L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 309.2 → 120.0 (bestatin) and 313.4 → 138.0 (granisetron) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 5 ng/mL, with good linearity (r 2 ≥ 0.999) over the linear range of 5–2000 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of bestatin in rats.
Keywords: Bestatin; LC–MS/MS; Pharmacokinetics;
Simultaneous profiling of N-glycans and proteins from human serum using a parallel-column system directly coupled to mass spectrometry by Yun-Gon Kim; Kyoung-Soon Jang; Hwang-Soo Joo; Hyun-Ki Kim; Chang-Soo Lee; Byung-Gee Kim (109-119).
A method for the rapid identification of proteins and their N-glycans was developed through the use of two parallel columns directly connected to a mass spectrometer. Both porous graphitic carbon (PGC) and C18 capillary columns were connected in parallel with two switching valves for the simultaneous analysis of glycans and peptides, respectively. To verify the efficiency of the analytical system, profiling of N-glycans and proteins from human serum was demonstrated. This method is suitable for high-throughput analysis and automation, is contamination-free for the identification of N-glycans and proteins in a complex biological sample, and can be applied to glycomics and proteomics.
Keywords: Human serum; C18; Porous graphite carbon; Electrospray mass spectrometry;
A sensitive method for quantitation of β-lyase metabolites of sulfur mustard as 1,1′-sulfonylbis[2-(methylthio)ethane] (SBMTE) in human urine by isotope dilution liquid chromatography–positive ion-electrospray-tandem mass spectrometry by James D. Daly; Colleen M. O’Hehir; George M. Frame (120-127).
A method for measurement of an important biological marker, 1,1′-sulfonylbis[2-(methylthio)ethane] (SBMTE) of sulfur mustard agent HD [bis-(2-chloroethyl)sulfide] in human urine, to quantify HD exposure, is presented. It employs TiCl3 reduction of β-lyase metabolites to SBMTE, and automated solid-phase extraction sample preparation, followed by isotope dilution liquid chromatography–positive ion-electrospray ionization-tandem mass spectrometry with 7.5 min/sample cycle time, to achieve SBMTE quantitation of up to 200 samples/24 h a day. Percent relative standard deviations over the calibration range varied from 12.0% at 0.1 ng/mL to 0.9% at 100 ng/mL, and the limit of detection from a 0.5 mL sample was below the lowest level calibration standard of 0.1 ng/mL.
Keywords: Sulfur mustard; SBMTE; Metabolites; Urine; Liquid chromatography; Tandem mass spectrometry;
Determination of valproic acid in human serum and pharmaceutical preparations by headspace liquid-phase microextraction gas chromatography-flame ionization detection without prior derivatization by Parvin Shahdousti; Abdorreza Mohammadi; Naader Alizadeh (128-133).
An efficient and fast extraction technique for the enrichment of valproic acid from human blood serum samples using the headspace liquid phase microextraction (HS-LPME) combined with gas chromatography (GC) analysis has been developed. The extraction was conducted by suspending a 2 μL drop of organic solvent in a 1 mL serum sample; following 20 min of extraction, withdrawing organic solvent into a syringe and injection into a GC with a flame ionization detector (FID), without any further pre-treatment. Four organic solvents, 1-decanole, benzyl alcohol, 1-octanol and n-dodecane, were studied as extractants, and n-dodecane was found to be the most sensitive solvent for valproic acid. The results revealed that HS-LPME is suitable for the successful extraction of valproic acid from human blood serum samples. Parameters like extraction time, ionic strength, pH, organic solvent volume, and temperature of the sample were studied and optimized to obtain the best extraction results. An enrichment factor of 27-fold was achieved in 20 min. The procedure resulted in a relative standard deviation of <13.2% (n = 7) and a linear calibration range from 2 to 20 μg mL−1 (r > 0.98), and the limit of detection was 0.8 μg mL−1 in serum blank samples. Overall, LPME proved to be a fast, sensitive and simple tool for the preconcentration of valproic acid from real samples. The proposed method was also applied to the analysis of valproate in pharmaceutical preparations.
Keywords: Headspace liquid phase microextraction; Valproic acid; Gas chromatography; Human serum analysis; Pharmaceutical preparations;
Positive and negative electrospray LC–MS–MS methods for quantitation of the antiparasitic endectocide drugs, abamectin, doramectin, emamectin, eprinomectin, ivermectin, moxidectin and selamectin in milk by David A. Durden (134-146).
Avermectin endectocides are used for the treatment of cattle against a variety of nematode and arthropod parasites, and consequently may appear in milk after normal or off-label use. The compounds abamectin, doramectin, and ivermectin, contain only C, H and O and may be expected to be detected by LC–MS in negative ion mode. The others contain nitrogen in addition and would be expected to be preferentially ionized in positive mode. The use of positive ion and negative ion methods with electrospray LC–MS–MS were compared. Using negative ion the compounds abamectin, doramectin, ivermectin, emamectin, eprinomectin, and moxidectin gave a curvilinear response and were quantified in raw milk by LC–MS–MS with a triethylamine–acetonitrile buffer over the concentration range 1–60 ppb (μg/kg) using selamectin as the internal standard. The limits of detection (LOD) were between 0.19 ppb (doramectin) and 0.38 ppb (emamectin). The compounds gave maximum sensitivity with positive ionisation from a formic acid–ammonium formate–acetonitrile buffer and were detected in milk (LC–MS–MS) also with a curvilinear response over the range 0.5–60 ppb. Although the positive ion signals were larger, with somewhat lower limits of detection (LOD between 0.06 ppb (doramectin) and 0.32 ppb (moxidectin) the negative ion procedure gave a more linear response and more consistent results. Comparison of spiked samples in the range 2–50 ppb showed a high degree of correlation between the two methods.
Keywords: Avermectin; Endectocide; Milk; LC–MS–MS; Electrospray ionization; Liquid chromatography; Tandem mass spectrometry; Residue analysis; Abamectin; Doramectin; Emamectin; Eprinomectin; Ivermectin; Moxidectin; Selamectin;
Simultaneous determination of 6 beta-blockers, 3 calcium-channel antagonists, 4 angiotensin-II antagonists and 1 antiarrhytmic drug in post-mortem whole blood by automated solid phase extraction and liquid chromatography mass spectrometry by Lena Kristoffersen; Elisabeth Leere Øiestad; Mimi Stokke Opdal; Mette Krogh; Elsa Lundanes; Asbjørg Solberg Christophersen (147-160).
A method for the simultaneous determination of the beta-blockers atenolol, sotalol, metoprolol, bisoprolol, propranolol and carvedilol, the calcium-channel antagonists diltiazem, amlodipine and verapamil, the angiotensin-II antagonists losartan, irbesartan, valsartan and telmisartan, and the antiarrythmic drug flecainide, in whole blood samples from forensic autopsies was developed. Sample clean-up was achieved by precipitation and solid phase extraction (SPE) with a mixed-mode column. Quantification was performed by reversed phase high performance liquid chromatography with positive electrospray ionization mass spectrometric detection (HPLC-MS). The method has been developed and robustness tested by systematically searching for satisfactory conditions using experimental designs including factorial and response surface designs. With the exception of amlodipine, the concentration limit of quantification (cLOQ) covered low therapeutic concentration levels for all the compounds. Within assay precisions and accuracies (bias) were 3.4–21% RSD and from −24 to 21% for the concentration range 1.00–5.00 μM, respectively. Between assay precisions were 4.4–28% RSD for the concentration range from 0.1 to 5 μM and recoveries varied from 9 to 103%. The method is used for determination of cardiovascular drugs in post-mortem whole blood samples from forensic autopsy cases.
Keywords: Cardiovascular drugs; Beta-blockers; Calcium-channel antagonists; Angiotensin-II antagonists; Antiarrythmic drug; Whole blood; Experimental design; SPE; HPLC-MS;
Determination of a potent non-competitive AMPA receptor antagonist in rat brain by M. Rizzo; D. Ventrice; F. Casale; G. De Sarro; R. Gitto; A. Chimirri (161-167).
N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative (PS3Ac) has been determined in brain tissues by high performance liquid chromatography (HPLC) coupled with a diode array detection. In a previous paper we presented a validation method for detecting PS3Ac and its metabolites in plasma samples after intraperitoneal administration to Wistar rats. In the present paper, we report the results of the determination of PS3Ac and its N-deacetyl (PS3) and O-demethyl (PS3OH) metabolites, in the brain after extraction based on a polymeric matrix with a high hydrophilic–lipophilic balance, using Oasis cartridges. The chromatographic separation was performed in an octadecylsilica stationary phase at 25 °C using a mixture of 10 mM potassium dihydrogen orthophosphate (pH 2.24) and acetonitrile in ratio of 30:70 (v/v) as mobile phase, with a flow rate of 0.8 ml/min. The method exhibited a large linear range from 0.05 to 2 μg/ml for all studied compounds (n = 6). In the within-day assay (n = 4), the accuracy ranged from 87.5% determined with 0.05 μg/ml of PS3 to 110.1% determined with 0.2 μg/ml of PS3OH. In the between-day assay the coefficient of variation ranged from 2.4 determined with 0.05 μg/ml of PS3 to 9.7 determined with 0.2 μg/ml of PS3OH. The extraction efficiency ranged from 77.8% for PS3OH at 0.2 μg/ml to 94.3 for PS3Ac at 0.5 μg/ml. The limit of detection for all the tetrahydroisoquinoline derivatives ranged around 50 ng/ml. The method proved to be highly sensitive and specific to determinate PS3Ac and its metabolites and has been successfully applied to value their concentrations in brain matrix over the time.
Keywords: AMPA receptor antagonist; SPE-HPLC; Tetrahydroisoquinoline; Brain;
Targeted quantitative analysis of fatty acids in atherosclerotic plaques by high sensitivity liquid chromatography/tandem mass spectrometry by Caterina Pettinella; Seon Hwa Lee; Francesco Cipollone; Ian A. Blair (168-176).
The quantitative analysis of fatty acid composition in atherosclerotic plaques provides a way to monitor the underlying etiology of atherosclerosis. Previously, the method of choice for analyzing fatty acids in biological samples was gas chromatography/mass spectrometry (GC/MS); however, recent developments in electrospray ionization (ESI)/liquid chromatography (LC)/tandem mass spectrometry have made it a superior alternative. Previous research has largely focused on global analyses of intact lipids rather than more targeted analysis of the fatty acids themselves. We have now developed a targeted, stable isotope dilution LC–electrospray ionization/multiple reaction monitoring/MS method for the quantitative analysis of 10 fatty acids (myristic, palmitic, stearic, oleic, linoleic, α-linolenic, γ-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids) using their trimethylaminoethyl ester (TMAE) derivatives to improve sensitivity. The method was validated, had a detection limit in the fmol range, and was used in the analysis of fatty acids in atherosclerotic plaques from carotid arteries.
Keywords: Trimethylaminoethyl ester; Fatty acids; Stable isotope dilution; Liquid chromatography/mass spectrometry; Electrospray ionization; Atherosclerosis;
Gas chromatographic–mass spectrometric determination of brain levels of α-cholest-8-en-3β-ol (lathosterol) by Berta Luzón-Toro; Alberto Zafra-Gómez; Oscar Ballesteros (177-182).
A gas chromatographic–mass spectrometric (GC–MS) method is proposed for the detection and quantification of lathosterol in rabbit brain. This compound is one of the most important precursors of the cholesterol synthesis. The interest in brain cholesterol metabolism is growing nowadays since it was described to play an important role in some neurodegerative disorders such as Alzheimer's disease and Multiple Sclerosis. The analytical methodology proposed involves a liquid–liquid extraction procedure (LLE) followed by a silylation step previous to the GC–MS analysis. The chromatographic separation was performed by using a low bleed HP5-MS fused silica capillary column. A clean up is not necessary when using single-ion monitoring (SIM) mode. The molecular ion appears at 458 m/z; being as well the base peak. α-Naphtol was used as an internal standard. The detection limit obtained was 0.09 μg mL−1. The method was applied to the determination of brain lathosterol levels in rabbits fed with different types of diets (control and atherogenic, supplemented or not with natural polyphenolic antioxidants). The quantification of the compound in samples showed a reduction, after 1 month, of this precursor of cholesterol synthesis in groups fed with antioxidant supplemented diets.
Keywords: Lathosterol; Cholesterol precursor; GC–MS analysis; Polyphenolic antioxidants; Brain;
Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in human plasma by HPLC-ESI/MS using a vancomycin chiral column by Wen Liu; Feng Wang; Huan-de Li (183-189).
A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS) method for simultaneous stereoselective analysis of venlafaxine (VEN) and its major metabolite O-desmethylvenlafaxine (ODV) enantiomers in human plasma has been developed and validated. Chiral chromatography is performed on the CHRIOBIOTIC V ™ (5 μm, 250 mm × 4.6 mm) column with mobile phase constituted of 30 mmol/l ammonium acetate–methanol (15:85, pH 6.0) at a flow rate of 1.0 ml/min and a postcolumn splitting ratio of 3:1. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and detected using the selected ion recording (SIR) mode. Calibration curves obtained from spiked plasma were linear in the range of 5.0–400 ng/ml for S-(+)-VEN and R-(−)-VEN, 4.0–280 ng/ml for S-(+)-ODV and R-(−)-ODV, respectively, with linear correlation coefficient all above 0.999. The average extraction recoveries for all the four analytes were above 76%. The methodology recoveries were higher than 92%. The limit of detection were 1.0 ng/ml for S-(+)-VEN and R-(−)-VEN, 1.5 ng/ml for S-(+)-ODV and R-(−)-ODV, respectively. The intra- and inter-day variation coefficients were less than 9%.
Keywords: Venlafaxine; Enantioseparation; Vancomycin chiral column; HPLC-MS/ESI;
High performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) assay for chiral separation of lactic acid enantiomers in urine using a teicoplanin based stationary phase by Dean Norton; Brian Crow; Michael Bishop; Kasey Kovalcik; Joe George; J. Alexander Bralley (190-198).
A novel method for the separation and simultaneous determination of urinary d- and l-lactic acid enantiomers by high performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) is presented. The chiral separation was optimized on a Chirobiotic teicoplanin aglyocone (TAG) column. Most interestingly, the addition of water in small volume fraction to the polar organic mobile phase was found to significantly improve the chromatography. Calibration curves were linear (r 2 > 0.9950) over the range 3–1000 mg/L for l-lactic acid and 0.5–160.8 mg/L for d-lactic acid. The limit of detection (LOD) (S/N = 3) and limit of quantification (LOQ) (S/N = 10) were determined experimentally (n = 3) to be 0.2 and 0.5 mg/L for l-lactic acid and 0.4 and 1.3 mg/L for d-lactic acid, respectively. The normal patient range of l-lactic acid was 1–20 μg/mg creatinine with an elevated value of 85 μg/mg creatinine. For d-lactic acid, the range of normal values were between 0 and 5 μg/mg creatinine with an elevated value of 40 μg/mg creatinine. Finally, the validated method allows for rapid analysis with a total run time of 7.5 min.
Keywords: Chiral separation; d-Lactic acid; l-Lactic acid; Enantiomers; HPLC/MS/MS; Urine; Chirobiotic TAG;
Determination of lefucoxib in rat plasma, urine, and feces by high-performance liquid chromatography with fluorescence detection: Application in pharmacokinetic studies by Xuezhi Bi; Zhiyun Meng; Guifang Dou (199-205).
A sensitive, specific, and reproducible high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of lefucoxib in rat plasma, urine, and feces. The method involved liquid–liquid extraction using methyl tert-butyl ether, and celecoxib was used as the internal standard. The chromatographic separation was performed on a Kromasil C18 column (250.0 mm × 4.6 mm, 5.0 μm) with a mobile phase gradient consisting of water and methanol at a flow rate of 1 ml min−1. The assay was linear in the range of 5.0–1000.0 ng ml−1 with a correlation coefficient (r) of 0.9994. The limit of quantification was 5.0 ng ml−1. Inter- and intra-assay precisions were ≤14.2% and 5.5%, respectively. Relative recoveries ranged from 97.9% to 108.1%, and absolute recoveries were about 70.0% both with and without internal standard. All biological matrices (plasma, urine, and fecal homogenate) containing lefucoxib were stable for 5 h at room temperature (about 20 °C) and they are also stable after freeze–thaw cycles. The method was successfully applied to the pharmacokinetic studies of lefucoxib in rats.
Keywords: Lefucoxib; High-performance liquid chromatography; Fluorescence; Pharmacokinetic;
Quantification of valproic acid and its metabolite 2-propyl-4-pentenoic acid in human plasma using HPLC-MS/MS by Hao Cheng; Zhongfa Liu; William Blum; John C. Byrd; Rebecca Klisovic; Michael R. Grever; Guido Marcucci; Kenneth K. Chan (206-212).
A specific and sensitive HPLC-MS/MS method for the quantitative determination of valproic acid (VPA) and its metabolite, 2-propyl-4-pentenoic acid in human plasma has been developed, using VPA-d15 as the internal standard. The method was based on pre-column derivatization using 4-dimethylaminobenzylamine dihydrochloride. The derivatives were separated with a gradient elution and quantified by positive electrospray ionization with multiple reaction monitoring. The assay provides routine quantification limits of 200 ng/mL for VPA and 20 ng/mL for 4-ene VPA with within- and between-day coefficients of variation of <10%. This method has been applied to the analysis of plasma samples obtained from patients treated with this drug.
Keywords: Valproic acid; Metabolites; Derivatization; LC-MS/MS; Clinical application;
Analysis of iodinated peptides by LC-DAD/ESI ion trap mass spectrometry by V. Vergote; S. Bodé; K. Peremans; H. Vanbree; B. Baert; G. Slegers; C. Burvenich; B. De Spiegeleer (213-220).
The analysis of iodinated peptides resulting from chloramine-T (CAT), Iodo-Beads®, Iodo-Gen® and lactoperoxidase iodination reactions in the preparation of nanomole quantities 125I and 123I labelled tracers is described. Seven different model peptides were evaluated, varying in molecular weight from 294 (LY-dipeptide) to 2518 (obestatin containing 23 amino acid residues). Two different RP-C18 columns were used, each with a different gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting components of the reaction mixtures, while UV (DAD) served quantitative purposes. Non-, mono-, di-, tri- and tetra-iodinated peptides (respectively NIP, MIP, DIP, 3IP and 4IP) eluted in that order and were well separated from each other. An empirical model was derived. The applicability of this approach was demonstrated by the analysis of different reaction mixtures.
Keywords: Peptide iodination; LC-DAD/MS; Radio-HPLC; Obestatin; Neurotensin; Enkephalin; GGYR; LLY; VYV; LY;
Characterization of volatile constituents of Scaligeria tripartita and studies on the antifungal activity against phytopathogenic fungi by Nurhayat Tabanca; Betul Demirci; K. Husnu Can Baser; Emil Mincsovics; Shabana I. Khan; Melissa R. Jacob; David E. Wedge (221-229).
The chemical composition of the essential oils obtained from stems and leaves, fruits and roots of Scaligeria tripartita oil was analyzed by gas chromatography–mass spectrometry. A total of 38 compounds were identified ranging 89–94% of the oil samples. Geijerene was found as a main compound in the oils of the stems and leaves (37%) and fruits (55%), whereas epoxypseudoisoeugenol angelate (37%) was found as a main compound in the root oil. Oils were subsequently evaluated for their antimalarial, antimicrobial against human pathogenic bacteria or fungi and antifungal activities against plant pathogens. Antifungal activity of Scaligeria oils was observed against the strawberry anthracnose-causing fungal plant pathogens Colletotrichum acutatum, C. fragariae and C. gloeosporioides using the direct overlay bioautography assay. Chemotaxonomically important pure compounds indicated in the bioautography assay were subsequently evaluated in a 96-well microdilution broth assay. The performance of overpressured layer chromatography (OPLC) and TLC for the analysis of Scaligeria essential oils was also compared.
Keywords: Scaligeria tripartita; Pimpinella species; Overpressured layer chromatography (OPLC); Essential oil; GC/MS; Apiaceae; Phenylpropanoids; Antimalarial activity; Antimicrobial activity;
Ethanol analysis from biological samples by dual rail robotic autosampler by Cynthia L. Morris-Kukoski; Eshwar Jagerdeo; Jason E. Schaff; Marc A. LeBeau (230-235).
Detection, identification, and quantitation of ethanol and other low molecular weight volatile compounds in liquid matrices by headspace gas chromatography–flame ionization detection (HS–GC–FID) and headspace gas chromatography–mass spectrometry (HS–GC–MS) are becoming commonly used practices in forensic laboratories. Although it is one of the most frequently utilized procedures, sample preparation is usually done manually. Implementing the use of a dual-rail, programmable autosampler can minimize many of the manual steps in sample preparation. The autosampler is configured so that one rail is used for sample preparation and the other rail is used as a traditional autosampler for sample introduction into the gas chromatograph inlet. The sample preparation rail draws up and sequentially adds a saturated sodium chloride solution and internal standard (0.08%, w/v acetonitrile) to a headspace vial containing a biological sample, a calibrator, or a control. Then, the analytical rail moves the sample to the agitator for incubation, followed by sampling of the headspace for analysis. Using DB-624 capillary columns, the method was validated on a GC–FID and confirmed with a GC–MS. The analytes (ethanol, acetonitrile) and possible interferences (acetaldehyde, methanol, pentane, diethyl ether, acetone, isopropanol, methylene chloride, n-propanol, and isovaleraldehyde) were baseline resolved for both the GC–FID and GC–MS methods. This method demonstrated acceptable linearity from 0 to 1500 mg/dL. The lower limit of quantitation (LOQ) was determined to be 17 mg/dL and the limit of detection was 5 mg/dL.
Keywords: Ethanol; Robotic autosampler; Automation; Blood alcohol;
A GC-based metabonomics investigation of type 2 diabetes by organic acids metabolic profile by Kailong Yuan; Hongwei Kong; Yufeng Guan; Jun Yang; Guowang Xu (236-240).
“Metabonomics” method requires the development of rapid, advanced analytical tools and GC will play an important role for its special advantage. In this study we show the application of GC-based metabonomics to investigate the control and type 2 diabetes (DM2) patients by urinary organic acids metabolic profile. After peak matching, multivariate statistical analysis methods: principal components analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used. The results showed that there was a relationship between organic acids metabolic profiles and DM2, and PLS-DA can distinguish the DM2 patients from the control. Five organic acids as potential biomarkers were identified.
Keywords: Metabonomics; GC; Organic acids; Peak matching algorithm; Type 2 diabetes; Biomarkers;
The study on the chromatographic fingerprint of Fructus xanthii by microwave assisted extraction coupled with GC–MS by Gui-Hua Ruan; Gong-Ke Li (241-248).
The chromatographic fingerprint of Fructus xanthii, a kind of Traditional Chinese Medicines (TCMs), was studied by microwave assisted extraction (MAE) coupled with gas chromatography-mass spectrometry (GC–MS). The optimized conditions of MAE were examined. The method of MAE was evaluated in contrast to heat reflux extraction (HRE) method and by the validation tests of precision and repeatability. The relative standard deviations (RSDs) of retention time and peak area of each component were less than 0.2% and 6%, respectively. Twenty-five different batches of samples collected from different producing areas and the toasting process of F. xanthii were studied. The characteristic differences in the producing areas and the chemical variances in the toasting process were obtained and studied by principal components analysis (PCA) and similarity analysis. The trends of main varying components were attempted to be described in order to specify the related pharmacology and toxicology in crude and toasted samples. The results suggest that the chromatographic fingerprint developed by MAE coupled with GC–MS provides useful information to reveal the quality of F. xanthii and evaluate the quality changes in the producing process.
Keywords: Microwave assisted extraction; GC-MS; Fructus xanthii; Chromatographic fingerprint; Traditional Chinese Medicine;
Monitoring of lopinavir and ritonavir in peripheral blood mononuclear cells, plasma, and ultrafiltrate using a selective and highly sensitive LC/MS/MS assay by Manuela Ehrhardt; Marion Möck; Walter E. Haefeli; Gerd Mikus; Jürgen Burhenne (249-258).
For the determination of the HIV protease inhibitors lopinavir and ritonavir in human plasma, plasma ultrafiltrate, and peripheral blood mononuclear cells (PBMCs) a highly sensitive and selective method has been developed, validated, and applied to samples of a healthy volunteer. BD Vacutainer® CPT™ and Amicon Centriplus® centrifugal filter devices were used for separation of PBMCs and for ultrafiltrate generation, respectively. After liquid/liquid-extraction extracts were chromatographed isocratically within 6 min on a Jupiter Proteo column. The drugs were quantified using 2H5-saquinavir as internal standard and electrospray tandem mass spectrometry in the selected reaction monitoring mode. Limits of quantification for both analytes were 4.0 ng/mL in plasma, 0.2 ng/mL in ultrafiltrate, and 0.1 ng/cell pellet (∼3 × 106 cells) in PBMCs. The calibration ranges were linear over more than three logs with an over-all accuracy varying between 98.7% and 111.5% and an over-all precision ranging from 6.2% to 14.0% (SD batch-to-batch). After a regular oral dose of Kaletra® (400 mg lopinavir, 100 mg ritonavir) analyte concentrations were detectable over a full dosing interval in plasma, ultrafiltrate, and PBMCs. The method is well suited for monitoring of free and total plasma, and intracellular lopinavir/ritonavir concentrations in samples from clinical trials.
Keywords: Kaletra®; Ritonavir; Lopinavir; PBMC; Protein binding; LC/MS/MS;
Sensitive and specific LC–ESI–MS/MS method for the determination of a styrylquinoline, BA011FZ041, a potent HIV anti-integrase agent, in rat plasma by Alain Pruvost; Mikaël Levi; Fatima Zouhiri; Isabelle Ménier; Henri Benech (259-266).
A LC–MS/MS method was validated for the determination of BA011FZ041, a styrylquinoline derivative. After addition of BA011FZ055 as internal standard (IS), the method involved solid phase extraction (SPE), LC separation with an ether-phenyl column and quantification by MS/MS after positive ESI. The calibration curve, ranging from 1 to 500 ng/mL was fitted to a 1/x-weighted quadratic regression model. Lower limit of quantification (LLOQ) was 1 ng/mL using 100 μL of plasma. Intra- and inter-assay precision and accuracy values were within the regulatory limits. The method was successfully applied to the determination of BA011FZ041 in rat plasma and PBMCs after i.v. dosing.
Keywords: HIV; Anti-integrase; LC–MS/MS; Plasma; PBMCs;
Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography by Jian-Feng NIU; Guang-Ce WANG; Xiang-zhi Lin; Bai-Cheng Zhou (267-276).
C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A 620/A 280 ratio) of C-phycocyanin isolated with STREAMLINE™ column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A 620/A 650 index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin.
Keywords: Spirulina platensis; Purification; C-phycocyanin; STREAMLINE™ column; Anion-exchange chromatography; Hydroxyapatite chromatography;
Higher order structure of Mucor miehei lipase and micelle size in cetyltrimethylammonium bromide reverse micellar system by Kazumitsu Naoe; Chihiro Takeuchi; Mikio Kawagoe; Kazuhito Nagayama; Masanao Imai (277-284).
The higher order structure of Mucor miehei lipase and micelle size in a cationic cetyltrimethylammonium bromide (CTAB) reverse micellar system was investigated. Circular dichroic (CD) measurement revealed that the lipase far-UV CD spectra changed markedly, going from buffer solution to the reverse micellar solution, and were very similar for any organic solvent used. The ellipticity of the solubilized lipase in the far-UV region markedly decreased with increasing water content (W 0: molar ratio of water to CTAB), indicating that the secondary structure of lipase changed with the water content. The linear correlation between the W 0 and the micelle size was obtained by measuring dynamic light scattering. From the linear correlation between the micelle size and W 0, the higher order structure of the solubilized lipase appears to be affected directly by the micellar interface. The species and concentration of alcohol as a cosurfactant had an inferior effect on lipase structure. Especially, at ratios of 1-pentanol to CTAB of less than 8, the secondary and tertiary structures of lipase were preserved in the reverse micelles. The CTAB concentration had little effect on the lipase structure in the micelles. The catalytic activity of the lipase solubilized in the CTAB reverse micelles increased with increasing the W 0.
Keywords: Reverse micelle; Lipase; Higher order structure; CTAB; Micelle size;
Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody by Chris Chumsae; Georgeen Gaza-Bulseco; Joanne Sun; Hongcheng Liu (285-294).
Methionine (Met) oxidation is a major degradation pathway of protein therapeutics. Met oxidation of a fully human recombinant monoclonal antibody was investigated under both chemically stressed conditions using tert-butylhydroperoxide (tBHP) and thermal stability conditions where the sample was incubated in formulation buffer at 25 °C for 12 months. This antibody has one Met residue on each of the light chains and four Met residues on each of the heavy chains. In the thermal stability sample, only Met residues 256 and 432 in the Fc region were oxidized to form methionine sulfoxide, while Met residues in the Fab region were relatively stable. The susceptibility of Met residues 256 and 432 was further confirmed by incubating samples with tBHP, which has been shown to induce Met oxidation. Further analysis revealed that the susceptible Met residues of each heavy chain were randomly oxidized in samples incubated with tBHP, while in the thermal stability sample, the susceptible Met residues of one heavy chain were preferentially oxidized.
Keywords: Recombinant monoclonal antibody; Methionine oxidation; Mass spectrometry;
Stir bar sorptive extraction and liquid chromatography with UV detection for determination of antidepressants in plasma samples by Andréa Rodrigues Chaves; Silvana Maciel Silva; Regina Helena Costa Queiroz; Fernando Mauro Lanças; Maria Eugênia Costa Queiroz (295-302).
A sensitive and reproducible stir bar sorptive extraction and liquid chromatography (SBSE/LC–UV) method is described for the determination of sertraline, mirtazapine, fluoxetine, citalopram, paroxetine, imipramine, nortriptyline, amitriptyne, and desipramine in plasma samples. Important factors in the optimization of SBSE efficiency are discussed, such as extraction time, pH, ionic strength, influence of plasma proteins, and desorption conditions: solvents, modes (magnetic stir, ultrasonic), time, and number of desorption steps. The SBSE/LC–UV method showed to be linear in a concentration ranging from the limit of quantification (LOQ) to 1000.0 ng mL−1. The LOQ values ranged from 10.0 ng mL−1 to 40.0 ng mL−1. The inter-day precision of the SBSE/LC–UV method presented coefficient of the variation lower than 15%. Based on figures of the merit results, the SBSE/LC–UV methodology showed to be adequate to the antidepressants analyses from therapeutic to toxic therapeutic levels. In order to evaluate the proposed method for clinical use, the SBSE/LC–UV method was applied to the analysis of plasma samples from elderly depressed patients.
Keywords: Stir bar sorptive extraction; Liquid chromatography; Antidepressants; Plasma samples;
Highly sensitive assay for the measurement of serotonin in microdialysates using capillary high-performance liquid chromatography with electrochemical detection by Sandrine Parrot; Laura Lambás-Señas; Sabine Sentenac; Luc Denoroy; Bernard Renaud (303-309).
A highly sensitive isocratic capillary high-performance liquid chromatographic (HPLC) method with electrochemical detection (ED) for the simultaneous measurement of serotonin (5-hydroxytryptamine, 5-HT) and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in microdialysates has been developed using a 0.5 mm i.d. capillary column and a 11-nL detection cell. This method, validated on both pharmacological and analytical bases, can be performed using injection volumes as low as 1 μL. The limits of detection were 5.6 × 10−11 mol/L and 3.0 × 10−9 mol/L for 5-HT and 5-HIAA. Several applications of the present method are given on microdialysates from rodent brain and human spinal cord.
Keywords: Capillary liquid chromatography; Electrochemical detection; Serotonin; Microdialysis;
Simultaneous measurement of intracellular triphosphate metabolites of zidovudine, lamivudine and abacavir (carbovir) in human peripheral blood mononuclear cells by combined anion exchange solid phase extraction and LC–MS/MS by Brian L. Robbins; Philip A. Poston; Erin F. Neal; Clive Slaughter; John H. Rodman (310-317).
All nucleoside reverse transcriptase inhibitors (NRTI) must first be metabolized to their triphosphate forms in order to be active against HIV. Zidovudine (ZDV), abacavir (ABC) and lamivudine (3TC) have proven to be an efficacious combination. In order simultaneously to measure intracellular levels of the triphosphates (-TP) of ZDV, ABC (carbovir, CBV) and 3TC, either together or individually, we have developed a cartridge-LC–MS/MS method. The quantitation range was 2.5–250 pg/μl for 3TC-TP, 0.1–10.0 pg/μl for ZDV-TP and 0.05–5.00 pg/μl for CBV-TP. This corresponds to 0.1–11.0 pmol 3TC-TP per million cells, 4–375 fmol ZDV-TP per million cells and 2–200 fmol CBV-TP per million cells, extracted from 10 million cells. Patient samples demonstrated measured levels in the middle regions of our standard curves both at pre-dose and 4 h post-dose times.
Keywords: Human immunodeficiency virus; Zidovudine triphosphate; Carbovir triphosphate; Lamivudine triphosphate; Antiviral drug metabolism;
Electrospray ionization LC–MS/MS validated method to quantify griseofulvin in human plasma and its application to bioequivalence study by Hiren N. Mistri; Arvind G. Jangid; Mallika Sanyal; Pranav Shrivastav (318-326).
A simple, sensitive and rapid liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated to quantify griseofulvin in human plasma using propranolol hydrochloride as internal standard (IS). Samples were prepared using solid phase extraction and analysed without drying and reconstitution. The analytes were chromatographed on Hypersil, hypurity C18 reverse phase column under isocratic conditions using 0.05% formic acid in water:acetonitrile (30:70, v/v) as the mobile phase. Total chromatographic run time was 3.0 min. Quantitation was done on a triple quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring (MRM) mode to detect parent → product ion transition for analyte and IS. The method was validated for sensitivity, matrix effect, accuracy and precision, linearity, recovery and stability studies. Linearity in plasma was observed over the concentration range 20–3000 ng/mL for griseofulvin. Lower limit of quantification (LLOQ) achieved was 20 ng/mL with precision (CV) less than 10% using 5 μL injection volume. The absolute recovery of analyte (87.36%) and IS (98.91%) from spiked plasma samples was consistent and reproducible. Inter-batch and intra-batch coefficients of variation across four validation runs (LLOQ, LQC, MQC and HQC) was less than 7.5%. The accuracy determined at these levels was within ±4.2% in terms of relative error. The method was applied to a pilot bioequivalence study of 500 mg griseofulvin tablet in six healthy human subjects under fed condition.
Keywords: Griseofulvin; LC–MS/MS; Solid phase extraction; Human plasma; Pilot bioequivalence;
Achiral–chiral LC/LC–FLD coupling for determination of carvedilol in plasma samples for bioequivalence purposes by Andrei Medvedovici; Florin Albu; Cristina Georgita; Daniela Iuliana Sora; Toma Galaon; Stefan Udrescu; Victor David (327-335).
Bioequivalence data for two pharmaceutical formulations (solid oral dosage forms) containing carvedilol is presented for both racemic and enantiomers of the active substance. This was achieved by on-line coupling of two liquid chromatographic separations followed by fluorescence detection. The first LC dimension was used for a fast separation of racemic carvedilol from propranolol (IS) and the endogenous matrix, by means of a reversed phase mechanism. The peak of racemic carvedilol was on-line transferred to the second enantioselective LC dimension, based on a reversed phase separation on cellulose tris(3,5-dimethyl-phenylcarbamate) stationary phase. Both stages were monitored over a single run by means of a fluorescence detector operated at an excitation wavelength of 285 nm and an emission wavelength of 355 nm. Automated shortcutting of the racemic carvedilol peak to the chiral column and simultaneous detection over the two LC dimensions have been obtained by using an experimental set-up based on two six-port rotative switching valves. Linearity was demonstrated on the interval 2–150 ng/mL for racemic carvedilol and on 1–75 ng/mL intervals for enantiomers. LLOQ fits between 0.7 and 1.4 ng/mL. Recoveries of the target compounds are 87 ± 4 and 81 ± 4% for the IS. Precision ranged from 0.6 to 2.5% and the mean accuracy obtained on quality control samples (measured as % bias) over the whole study falls between −0.8 and 6.3%.
Keywords: Carvedilol; Racemate; Enantiomers; Bidimensional LC; Achiral and chiral RPLC separation mechanisms; Cellulose tris(3,5-dimethyl-phenylcarbamate); Simultaneous monitoring; Fluorescence detection; Bioequivalence study; Solid oral dosage forms; Pharmacokinetic parameters;
Proteomic analyses of amniotic fluid: Potential applications in health and diseases by Pierre-Alain Queloz; David Crettaz; Lynne Thadikkaran; Vincent Sapin; Denis Gallot; Jacques Jani; Jan Deprest; Didier Lémery; Stefano Barelli; Jean-Daniel Tissot (336-342).
Amniotic fluid (AF) is a potential source of biomarkers for many disorders which may occur during pregnancy. The purpose of this study was to evaluate the place of two-dimensional gel electrophoresis (2-DE) technologies to compare AF in both normal and pathological situations. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE; Ettan™ DIGE) as well as two-dimensional gel electrophoresis and silver staining followed by image analysis were used. Differentially expressed proteins were identified by mass spectrometry. This approach was used to study electrophoregrams of normal AF obtained at 17 weeks of gestation and at term, as well as AF from fetuses presenting with congenital diaphragmatic hernia. Finally, the potential of two-dimensional electrophoresis was assessed by studying the protein profile of plasma containing AF proteins in a model of premature rupture of the membranes (PROM). Our results clearly show that two-dimensional electrophoresis technologies still have place for analyzing biological fluids such as AF.
Keywords: Amniotic fluid; Congenital diaphragmatic hernia; Proteomics; Two-dimensional electrophoresis;
Monitoring the reaction of hemoglobin with hydrogen peroxide by capillary electrophoresis-chemiluminescence detection by Shi-Lai Zhou; Jun-Hua Wang; Wei-Hua Huang; Xin Lu; Jie-Ke Cheng (343-347).
The reaction of hemoglobin (Hb) with hydrogen peroxide (H2O2) leads to fluorescent product and heme degradation. We applied capillary electrophoresis-chemiluminescence (CE-CL) detection to monitor the course of Hb reacting with H2O2. Hb and released free iron ion (Fe3+) were detected based on their enhancement effects on CL of the luminol-H2O2 system. In this study, we discovered an intermediate of this reaction which intensely enhances the luminol-H2O2 CL system. The ratio of max CL signals of Fe3+, Hb and this intermediate is circa 1:10:60.
Keywords: Capillary electrophoresis; Chemiluminescence; Hemoglobin; Hydrogen peroxide; Heme degradation;
Sensitive quantification of rifaximin in human plasma by liquid chromatography–tandem mass spectrometry by Xianhua Zhang; Jingli Duan; Ke Li; Liya Zhou; Suodi Zhai (348-355).
A liquid chromatography–mass spectrometry method (LC–MS/MS) for the quantitative determination of rifaximin in human plasma was developed and validated. In the developed procedure, metoprolol was added to human plasma as an internal standard (IS) and acetonitrile was used to precipitate the plasma proteins before LC–MS/MS analysis. Chromatographic separation was obtained on a RESTEK Pinnacle C18 column (50 mm × 2.1 mm, 5 μm) with a mobile phase consisted of ammonium acetate solution (15 mM, pH 4.32) as buffer A and methanol as mobile phase B. Quantification was performed in positive mode using multiple reaction monitoring (MRM) of the transitions m/z 786.1 → 754.1 for rifaximin and m/z 268.3 → 116.1 for the IS. The assay has been validated over the concentration range of 0.5–10 ng/ml (r = 0.9992) based on the analysis of 0.2 ml of plasma. The assay accuracy was between 98.2% and 109%. The within-day and between-day precision was better than 3.9% and 8.9% at three concentration levels. The freeze–thaw stability was also investigated and it was found that both rifaximin and the IS were quite stable. This method provides a rapid, sensitive, specific and robust tool for the quantitative determination of rifaximin in human plasma, which is especially useful for the pharmacokinetic study of rifaximin.
Keywords: Rifaximin; Metoprolol; LC–MS/MS; Human plasma;
A new high performance liquid chromatographic method for quantification of atomoxetine in human plasma and its application for pharmacokinetic study by Chaula Patel; Minal Patel; Shubha Rani; Manish Nivsarkar; Harish Padh (356-360).
Atomoxetine is the first, non-stimulant alternative to other stimulant medications used for the treatment of Attention-Deficit/Hyperactivity Disorder (ADHD). Reported methods for the determination of atomoxetine include expensive liquid chromatography tandem mass spectrometry (LCMS) and high performance liquid chromatography (HPLC) with liquid scintillation counting (LSC) detection. Till date, no method has been reported in literature to determine atomoxetine using HPLC with UV detection. In this paper, we describe a new HPLC method for the determination of atomoxetine using liquid–liquid extraction with tertiary butyl methyl ether and UV detector. This method was found to be linear over the concentration range of 0.05–3.0 μg/ml. The limit of quantification was 0.05 μg/ml. Intra- and inter-day precision was <15% and accuracy was in the range of 95.67–108.80%. Stability studies showed that atomoxetine was stable in human plasma for short- and long-term period for sample preparation and analysis. This method was used for sample analysis in a pharmacokinetic study of atomoxetine (25 mg) in five healthy adult female volunteers. The observed mean ± S.D. pharmacokinetic parameters C max, T max and AUC0–t were 0.40 ± 0.06 μg/ml, 3.40 ± 0.42 h and 1.34 ± 0.52 μg h/ml, respectively.
Keywords: Atomoxetine; Pharmacokinetics; Human plasma; UV detection; Indian females;
Sample clean-up with sol–gel enzyme and immunoaffinity columns for the determination of bisphenol A in human urine by Kerstin Schöringhumer; Margit Cichna-Markl (361-369).
The paper describes the development of a simple and highly selective analytical method for the determination of free and total bisphenol A in urine samples. Free bisphenol A levels can be determined after sample clean-up using sol–gel immunoaffinity columns containing anti-bisphenol A antibodies. In determining total bisphenol A levels, the sample pre-treatment procedure consists of sample preparation using an on-line combination of two sol–gel columns, an enzyme column containing glucuronidase and arylsulfatase, and an immunoaffinity column. Bisphenol A can then be quantified by high-performance liquid chromatography and fluorescence detection. The mean recovery was found to be 78% with a standard deviation of 3.4%, the LOD (S/N = 3) was 0.2 ng/ml. The method was applied to determine free and total urinary BPA levels of healthy adults and dialysis patients.
Keywords: Bisphenol A; Sol–gel; Enzyme column; Immunoaffinity column; Urine; Dialysis patients;
Determination of meperidine, tramadol and oxycodone in human oral fluid using solid phase extraction and gas chromatography–mass spectrometry by Christine Moore; Sumandeep Rana; Cynthia Coulter (370-375).
Analytical procedures for the determination of meperidine, tramadol and oxycodone in oral fluid have been developed and validated using gas chromatography–mass spectrometry (GC/MS) following initial screening with enzyme linked immunosorbent assay (ELISA). The oral fluid samples were collected using the Quantisal™ device, and any drugs present were quantified using mixed mode solid-phase extraction and electron impact GC/MS. For confirmation, three ions were monitored and two ion ratios determined, which were within 20% of those of the known calibration standards. The limits of quantitation were 10 ng/mL; the intra-day precision of the assays (n = 5) was 2.33%, 1.00% and 7.61%; inter-day precision 2.48%, 2.44% and 5.8% (n = 10) for meperidine, tramadol and oxycodone, respectively. The percentage recovery of the drugs from the collection pads was 86.7%, 87.7% and 96.6%, respectively (n = 6). The methods were applied to specimens obtained during research studies in the USA.
Keywords: Oral fluid; Meperidine; Tramadol; Oxycodone;
Determination of 19 antiretroviral agents in pharmaceuticals or suspected products with two methods using high-performance liquid chromatography by Hervé Rebiere; Bernard Mazel; Corinne Civade; Pierre-Antoine Bonnet (376-383).
Three classes of antiretroviral agents are usually available for the treatment of HIV infection: nucleoside reverse transcriptase inhibitors (IN), non-nucleoside reverse transcriptase inhibitors (INN) and protease inhibitors (IP). Two methods by reversed-phase liquid chromatography were developed for the analysis of 19 antiretroviral molecules belonging to these three therapeutic classes and used in medicinal products. Both of these HPLC techniques use a C18 column and UV detection. The first method is for IN family analysis and allows eight molecules to be separated: zalcitabine, lamivudine, amdoxovir, emtricitabine, didanosine, stavudine, zidovudine and abacavir. The second method is for INN and IP family analysis and allows 11 molecules to be separated: fosamprenavir, nevirapine, indinavir, amprenavir, saquinavir, atazanavir, ritonavir, lopinavir, efavirenz, nelfinavir and tipranavir. The combination of these two methods makes possible the quality control of mono-, bi- or tri-therapy pharmaceutical products and the detection of illegal products sold particularly in developing countries.
Keywords: HIV; Antiretroviral agent; Nucleoside reverse transcriptase inhibitors; Non-nucleoside reverse transcriptase inhibitors; Protease inhibitors; HPLC–UV; Illegal products;
Hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC) based facile purification of recombinant Streptokinase from E. coli inclusion bodies by Deepika Goyal; Debendra K. Sahoo; Girish Sahni (384-391).
The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD600 = 40) culture of recombinant E. coli, and an SK protein concentration of 1080 mg l−1 was achieved. The wet cell pellet after centrifugation was re-suspended in 8 M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1 M NaCl during loading, 82.7% rSK activity could be recovered in the 10 mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18 × 105 in cell lysate to 0.53 × 105 IU mg−1 resulted after this step. rSK was further purified to near-homogeneity (specific activity = 0.96 × 105 IU mg−1) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps.
Keywords: Streptokinase; Escherichia coli; Downstream processing; Expanded bed adsorption chromatography; Hydrophobic interaction chromatography;
Simultaneous quantification of flavonoids and triterpenoids in licorice using HPLC by Yuan-Chuen Wang; Yi-Shan Yang (392-399).
Numerous bioactive compounds are present in licorice (Glycyrrhizae Radix), including flavonoids and triterpenoids. In this study, a reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous quantification of three flavonoids (liquiritin, liquiritigenin and isoliquiritigenin) and four triterpenoids (glycyrrhizin, 18α-glycyrrhetinic acid, 18β-glycyrrhetinic acid and 18β-glycyrrhetinic acid methyl ester) from licorice was developed, and further, to quantify these 7 compounds from 20 different licorice samples. Specifically, the reverse-phase HPLC was performed with a gradient mobile phase composed of 25 mM phosphate buffer (pH 2.5)–acetonitrile featuring gradient elution steps as follows: 0 min, 100:0; 10 min, 80:20; 50 min, 70:30; 73 min, 50:50; 110 min, 50:50; 125 min, 20:80; 140 min, 20:80, and peaks were detected at 254 nm. By using our technique, a rather good specificity was obtained regarding to the separation of these seven compounds. The regression coefficient for the linear equations for the seven compounds lay between 0.9978 and 0.9992. The limits of detection and quantification lay in the range of 0.044–0.084 and 0.13–0.25 μg/ml, respectively. The relative recovery rates for the seven compounds lay between 96.63 ± 2.43 and 103.55 ± 2.77%. Coefficient variation for intra-day and inter-day precisions lay in the range of 0.20–1.84 and 0.28–1.86%, respectively. Based upon our validation results, this analytical technique is a convenient method to simultaneous quantify numerous bioactive compounds derived from licorice, featuring good quantification parameters, accuracy and precision.
Keywords: Licorice; Flavonoids; Triterpenoids; Simultaneous quantification;
A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent by Gholamreza Bahrami; Bahareh Mohammadi (400-404).
A new, sensitive and simple high-performance liquid chromatographic method for analysis of topiramate, an antiepileptic agent, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent is described. Following liquid–liquid extraction of topiramate and an internal standard (amlodipine) from human serum, derivatization of the drugs was performed by the labeling agent in the presence of dichloromethane, methanol, acetonitrile and borate buffer (0.05 M; pH 10.6). A mixture of sodium phosphate buffer (0.05 M; pH 2.4): methanol (35:65 v/v) was eluted as mobile phase and chromatographic separation was achieved using a Shimpack CLC-C18 (150 × 4.6 mm) column. In this method the limit of quantification of 0.01 μg/mL was obtained and the procedure was validated over the concentration range of 0.01 to 12.8 μg/mL. No interferences were found from commonly co-administrated antiepileptic drugs including phenytoin, phenobarbital carbamazepine, lamotrigine, zonisamide, primidone, gabapentin, vigabatrin, and ethosuximide. The analysis performance was carried-out in terms of specificity, sensitivity, linearity, precision, accuracy and stability and the method was shown to be accurate, with intra-day and inter-day accuracy from −3.4 to 10% and precise, with intra-day and inter-day precision from 1.1 to 18%.
Keywords: Reverse phase chromatography; Topiramate; Serum; 4-Chloro-7-nitrobenzofurazan; NBD-Cl;
High performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine and its three metabolites in human plasma by Wen Liu; Hua-lin Cai; Huan-de Li (405-411).
A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine (VEN) and its three metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV) in human plasma has been developed and validated. Estazolam was used as the internal standard. The compounds and internal standard were extracted from plasma by a liquid–liquid extraction. The HPLC separation of the analytes was performed on a Thermo BDS HYPERSIL C18 (250 mm × 4.6 mm, 5 μm, USA) column, using a gradient elution program with solvents constituted of water (ammonium acetate: 30 mmol/l, formic acid 2.6 mmol/l and trifluoroacetic acid 0.13 mmol/l) and acetonitrile (60:40, V/V) at a flow-rate of 1.0 ml/min. All of the analytes were eluted within 6 min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. Calibration curves in spiked whole blood were linear from 4.0–700 ng/ml, 2.0–900 ng/ml, 3.0–800 ng/ml and 2.0–700 ng/ml for VEN, ODV, NDV and DDV, respectively, all of them with coefficients of determination above 0.9991. The average extraction recoveries for all the four analytes were above 77%. The methodology recoveries were higher than 91%. The limits of detection were 0.4, 0.2, 0.3, and 0.2 ng/ml for VEN, ODV, NDV and DDV, respectively. The intra- and inter-day variation coefficients were less than 11%. The method is accurate, sensitive and reliable for the pharmacokinetic study of venlafaxine as well as therapeutic drug monitoring (TDM).
Keywords: Venlafaxine; Metabolite; HPLC-MS/ESI;
Analysis of alkylphenol and bisphenol A in eggs and milk by matrix solid phase dispersion extraction and liquid chromatography with tandem mass spectrometry by Bing Shao; Hao Han; Xiaoming Tu; Lei Huang (412-416).
A method based on matrix solid phase dispersion (MSPD) using C18 as dispersant, and a subsequent cleanup step with amino-propyl solid phase extraction cartridges and liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) has been developed for the simultaneous determination of nonylphenol (NP), octylphenol (OP) and bisphenol A (BPA) in eggs and milk. Recovery studies were performed at different fortification levels. Average recoveries by MSPD varied from 79% of BPA to 98% of NP and relative standard deviations were equal or lower than 15% for egg samples. The average recoveries in milk ranged from 86 to 84% for BPA, 90 to 99% for NP and 82 to 103% for OP and relative standard deviations were equal to or lower than 8%. The limits of detection (LODs) in eggs were 0.10, 0.10 and 0.25 μg/kg for BPA, NP and OP, respectively and LODs for milk were 0.10, 0.05 and 0.10 μg/kg for BPA, NP and OP, respectively. Investigation of the levels in commercial samples indicated that NP was ubiquitous in milk and eggs at levels ranging from 4.24 to 17.60 μg/kg, and the milk samples were more heavily contaminated by NP than were the egg samples.
Keywords: Alkylphenol; Bisphenol A; Matrix solid phase dispersion; LC–ESI–MS/MS;
Determination of clarithromycin in human serum by high-performance liquid chromatography after pre-column derivatization with 9-fluorenylmethyl chloroformate: Application to a bioequivalence study by Gholamreza Bahrami; Bahareh Mohammadi (417-422).
A sensitive liquid chromatographic method for the analysis of clarithromycin, a macrolide antibiotic, in human serum using pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) is described. The method involved liquid–liquid extraction of the drug and an internal standard (amantadine) followed by pre-column derivatization of the analytes with FMOC-Cl. A mixture of 0.05 M phosphate buffer containing triethylamine (2 mL L−1; pH 3.8) and methanol (17:83, v/v) was used as mobile phase and chromatographic separation was achieved on a Shimpack CLC-ODS column. The eluate was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The analytical method was linear over the concentration range of 0.025–10 μg mL−1 of clarithromycin in human serum with a limit of quantification of 0.025 μg mL−1. The assay is sensitive enough to measure drug levels obtained in human single dose studies. In the present method, sensitivity and run time of analysis have been improved, and successfully applied in a bioequivalence study of three different clarithromycin preparations in 12 healthy volunteers.
Keywords: Clarithromycin; Reverse phase chromatography; Bioequivalence study; Macrolide antibiotics;
Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC–MS by Rosa Cordero; Sue Paterson (423-431).
A method is described for the simultaneous identification and quantification of opiates, amphetamines, cocainics, diazepam and nordiazepam from one hair extract (typically 10–50 mg hair). After decontamination by washing with shampoo, dichloromethane, isopropanol and acetone, drugs were extracted using 0.1 M HCl followed by SPE clean-up using mixed-mode extraction cartridges. The SPE extracts were submitted to a two-step derivatisation using MBTFA and MSTFA + 1% TCMS and analysis was performed by GC–MS using both SIM and scan modes. Four deuterated standards were used to monitor 14 compounds. The limit of quantification was the total drug detected from the sample. This was 5 ng for amphetamines and 10 ng for remaining drugs which is equivalent to 0.1 and 0.2 ng/mg from a 50 mg sample.Standard curves for the range 5–400 ng total drug concentration for all drugs had regression coefficients greater than 0.98. An authentic hair sample was used to validate the method and gave R.S.D.s <25% for both inter and intra-day reproducibility.The results of the analysis of hair taken from four patients attending a drug treatment clinic and six hair samples including head hair, pubic hair, axial hair and beard taken at post-mortem are presented.
Keywords: GC–MS; Hair; Drugs of abuse;
Rapid online-SPE-MS/MS method for ketoprofen determination in dermal interstitial fluid samples from rats obtained by microdialysis or open-flow microperfusion by Karin E. Pickl; Christoph Magnes; Manfred Bodenlenz; Thomas R. Pieber; Frank M. Sinner (432-439).
Pharmacokinetic studies of topical ketoprofen formulations using continuous sampling techniques such as microdialysis (MD) or open-flow microperfusion (OFM) require sensitive assays due to small sample volumes. A simple and easy online-SPE-MS/MS method for ketoprofen analysis was developed for both MD and OFM samples obtained from rat dermal tissue. The quantification range is 25–5000 ng/ml with a limit of detection of 3 ng/ml using only 10 μl sample volume. The method is characterized by a simple setup using a short polymeric SPE column (OASIS HLB) for desalting with 1.5 min run times in combination with a sensitive MS detection in negative ESI MRM mode. An easy sample workup procedure was used which enables high throughput analysis of a large number of samples for pharmacokinetic studies. In addition, a commercial available (fenoprofen) as well as an isotopically labelled (deuterated ketoprofen) standard were investigated as potential internal standards. The method was validated according to FDA guidelines for bioanalytical validation in terms of accuracy, intra-batch and inter-batch precision, linearity, matrix effect, recovery and stability for both internal standards. Accuracies were 98–113% (fenoprofen) and 95–108% (deuterated ketoprofen), intra-batch precision was 2–3% R.S.D. (fenoprofen) and 2–6% R.S.D. (deuterated ketoprofen), and inter-batch precision was 2–6% R.S.D. (fenoprofen) and 3–6% R.S.D. (deuterated ketoprofen) over the entire quantification range. The presented method was applied to dermal interstitial fluid samples obtained in a topical administration study of ketoprofen in rats.
Keywords: Ketoprofen; Interstitial fluid; Microdialysis; Open-flow microperfusion;
Liquid chromatography mass spectrometry profiling of histones by Xiaodan Su; Naduparambil K. Jacob; Ravindra Amunugama; David M. Lucas; Amy R. Knapp; Chen Ren; Melanie E. Davis; Guido Marcucci; Mark R. Parthun; John C. Byrd; Richard Fishel; Michael A. Freitas (440-454).
Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RPLC–MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC–MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC–MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU–PAGE separation and nano-LC–MS/MS.
Keywords: Histone; RPLC–MS; Post-translational modification; AU–PAGE; Nano-LC–MS/MS;
Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery by Danielle Smith; Nalini Sadagopan; Michael Zientek; Anita Reddy; Lucinda Cohen (455-463).
The use of a cassette incubation of probe substrates with human liver microsomes (HLM) – also known as the ‘cocktail’ approach – is becoming a widely accepted approach to determine the interaction of new chemical entities (NCEs) with cytochrome P450 enzymes (CYP450) in early drug discovery. This article describes two LC–MS/MS-based analytical methods used at the high-throughput (HT) stage and late discovery (LD) stage for analysis of ‘cocktail’ incubates to analyze the probe metabolites 1′-hydroxymidazolam (CYP3A4), 4′-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), 1′-hydroxytacrine (CYP1A2) and 4′-hydroxymephenytoin (CYP2C19). The analytical methods are advantageous over currently reported methods due to their sensitivity, shorter analyses times (<2 min/sample for the HT method and 4 min/sample for the LD method) and their ability to monitor a unique set of clinically relevant probe metabolites from a biological incubate containing low microsomal protein (0.1 mg/mL). The analytical methods employ the same mobile phase, acetonitrile and 0.1% formic acid, under similar LC–MS/MS conditions. In the HT method, the chromatographic method consists of a short robust step-gradient where the probe metabolites are simultaneously and quickly eluted to enhance throughput. The probe metabolites are chromatographically resolved in the LD stage by utilizing a true linear gradient to obtain optimal peak separation. The IC50 data generated by both analytical methods using single incubations versus cocktail incubations for various test compounds are in good agreement (correlation coefficient (r 2) ≥ 0.98). The scientist conducting the analysis is provided with a choice of method selection depending on the stage of the test compound and on whether throughput or minimizing interference from other co-eluting metabolites is the most important criterion.
Keywords: In vitro cytochrome P450 inhibition assay; LC–MS/MS; DDI; Cocktail DDI analysis; Bioanalysis; CYP3A4; CYP2D6; CYP2C19; CYP2C9; CYP1A2; High-throughput analysis; Drug discovery;
Quantitative determination of the macrolide antibiotic tulathromycin in plasma and broncho-alveolar cells of foals using tandem mass spectrometry by Eberhard Scheuch; Janina Spieker; Monica Venner; Werner Siegmund (464-470).
The long-acting antibiotic tulathromycin is on the marked for treatment of pulmonary infection of cattle, swine and horses. To measure disposition and distribution of tulathromycin in foals, a high throughput method was developed for horse plasma (calibration range: 0.006–0.8 μg/mL) and broncho-alveolar cells (calibration range: 0.1–4.0 μg/109 cells) using tandem mass spectrometry. Tulathromycin was extracted from plasma and broncho-alveolar fluid using cation exchange cartridges with acetonitrile/ammonia (95:5, v/v). The chromatography was performed isocratically with a mobile phase consisting of 5 mM ammonium formiate buffer/acetonitrile (30:70, v/v). The mass spectrometer operated in selected ion mode with atmospheric pressure chemical ionization to monitor the respective MH+ ions m/z 577.3 for tulathromycin and m/z 679.3 for the internal standard roxithromycin. All quality parameters fulfilled the international criteria for bioanalytical method validation and were successfully applied to the determination of tulathromycin in plasma of foals and broncho-alveolar cells. In foals, tulathromycin after intramuscular administration was rapidly absorbed, widely distributed and slowly eliminated. It cumulated manifold in broncho-alveolar cells.
Keywords: Tulathromycin; Drug assay; Pharmacokinetics; Broncho-alveolar cells; LC–MS/MS;
Simultaneous LC–MS–MS determination of cyclosporine A, tacrolimus, and sirolimus in whole blood as well as mycophenolic acid in plasma using common pretreatment procedure by Maciej J. Bogusz; Eid Al Enazi; Huda Hassan; Jamil Abdel-Jawaad; Jamal Al Ruwaily; Mohammed Al Tufail (471-480).
The purpose of the study was to develop rapid and simple procedure for simultaneous determination of cyclosporine A (CsA), tacrolimus (TCR), and sirolimus (SIR) in whole blood and mycophenolic acid (MPA) in plasma. Ascomycin (ASCO), cyclosporine D (CsD), and desmethoxysirolimus (DMSIR) were used as internal standards (IS) for TCR, CsA and MPA, and SIR, respectively. In the method development, six-level blood calibrators were used for CsA (range 47–1725 ng/ml), TCR (range 2.1–38.8 ng/ml), and SIR (range 2.4–39.6 ng/ml). Four-level calibrators were used for MPA (range 0.15–5.48 μg/ml). Four levels of quality control (QC) standards were used for blood samples, together with two levels of QC standards in plasma. All QC standards and calibrators were obtained from commercial sources. Sample preparation based on precipitation of 50 μl of sample in zinc sulfate–methanol–acetonitrile mixture containing IS, followed by centrifugation. HPLC was performed on ChromSpher π column, 30 mm × 3 mm, in ballistic gradient of ammonium formate buffer–methanol at 0.8 ml flow rate. Following gradient elution profile was applied: 0–1.2 min at 30% methanol (divert valve to waste), 1.21–3.1 min 97% methanol (divert valve to detector), 3.11–3.7 min 30% methanol (divert valve to waste). ESI–MS–MS (MRM) was done on TSQ Quantum instrument with ESI source in positive ion mode. Ammoniated adducts of protonated molecules were used as precursor ions for all analytes but MPA. For this compound sodium adduct was used. Following transitions were monitored: for CsA m/z 1220–1203; for CsD 1234–1217; for SIR 931.6–864.5 and 882.6; for DMSIR 902–834.5; for TCR 821.5–768.5 and 785.5; for ASCO 809.5–756; for MPA 343–211.6; for MPA-glucuronide 514–306 and 211.6. The limits of quantitation were: 1 ng/ml for TCR and SIR, 20 ng/ml for CsA, and 0.1 μg/ml for MPA. Post-column infusion experiments showed that no positive or negative peaks appeared after injection of matrix in the elution range of target compounds. General signal suppression caused by matrix ranged from 20–40%, and was caused mainly by zinc sulfate present in deproteinizing solution. Extracted samples were stable for 2 days at 4 °C and for at least 20 days at −20 °C. MPA was fully separated from its glucuronide, which was eluted at around 0.7–0.8 min and directed to the waste. Some mutual cross-contribution of CsD and CsA was observed (below 1%), other IS did not contribute to target compounds and vice versa. Observations of chromatograms from patients taken single therapy demonstrated that possible metabolites of CsA, TCR, or SIR did not interfere with target compounds or IS.
Keywords: Immunosuppressants; TDM; LC–MS–MS;
Very small injected samples to study chloroquine and quinine in human serum using capillary-LC and native fluorescence by H. Ibrahim; J. Bouajila; N. Siri; G. Rozing; F. Nepveu; F. Couderc (481-487).
A comparison between HPLC with conventional fluorescence detection and capillary-LC (μHPLC) with native laser-induced fluorescence (LIF) detection was done to determine chloroquine (CQ) and quinine (Q) in human serum. HPLC experiments were run with parameters of the conventional fluorimeter set at the highest level of sensitivity. Results were compared with those obtained on μHPLC coupled to a ZETALIF (He–Cd 325 nm) detector which provided a 50-fold increase in sensitivity. In μHPLC-LIF injection volumes were 200 nL instead of 10 μL in conventional HPLC. The separation was completed within 3 min (6 min on HPLC). The limit of detection on μHPLC-LIF was 1.9 and 1.3 fmol for CQ and Q, respectively. Both experiments were validated on serum samples. The mean recovery was more than 95% for CQ and Q. The intra- and inter-day precision and accuracy were found to be within the acceptable limits (<10%).
Keywords: μHPLC; Laser-induced fluorescence detection; Chloroquine; Quinine;
Determination of alginate copolymer in pharmaceutical formulations by micellar electrokinetic chromatography by Nevin Öztekin; Selda Başkan; F. Bedia Erim (488-492).
A micellar electrokinetic chromatography method was developed for the determination and quantification of sodium alginate. The alginate peak migrated in the very short time of 1.33 min and calibrated easily though the polydisperse properties of alginates. The minimum detection limit (LOD) of the method was calculated as 0.393 mg/ml. This analysis method was successfully applied to the alginate quantification in an antacid pharmaceutical formulation. Precise and reproducible analysis results were obtained, with liquid formulations injected directly without any pre-separation process.
Keywords: Capillary electrophoresis; MEKC; Alginate; Antacid;
Direct quantification of dimethylsulfoniopropionate (DMSP) in marine micro- and macroalgae using HPLC or UPLC/MS by Theresa Wiesemeier; Georg Pohnert (493-498).
A simple method for the direct quantification of dimethylsulfinopropionate (DMSP) using HPLC or UPLC coupled to UV and/or MS detection is introduced. The protocol is applied for the determination of DMSP from marine micro- and macroalgae. The method is based on the derivatisation of DMSP using 1-pyrenyldiazomethane followed by reversed phase HPLC or UPLC separation. The detection limit is 590 nM, corresponding to 1 ng DMSP per injection. Using a combination of UV and MS detection the calibration curves were linear in the range of 2.93 μM to 11.7 mM concentrations. We show that direct determination of DMSP is possible from macroalgal tissue and microalgal cultures if DMSP-lyase activity is suppressed during work-up.
Keywords: HPLC/MS; UPLC/MS; Marine plankton; Dimethylsulfide (DMS); Dimethylsulfinopropionate (DMSP);
Conjugation of enzyme on superparamagnetic nanogels covered with carboxyl groups by Jun Hong; Dongmei Xu; Peijun Gong; Hongjuan Ma; Li Dong; Side Yao (499-506).
α-Chymotrypsin (CT) as model enzyme was conjugated onto the novel carboxyl-functionalized superparamagnetic nanogels, prepared via facile photochemical in situ polymerization, by using 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC) as coupling reagent. The obtained magnetic immobilized enzyme was characterized by use of photo correlation spectroscopy (PCS), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) measurement, thermogravimetric (TG) analysis and vibrating sample magnetometer (VSM) measurement. PCS result showed that the immobilized enzyme was 68 nm in diameter while the magnetic nanogels with carboxyl groups were only 38 nm; enzyme immobilization led to pronounced change in size. Superparamagnetic properties were retained for Fe3O4 after enzyme immobilization while slightly reducing its value of saturation magnetization. Immobilization and surface coating did not induce phase change of Fe3O4 by XRD analysis. The binding capacity was 30 mg enzyme/g and 37.5 mg enzyme/g nanogel determined by TG analysis and BCA protein assay, respectively. Specific activity of the immobilized CT was calculated to be 0.77 U/(mg min), 82.7% as that of the free form.
Keywords: Carboxyl-functionalized magnetic nanogels; Immobilized enzyme; Magnetic separation; Photochemical in situ polymerization;
A rapid method for the quantification of the enantiomers of Warfarin, Phenprocoumon and Acenocoumarol by two-dimensional-enantioselective liquid chromatography/electrospray tandem mass spectrometry by Gennaro Vecchione; Bruno Casetta; Michela Tomaiuolo; Elvira Grandone; Maurizio Margaglione (507-514).
We describe a new fully validated enantioselective LC–MS/MS method for stereospecific quantification of both the racemic forms of Warfarin (WF), Phenprocoumon and Acenocoumarol in human plasma. Measurement specificity was assessed by using different blank donor plasma samples, where no interfering reagent peak appeared at the retention time (RT) of the targeted analytes. Response was linear for all analytes. Typical linear regression coefficients have >0.99. The recoveries ranged from 98% to 118%. Determinations in 10 normal healthy individuals revealed a high reproducibility of RTs. These findings confer to the method suitability for large population studies.
Keywords: Vitamin K-dependent factors; Venous thrombosis; Warfarin; Phenprocoumon; Acenocoumarol; enantiomers; LC–MS–MS;
Development of a LC/MS/MS method for the analysis of cannabinoids in human EDTA-plasma and urine after small doses of Cannabis sativa extracts by Sandra B. Grauwiler; André Scholer; Jürgen Drewe (515-522).
A novel high-performance liquid chromatographic separation method with tandem-mass spectrometry detection was developed for the simultaneous determination of Δ9-tetrahydrocannabinol (THC) and its major metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) as well as the components cannabidiol (CBD) and cannabinol (CBN) in human EDTA-plasma and urine. Run time was 25 min. Lower limit of quantification was 0.2 ng/ml. The coefficients of variation of all inter- and intra-assay determinations were between 1.3 and 15.5%. The method was successfully applied to the determination of cannabinoids in human plasma and human urine after administration of Δ9-tetrahydrocannabinol or Cannabis sativa extracts.
Keywords: Δ9-Tetrahydrocannabinol; 11-Hydroxy-Δ9-tetrahydrocannabinol; 11-Nor-Δ9-tetrahydrocannabinol-9-carboxylic acid; Cannabidiol; Cannabinol; Cannabinoids; LC/MS/MS; APCI; Human EDTA-plasma; Human urine;
The isolation of 1,2,3,4,6-penta-O-galloyl-beta-d-glucose from Acer truncatum Bunge by high-speed counter-current chromatography by Wen-Hua Zhao; Chun-Chun Gao; Xiao-Feng Ma; Xiao-Yu Bai; Ying-Xia Zhang (523-527).
High-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of 1,2,3,4,6-penta-O-galloyl-beta-d-glucose from the ethyl acetate extract of the leaves of Acer truncatum Bunge using a two-phase system composed of n-hexane-ethyl acetate-methanol-water at a volume ratio of (0.25:5:1:5, v/v/v/v) for the first time. Each injection of 80 mg crude extract yielded 7.25 mg of pure 1,2,3,4,6-penta-O-galloyl-beta-d-glucose. High-performance liquid chromatography (HPLC) analyses of the CCC fraction revealed that the purity of 1,2,3,4,6-penta-O-galloyl-beta-d- glucose was over 95%.
Keywords: 1,2,3,4,6-penta-O-galloyl-beta-d-glucose; Acer truncatum Bunge; Counter-current chromatography;
A TLC bioautographic assay for the detection of nitrofurantoin resistance reversal compound by Ahmad R. Shahverdi; Farid Abdolpour; Hamid R. Monsef-Esfahani; Hasan Farsam (528-530).
A simple TLC bioautographic method was developed for detection of antibiotic resistance reversal agents. In this study, the retention factor values of the components of some essential oils not previously shown to have any antibacterial activity were evaluated on nitrofurantoin supplemented agar media. The active component of Artemisia annua, Artemisia dracunculus and Eucalyptus globulus essential oils was piperitone which increased the antibacterial activity of nitrofurantoin against Enterobacter cloacae. Piperitone was not detected in the essential oil of Humulus lupulus and we could not observe any clear areas in this bioautographic method.
Keywords: Bioautographic method; Antibiotic resistance; Essential oils; Piperitone;
Sensitive electrochemical detection method for α-acids, β-acids and xanthohumol in hops (Humulus lupulus L.) by Javor Kac; Tomaž Vovk (531-537).
A new HPLC method with coulometric detection for the quantification of xanthohumol, α-acids and β-acids in hops was developed. The separation of compounds was accomplished with a C18 column and isocratic elution with methanol: 50 mM potassium phosphate: ortho-phosphoric acid = 80:20:0.25 (v/v/v). The method was validated and UV and electrochemical detectors (ECD) were compared. The HPLC method with ECD was precise, accurate and very sensitive for detection of xanthohumol and α- and β-acids. The detection limits of analytes were at least 8.8 to 24 times lower with ECD than those of the UV detector. The ECD method was successfully applied for quantification of studied compounds in hop pellets. The concentrations of all compounds obtained with ECD and UV were found to be equivalent. This is the first study demonstrating a very sensitive and validated method for the quantification of xanthohumol, α- or β-acids in hop samples with the use of the electrochemical detector.
Keywords: Hops; Xanthohumol; Humulones; Lupulones; Electrochemical detector; UV detector;
Simultaneous determination of buprenorphine, norbuprenorphine and the enantiomers of methadone and its metabolite (EDDP) in human plasma by liquid chromatography/mass spectrometry by Maria Esther Rodriguez-Rosas; Michelle R. Lofwall; Eric C. Strain; Danuta Siluk; Irving W. Wainer (538-543).
A previously reported enantioselective LC–MS assay for the determination of (R)- and (S)-methadone [Met] and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine [EDDP] (the primary metabolite of Met) has been adapted for use in the simultaneous determination of the plasma concentrations of Met, EDDP, buprenorphine (Bu) and norbuprenorphine (norBu). All of the target compounds were separated within 15 min using an α1-acid glycoprotein chiral stationary phase, a mobile phase composed of acetonitrile: ammonium acetate buffer [10 mM, pH 7.0] in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min at 25 °C. Deuterium labeled compounds were used as internal standards [d4-Bu, d3-norBu, (R,S)-d3-Met and (R,S)-d3-EDDP] and linear relationships between peak height ratios and drug concentrations were obtained for Bu and norBu in the range 0.2–12 ng/ml with correlation coefficients greater than 0.999. The relative standard deviations (%R.S.D.) for the intra- and inter-day precision of the method were <4.5% and for accuracy was <4.0%. The method was validated and used to analyze plasma samples obtained from opioid dependent methadone-maintained adults enrolled in a research study.
Keywords: Buprenorphine; Norbuprenorphine; (S)-methadone; (R)-methadone; (S)-EDDP; (R)-EDDP; Chiral chromatography; AGP chiral stationary phase; LC–MS;
Quantification of l-stepholidine in rat brain and plasma by high performance liquid chromatography combined with fluorescence detection by John Odontiadis; Erin M. MacKenzie; Sridhar Natesan; David Mamo; Shitij Kapur; Glen B. Baker (544-547).
A sensitive and reliable assay for the quantification of l-stepholidine (SPD) in rat plasma and brain was developed using high performance liquid chromatography (HPLC) combined with fluorescence detection. Brain regions (prefrontal cortex, striatum, and cerebellum) and plasma from rats treated with SPD (10 mg/kg s.c.) 20, 40, 60, or 90 min prior to euthanasia were analyzed for SPD levels. Brain samples were homogenized in ice-cold 0.1 M perchloric acid and centrifuged to remove proteins. The supernatants and diluted plasma samples, to which O-desmethylvenlafaxine was added as a process standard, were basified and extracted with ethyl acetate. The organic phase was taken to dryness and the residue taken up in mobile phase. The samples were then injected into an HPLC equipped with a fluorescence detector (excitation and emission wavelengths set at 280 and 320 nm, respectively). The mean recovery of SPD was 74.6%, and reliability studies confirmed the reproducibility of the assay (intra- and inter-assay coefficients of variation of 4.8% and 5.3%, respectively). The assay was readily applicable to the brain and plasma samples obtained from rats injected with SPD as described above; the levels and patterns of disappearance of SPD in brain regions and plasma are shown.
Keywords: l-Stepholidine; High performance liquid chromatography; Antipsychotic; Schizophrenia;
Determination of the DNA methylation level of the marbled crayfish: An increase in sample throughput by an optimised sample preparation by Ralf Schiewek; Michaela Wirtz; Markus Thiemann; Kay Plitt; Günter Vogt; Oliver J. Schmitz (548-552).
Using a previously described capillary electrophoretic method with laser-induced fluorescence detection the genomic methylation level can be determined exactly. We present a sample preparation that eliminates the surplus of fluorescence marker used for coupling resulting in an increase of sample throughput from 75 to 250 analyses per week. The sensitivity of the method was also increased, which allows the determination of methylation levels under 1%. With these changes in sample preparation a methylation level of 1.64 ± 0.03% in hepatopancreas DNA of the recently discovered marbled crayfish could be determined.
Keywords: DNA methylation; 5-Methylcytosine; Epigenomics; CE-LIF; Marbled crayfish;
Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies by Gustavo D. Mendes; Daniele Hamamoto; Jaime Ilha; Alberto dos Santos Pereira; Gilberto De Nucci (553-559).
A rapid, sensitive and specific method for quantifying the aromatase inhibitor (anastrozole) in human plasma using dexchlorpheniramine as the internal standard (I.S.) is described herein. The analyte and the I.S. were extracted from 200 μl of human plasma by liquid–liquid extraction using a mixture of diethyl ether:dichloromethane (70:30, v/v) solution. Extracts were removed and dried in the organic phase then reconstituted with 200 μl of acetonitrile:water (50:50; v/v). The extracts were analyzed by high performance liquid chromatography coupled with photospray tandem mass spectrometry (HPLC–MS–MS). Chromatography was performed isocratically on a Genesis, C18 4 μm analytical column (100 mm × 2.1 mm i.d.). The method had a chromatographic run time of 2.5 min and a linear calibration curve ranging from 0.05–10 ng ml−1. The limit of quantification (LOQ) was 0.05 ng ml−1. This HPLC–MS–MS procedure was used to assess pharmacokinetic studies.
Keywords: Anastrazole; Photospray; Healthy volunteer; Plasma; Pharmacokinetics;
Enantioselective separation of phenylglycidates by capillary electrophoresis employing sulfated β-cyclodextrin as chiral selector by Jianjun Wang; Qipeng Yuan; David G. Evans; Liu Yang; Guojun Zheng; Wanru Sun (560-563).
A capillary electrophoresis (CE) method for the enantioseparation of phenylglycidates has been developed. Successful enantioseparation was achieved using sulfated β-cyclodextrin as chiral selector in a phosphate buffer. The effects of varying pH, sulfated β-cyclodextrin concentration and electrophoresis voltage were systematically investigated and the optimized separation conditions were thus obtained. When the migration time was set at the threshold value, it was found that the best enantioseparation was obtained at 10 kV with 3% (w/v) sulfated β-cyclodextrin at pH 6.5. A range of substituted phenylglycidates were successfully separated using the method and the results shown to be superior to those obtained using gas chromatography (GC).
Keywords: Enantioseparation; Sulfated β-cyclodextrin; Phenylglycidates;
Rapid and sensitive liquid chromatography–tandem mass spectrometry method for the quantitation of colchicine in human plasma by Yao Jiang; Jiang Wang; Yingwu Wang; Hao Li; J. Paul Fawcett; Jingkai Gu (564-568).
A rapid and sensitive method to determine colchicine in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed. Colchicine and the internal standard (I.S.), tegafur, were extracted from the matrix with n-hexane:dichloromethane:isopropanol (300:150:15, v/v/v) and separated by reversed-phase high-performance liquid chromatography (HPLC) using formic acid:10 mM ammonium acetate:methanol (1:49:75, v/v/v) as the mobile phase in a run time of 2.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 0.050–10 ng/ml with intra- and inter-day precision (as relative standard deviation (R.S.D.)) of <2 and <7%, respectively. The method was applied to a pharmacokinetic study of colchicine in healthy volunteers given an oral dose of 2.0 mg.
Keywords: Colchicine; LC–MS/MS; Pharmacokinetics; Human;
Determination of treosulfan in plasma and urine by HPLC with refractometric detection; pharmacokinetic studies in children undergoing myeloablative treatment prior to haematopoietic stem cell transplantation by Franciszek K. Główka; Marta Karaźniewicz Łada; Grzegorz Grund; Jacek Wachowiak (569-574).
A direct and selective HPLC method with refractometric detection was worked out for determination of treosulfan in plasma and urine of children. Before injection onto reverse phase column plasma samples with treosulfan and barbital (I.S.) were clarified using filtration. The mobile phase was composed of phosphate buffer, pH 5 and acetonitrile. The linear range of the standard curve of treosulfan spanned concentrations of 10.0–2000.0 μg/ml and 50.0–10000.0 μg/ml in plasma and urine, respectively, and covered the levels found in biological fluids after infusion of the drug. The limit of detection amounted to 5 μg/ml for plasma and 25 μg/ml for urine. Intra- and inter-day precision and accuracy of the measurement fulfilled analytical criteria accepted in pharmacokinetic studies. Recovery of treosulfan as well as stability in biological fluids was also calculated. The validated method was successfully applied in pharmacokinetic studies of treosulfan administered to children prior to haematopoietic stem cell transplantation. Differences between pharmacokinetics of treosulfan in children and adults were also studied.
Keywords: Alkylating compounds; Infusion; Microfiltration; Pharmacokinetic study; Pediatric patients;
Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: Application to cellular metabolism and accumulation studies by Ze-Ping Hu; Xiao-Xia Yang; Xiao Chen; Eli Chan; Wei Duan; Shu-Feng Zhou (575-580).
A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile–50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm × 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9–108.3% for CPT-11 in culture media and 94.3–107.2% for CPT-11 in cell lysates; and 87.7–106.8% for SN-38 in culture media and 90.1–105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.
Keywords: CPT-11; SN-38; Metabolism; Accumulation; HPLC;
Ultra-performance liquid chromatography–tandem mass spectrometry for the determination of atypical antipsychotics and some metabolites in in vitro samples by Kun-Yan Li; Yan-Gang Zhou; Hua-Yi Ren; Feng Wang; Bi-Kui Zhang; Huan-De Li (581-585).
The ultra-performance™ liquid chromatography–electrospray tandem mass spectrometry (UPLC–ESI-MS/MS) method has been developed to perform the determination of quetiapine, perospirone, aripiprazole and quetiapine sulfoxide in in vitro samples in less than 3 min. The UPLC separation was carried out using an Acquity UPLC™ BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size) that provided high efficiency and resolution in combination with high linear velocities. The UPLC system was coupled to a Waters Micromass Quattro Premier XE tandem quadrupole mass spectrometer. This system permits high-speed data acquisition without peak intensity degradation, and produces sharp and narrow chromatographic peaks ( w h about 2.5 s) of compounds. The determination was performed in multiple reaction monitoring (MRM) mode. The quantification parameters of the developed method were established, obtaining instrumental LODs lower than 0.005 μg/l and a repeatability at a low concentration level lower than 10% CV (n = 10). Finally, the method was successfully applied to the analysis of atypical antipsychotics and some metabolites in in vitro samples.
Keywords: UPLC–MS/MS; Quetiapine; Perospirone; Aripiprazole; Quetiapine sulfoxide;
Asymmetric dimethylarginine—comparison of HPLC and ELISA methods by R. Široká; L. Trefil; D. Rajdl; J. Racek; R. Cibulka (586-587).
Keywords: ADMA; ELISA; HPLC;