Journal of Chromatography B (v.864, #1-2)

A sensitive, rapid liquid chromatographic–electrospray ionization mass spectrometric method for determination of erythromycylamine in human plasma was developed and validated. Erythromycylamine in plasma (0.2 mL) was extracted with ethyl acetate, the organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness under gentle nitrogen stream at 45 °C, and the residue was dissolved in 100 μL of mobile phase. The samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm × 2.1 mm I.D., 5 μm). A mobile phase containing 10 mM of ammonium acetate (pH = 6.4)-acetonitrile-methanol (50:10:40, v/v/v) was used isocratically eluting at a flow rate of 0.2 mL/min. Erythromycylamine and its internal standard (IS), midecamycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 4.5 to 720 ng/mL with r  = 0.9997. The limit of quantification for erythromycylamine in plasma was 4.5 ng/mL with good accuracy and precision. The mean extraction recovery of the method was higher than 75.1% and 72.7% for erythromycylamine and IS, respectively. The intra-day and inter-day precision ranged from 5.2% to 6.4% and 5.6–9.3% (relative standard deviation, RSD), respectively. The established method has been successfully applied to a bioequivalence study of two dirithromycin formulations for 18 healthy volunteers.
Keywords: Dirithromycin; Erythromycylamine; LC–MS; Electrospray ionization; Bioequivalence;

A sensitive and specific liquid chromatography–mass spectrometry method for determination of metacavir in rat plasma by Zhan Li; Xin Huang; Zhenzhou Jiang; Yajie Xiao; Chunhui Liu; Luyong Zhang; Bin Shu; Jingfeng Huang; Tao Li; Tao Wang; Feng Wang (9-14).
A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) method has been developed and validated for the quantification of metacavir in rat plasma using tinidazole as an internal standard (I.S.). Following ethyl acetate extraction, the analytes were separated on a Shim-pack ODS (4.6 μm, 150 mm × 2.0 mm I.D.) column and analyzed in selected ion monitoring (SIM) mode with a positive ESI interface using the respective [M  + H]+ ions, 266 for metacavir and 248 for tinidazole. The method was validated over the concentration range of 1–600 ng/mL for metacavir. Between and within-batch precisions (R.S.D.%) were all within 15% and accuracy (%) ranged from 92.2 to 105.8%. The lower limit of quantification (LLOQ) was 1 ng/mL. The extraction recovery was on average 89.8%. The validated method was used for the pharmacokinetic study of metacavir in rats.
Keywords: Liquid chromatography/mass spectrometry; HBV; Metacavir; Rat plasma; Pharmacokinetics;

Flow field-flow fractionation/multiangle light scattering of sodium hyaluronate from various degradation processes by Myeong Hee Moon; Da Young Shin; Namkoo Lee; Euijin Hwang; Il-Hwan Cho (15-21).
Sodium hyaluronate (NaHA) is an ultrahigh molecular weight polysaccharide that is found in body tissues, synovial fluid, the vitreous humor, and the umbilical cord, and the size characterization of NaHA is important in pharmaceutical applications. On-line field-flow fractionation/multiangle light scattering/differential refractive index (FlFFF/MALS/DRI) has been applied for the study of degradation efficiency of sodium hyaluronate (NaHA). A NaHA raw sample was degraded by different chemical or physical methods and the degraded NaHA samples were separated using field-programming FlFFF, in which separation is achieved by differences in diffusion coefficients or hydrodynamic diameters. Separation was followed by serial detection using MALS and DRI. Molecular weight distribution (MWD) and information relating to the radius of gyration of the NaHA samples were examined for the raw and degraded NaHA samples. Samples studied include: two different products of ultrasonic degradation, two products of alkaline degradation, and four different products of enzymatic degradation. While alkaline degradation showed a moderate degradation compared to ultrasonic and enzymatic methods in reducing average MW, the latter two degradation methods showed significant changes in average molecular weight and in conformation of NaHA.
Keywords: Flow field-flow fractionation; Multiangle light scattering; Sodium hyaluronate; Molecular weight distribution; Degraded sodium hyaluronate;

Lysophosphatidic acid (LPA) is a class of lipids that play multiple biological functions. Several reports show that they are potential biomarkers for diagnosing ovarian cancer. Therefore, it is necessary to accurately quantify their levels in biological samples. Here we report a high throughput LC/ESI/MS/MS (liquid chromatography electrospray tandem mass spectrometry) method employing a reversed phase C18 column to quantify LPA. In this method, a [13C16] labeled 16:0 LPA is used as the internal standard and the lipids are extracted out from biological samples using Bligh–Dyer method under highly acidic condition. The total run time is 8 min. The detection limits of the assay reach fmol level and the CV% of the assay are within 10%. Using this method, we quantify the levels of six LPA species (16:0, 18:2, 18:1, 18:0, 20:4, and 22:6 LPA) in plasma samples. We find that some unknown compounds present in plasma can interfere with the quantification of LPA if they are not well separated from LPA. These unknown compounds are more hydrophobic than LPA and can be removed by thin-layer chromatography (TLC). We also find that the levels of LPA species in human plasma generally follow the order: 18:2 LPA > 16:0 LPA, 20:4 LPA > 18:1 LPA, 22:6 LPA, and 18:0 LPA.
Keywords: LPA; Lysophosphatidic acid; LC/ESI/MS/MS; LPA quantification;

Determination of the oxido-redox status of plasma albumin in hemodialysis patients by Maurizio Bruschi; Andrea Petretto; Giovanni Candiano; Luca Musante; Ezio Movilli; Laura Santucci; Andrea Urbani; Rosanna Gusmano; Enrico Verrina; Giovanni Cancarini; Francesco Scolari; Gian Marco Ghiggeri (29-37).
The oxido-redox status of plasma albumin in patients treated with hemodialysis was characterized with LC–ESI-MS/MS and was compared with models of oxidative stress. Oxidised albumin was characterized by sulfonation (SO3 ) of the SH at Cys 34, unfolding and acidification of the molecule. Albumin in hemodialysis patients presented, instead, only intermediate oxidation products such as sulfenic (SO2), sulfonic (SO) and methionine sulfoxide (C5H9NO2S) involving Cys 165–269 and Met 329–548 but did not present SO3 at Cys 34. Absence of charge and structural alterations compared to the oxidised templates was also confirmed with electrophoretic titration and calorimetry. In conclusion, the oxido-redox status of plasma albumin in hemodialysis patients lacks the hallmarks of the advanced oxidation products. LC–ESI-MS/MS was crucial to characterize albumin in conditions of oxidation stress; surrogate techniques can mirror conformational changes induced by oxidation.
Keywords: Plasma albumin; Oxidation stress; Oxidation markers; Hemodialysis;

This study was aimed at developing a fast and sensitive method for determination of oseltamivir carboxylic acid (OCA), the active moiety of anti-influenza agent, oseltamivir phosphate, in human serum by high performance liquid chromatography and UV detection. The analyte and an internal standard (vanillin) were extracted from human serum by a solid phase extraction (SPE) procedure. Chromatographic separation was achieved using a reverse phase C18 column with a mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (1 mL/L; pH 3.0) and acetonitrile (70:30, v/v). The detection wavelength was set at 215 nm. The average recoveries of the drug and internal standard were 98 and 85%, respectively. The calibration curve was linear over a concentration range of 15–6400 ng/mL of OCA in human serum. The lower limits of detection and quantification were 5 and 15 ng/mL, respectively. The coefficient variation values of both inter- and intra-day analysis were less than 12% whereas the percentage error was less than 4.5. The stability of the drug at the serum samples maintained at −40 °C for 60 days was found to be 100% from the initial value and no interferences were found from either endogenous components in serum or commonly co-administrated antiviral drugs. The validated method was applied to a randomized cross-over bioequivalence study of two different oseltamivir phosphate preparations in 24 healthy volunteers.
Keywords: HPLC; Oseltamivir; Influenza; Bioequivalence study;

Simultaneous determination of adenosine triphosphate and its metabolites in human whole blood by RP-HPLC and UV-detection by Erik J.C.M. Coolen; Ilja C.W. Arts; Els L.R. Swennen; Aalt Bast; Martien A. Cohen Stuart; Pieter C. Dagnelie (43-51).
To obtain insight in mechanisms of action of extracellular adenosine triphosphate (ATP) and adenosine, a simple HPLC method has been optimized and applied to investigate ATP metabolism in human whole blood ex vivo. This method provided good chromatographic resolution and peak shape for all eight compounds within a 19 min run time. The baseline was clean, the lower limit of quantification was below 0.3 μmol/L for all adenine nucleotides and the method demonstrated good linearity. Within-day precision ranged from 0.7 to 5.9% and between-days from 2.6 to 15.3%. Simplicity and simultaneous detection of ATP and its metabolites make this method suitable for clinical pharmacokinetic studies.
Keywords: ATP; Blood; HPLC; Adenosine;

In the present studies, to give momentum to traditionally low throughput pharmacokinetic screening, a bioanalytical method based on the concept of sample pooling for simultaneous bioanalysis of multiple compounds is discussed. A sensitive, selective, specific and rapid HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of three novel trioxane antimalarials (99–357, 99–408 and 99–411) in rat plasma using trioxane analogue as internal standard. The suitably validated bioanalytical method was then further extrapolated to rabbit and monkey plasma by performing partial validation. Extraction from the plasma involves a simple two-step liquid–liquid extraction with n-hexane. The analytes were chromatographed on a cyano column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 6) (85:15, v/v) and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) mode. The chromatographic run time was 5.5 min and the weighted (1/x 2) calibration curves were linear over a range of 1.56–200 ng/ml. The limit of detection (LOD) and lower limit of quantification (LLOQ) in rat plasma, rabbit plasma and monkey plasma were 0.78 and 1.56 ng/ml, respectively, for all three analytes. The intra- and inter-batch accuracy and precision in terms of % bias and % relative standard deviation were found to be well within the acceptable limits (<15%). The average absolute recoveries of 99–357, 99–408 and 99–411 from spiked plasma samples were >90%, >70% and >60%, respectively. The assay method described here could be applied to study the pharmacokinetics of 99–357, 99–408 and 99–411 using sample-pooling technique.
Keywords: Electrospray ionization; Tandem mass spectrometry; Sample pooling; Biomatrices; Trioxane antimalarials;

Validation of a gas chromatography–mass spectrometry method for the analysis of sterol oxidation products in serum by María Menéndez-Carreño; Cecilia García-Herreros; Iciar Astiasarán; Diana Ansorena (61-68).
A validated gas chromatography–mass spectrometry (GC–MS) detection method for the quantitative analysis of sterol oxidation products (SOPs) in serum is described. After a lipid extraction procedure with chloroform–methanol, a cold saponification and purification by solid phase extraction, oxysterols were derivatizated to form trimethyl-sylil-ethers which were subjected to GC–MS analysis. Calibration curves for cholesterol oxidation products showed determination coefficient (R 2) of 1.0, with low values for the coefficient of variation of the response factors (<1%). Detection and quantification limits were below 5 ng/mL and 10 ng/mL, respectively. Recovery data were between 77.65% and 110.29% (CV < 10% for all compounds). Good results were obtained for within- and between-day repeatability, with values below 10%. In conclusion, the method performed is suitable for the determination and quantification of SOPs in serum.
Keywords: Serum; Sterols; COPs; SOPs; Linearity; Recovery; Repeatability;

Combined with preparative high-performance liquid chromatography, high-speed counter-current chromatography was employed for isolation and purification of saponins from Gypsophila paniculata L. n-Hexane–n-butanol–methanol–0.02% TFA (1:9:1:9, v/v) was employed as solvent system and 210 nm was chosen as the wavelength of ultraviolet detection for the first time. The research tried to compare HSCCC with prep-HPLC, and further integrated their advantages to improve separation efficiency. Five known triterpene saponins were identified by 13C NMR and ESI-MS and their purities were all above 96%. The results demonstrated that adopted method was a feasible, economical and efficient technique for rapid preparative isolation of saponins.
Keywords: High-speed counter-current chromatography; Preparative high-performance liquid chromatography; Gypsophila paniculata L.; Saponins;

A rapid, sensitive and specific method was developed for the quantification of valacyclovir and acyclovir in human plasma. Sample preparation was performed by protein precipitation with acetonitrile followed by filtration. Valacyclovir, acyclovir and ganciclovir (internal standard) were separated isocratically on a reversed-phase porous graphitized carbon analytical column (2.1 mm × 125.0 mm i.d., particle size 5 μm), using a mobile phase of acetonitrile/water with 0.05% (v/v) diethylamine (50:50, v/v) at a flow rate of 0.15 mL min−1 in 4.0 min. Detection was performed by negative electrospray ionization using the selected ion monitoring mode of the deprotonated molecular ions at m/z 323.0 for valacyclovir, 224.0 for acyclovir and 254.0 for ganciclovir. The assay had linear calibration curves over the range 0.020–0.800 μg mL−1 for valacyclovir and 0.100–20.00 μg mL−1 for acyclovir. Accuracy and precision were within the acceptance limit of 15%. The method was successfully applied to the analysis of plasma samples obtained from patients after oral administration of valacyclovir.
Keywords: LC–ESI/MS; Valacyclovir; Acyclovir; Ganciclovir; Human plasma;

A new liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of ginkgolides (includes ginkgolide C for the first time) and bilobalide in plasma is presented. Ketoprofen was used as an internal standard, and sample pre-treatment consisted of a liquid–liquid extraction. Chromatographic separation was achieved on a 5 μm Shiseido C8 column (150 mm × 2.0 mm i.d., particle size 5 μm) with a mobile phase consisting of methanol/6 mM ammonium acetate (60/40, v/v) at a flow rate of 0.3 ml/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under negative ionization mode with an atmospheric pressure chemical ionization (APCI) interface. The method was validated in terms of intra- and inter-day precision (<12.7%), accuracy (within ±7.0%), linearity, specificity and stability. In addition, matrix effects of ginkgolides and bilobalide in plasma were evaluated in different reconstitution solvents. Smaller matrix effects were observed for reconstitution solvents containing less organic solvent. The method has been successfully applied to a pharmacokinetic study of Ginkgo biloba extract in rats after intravenous administration. This is the first report of pharmacokinetic data for ginkgolide C.
Keywords: Ginkgolides; Bilobalide; LC–MS/MS; Simultaneous analysis; Matrix effect; Pharmacokinetic;

Characterization of the mouse bronchoalveolar lavage proteome by micro-capillary LC–FTICR mass spectrometry by Joel G. Pounds; Jason W. Flora; Joshua N. Adkins; K. Monica Lee; Gaurav S.J.B. Rana; Tapas Sengupta; Richard D. Smith; Willie J. McKinney (95-101).
Bronchoalveolar lavage fluid (BALF) contains proteins derived from various pulmonary cell types, secretions and blood. As the characterization of the BALF proteome will be instrumental in establishing potential biomarkers of pathophysiology in the lungs, the objective of this study was to contribute to the comprehensive collection of Mus musculus BALF proteins using high resolution and highly sensitive micro-capillary liquid chromatography (μLC) combined with state-of-the-art high resolution mass spectrometry (MS). BALF was collected from ICR and C57BL/6 male mice exposed to nose-only inhalation to either air or cigarette smoke. The tandem mass spectra were analyzed by SEQUEST for peptide identifications with the subsequent application of accurate mass and time tags resulting in the identification of 1797 peptides with high confidence by high resolution MS. These peptides covered 959 individual proteins constituting the largest collection of BALF proteins to date. High throughput monitoring profiles of this extensive collection of BALF proteins will facilitate the discovery and validation of biomarkers that would elucidate pathogenic or adaptive responses of the lungs upon toxic insults.
Keywords: BAL; BALF; Bronchoalveolar; Lavage; Mus musculus; Proteome; FTICR; Cigarette smoke;

Methods based on reversed-phase liquid chromatography with UV detection of 4-nitrobenzoyl- or 3,5-dinitrobenzoyl azide derivatives lack in accuracy and stability of derivatives to be applied for azide determination in pharmaceutical protein samples with high sodium chloride concentrations. This paper describes a sensitive and selective ion chromatographic method, with simple sample preparation and suppressed conductivity detection, developed for trace determination of azide in protein samples containing sodium chloride in concentrations as high as 11.6 g L−1. Anion exchange stationary phase with quaternary alkyl amine functional groups and gradient elution with sodium hydroxide enabled good resolution of anions with similar retention times: azide, bromide and nitrate, as well as chloride whose retention time was shorter than azide's. Anions with high affinity to stationary phase (phosphate and citrate) were also eluted within acceptable analysis time of 32 min. The stability of sample solutions and the method selectivity, accuracy, precision and sensitivity satisfied the validation criteria of international organizations competent for pharmaceutical industry. The detection and quantitation limit ranges of sodium azide in protein samples were 0.007–0.02 mg L−1 and 0.02–0.06 mg L−1, respectively. Both limits increased with the concentration of sodium chloride.
Keywords: Ion chromatography of azide; Suppressed conductivity detection; Pharmaceutical protein samples; Nitrobenzoyl azide derivatives;

A sensitive and relatively rapid reversed-phase HPLC method was applied to the enantiomeric separation of tramadol and its two main metabolites, O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in plasma samples. Chromatography was performed on an AGP column containing α1-acid glycoprotein as chiral selector with a mobile phase of 30 mM diammonium hydrogen phosphate buffer–acetonitrile–triethylamine (98.9:1:0.1, v/v), adjusted to pH 7 by phosphoric acid, and a flow rate of 0.5 ml/min. The fluorescence of analytes was detected at excitation and emission wavelengths of 200 and 301 nm, respectively. The sample preparation was a simple extraction with ethyl acetate using fluconazol as internal standard (IS). The enantiomers of all analytes and IS peaks eluted within 32 min, without any endogenous interference. The calibration curves were linear (r 2  > 0.993) in the concentration range of 2–200, 2.5–100 and 2.5–75 ng/ml for tramadol, M1, and M2 enantiomers, respectively. The within- and between-day variation determined by the measurement of quality control samples at four tested concentrations, showed acceptable values. The lower limit of quantitation was 2 ng/ml for tramadol enantiomers and 2.5 ng/ml for M1 or M2 enantiomers. Mean recoveries of enantiomers from plasma samples were >81% for all analytes. The procedure was applied to assess the pharmacokinetics of the enantiomers of tramadol and its two main metabolites following oral administration of single 100-mg doses to healthy volunteers.
Keywords: Enantioselective assay; Tramadol; Metabolite; AGP column; Pharmacokinetics;

A new immobilized metal ion affinity (IMA) adsorbent containing superparamagnetic nanoparticles and coated with hydrophilic resins are proposed here to improve the purification of His-tagged proteins. The magnetic chelating resin was prepared by radical polymerization of magnetite (Fe3O4), styrene, divinyl benzene (DVB) and glycidyl methacrylate–iminodiacetic acid (GMA–IDA) in ethanol/water medium. IDA is immobilized on magnetite as a ligand and pre-charged Cu2+, Zn2+ and Ni2+ as metal ions. To identify the GMA–IDA magnetic particles easily, we named these particles MPGI. The MPGI adsorbent was used to test their suitability for the direct recovery of an intracellular, polyhistidine-tagged protein, enhanced green fluorescent protein [EGFP-(His)6], from Escherichia coli lysates in a single step. Parameters influencing the purification efficiencies such as pH, ionic strength and imidazole concentration were optimized to achieve improved separation. The optimal selectively was observed in binding buffer (0.2 M NaCl, 0.02 M imidazole), washing buffer (0.4 M NaCl, 0.03 M imidazole) and elution buffer (0.50 M imidazole). The Cu2+-charged MPGI adsorbent had the highest yield and purification factor at 70.4% and 12.3, respectively. The calculated isotherm parameters (Q m  = 53.5 mg/g, K d  = 5.84 mg/mL and Q m/K d  = 9.2 mL/g) indicated that the MPGI adsorbent could be used as a suitable adsorbent for EGFP from an aqueous solution.
Keywords: Immobilized metal ion affinity; Enhanced green fluorescent protein; Superparamagnetic nanoparticles;

Determination of rimantadine in rat plasma by liquid chromatography/electrospray mass spectrometry and its application in a pharmacokinetic study by Meijuan Xu; Wenzheng Ju; Xiaoyan Xia; Hengshan Tan; Min Chen; Jun Zhang; Ningning Xiong; Meng Jiang; Lei Chen; Lixiu Gong (123-128).
A rapid and sensitive liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of rimantadine in rat plasma. Rimantadine was extracted by protein precipitation with methanol, and the chromatographic separation was performed on a C18 column. The total analytical run time was relatively short (4.6 min), and the limit of assay quantification (LLOQ) was 2 ng/mL using 50 μL of rat plasma. Rimantadine and the internal standard (amantadine) were monitored in selected ion monitoring (SIM) mode at m/z 180.2 and 152.1, respectively. The standard curve was linear over a concentration range from 2 to 750 ng/mL, and the correlation coefficients were greater than 0.999. The mean intra- and inter-day assay accuracy ranged from 100.1–105.0% to 100.3–104.0%, respectively, and the mean intra- and inter-day precision was between 1.3–2.3% and 1.8–3.0%, respectively. The developed assay method was successfully applied to a pharmacokinetic study in rats after oral administration of rimantadine hydrochloride at the dose of 20 mg/kg.
Keywords: Rimantadine; Amantadine; LC/MS; Pharmacokinetic study;

A sensitive and enantioselective method was developed and validated for the determination of ondansetron enantiomers in human plasma using enantioselective liquid chromatography-tandem mass spectrometry. The enantiomers of ondansetron were extracted from plasma using ethyl acetate under alkaline conditions. HPLC separation was performed on an ovomucoid column using an isocratic mobile phase of methanol–5 mM ammonium acetate–acetic acid (20:80:0.02, v/v/v) at a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 294 → 170 for ondansetron enantiomers, and m/z 285 → 124 for tropisetron (internal standard). The method was linear in the concentration range of 0.10–40 ng/mL for each enantiomer using 200 μL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.10 ng/mL. The intra- and inter-assay precision was 3.7–11.6% and 5.6–12.3% for R-(−)-ondansetron and S-(+)-ondansetron, respectively. The accuracy was 100.4–107.1% for R-(−)-ondansetron and 103.3–104.9% for S-(+)-ondansetron. No chiral inversion was observed during the plasma storage, preparation and analysis. The method was successfully applied to characterize the pharmacokinetic profiles of ondansetron enantiomers in healthy volunteers after an intravenous infusion of 8 mg racemic ondansetron.
Keywords: Ondansetron; Enantiomers; Stereoselective pharmacokinetics; Enantioselective LC-MS/MS;

A simple, selective and sensitive isocratic HPLC method with triple quadrupole mass spectrometry detection has been developed and validated for simultaneous quantification of zopiclone and its metabolites in human plasma. The analytes were extracted using solid phase extraction, separated on Symmetry shield RP8 column (150 mm × 4.6 mm i.d., 3.5 μm particle size) and detected by tandem mass spectrometry with a turbo ion spray interface. Metaxalone was used as an internal standard. The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 0.5–150 ng/mL for both zopiclone and N-desmethyl zopiclone and 1–150 ng/mL for zopiclone-N-oxide. The intra-batch and inter-batch accuracy and precision evaluated at lower limit of quantification and quality control levels were within 89.5–109.1% and 3.0–14.7%, respectively, for all the analytes. The recoveries calculated for the analytes and internal standard were ≥90% from spiked plasma samples. The validated method was successfully employed for a comparative bioavailability study after oral administration of 7.5 mg zopiclone (test and reference) to 16 healthy volunteers under fasted condition.
Keywords: Zopiclone; N-desmethyl zopiclone; Zopiclone-N-oxide; LC–MS/MS; Solid phase extraction;

Determination of leukocyte DNA 6-thioguanine nucleotide levels by high-performance liquid chromatography with fluorescence detection by Karen-Marie Olesen; Steen Honoré Hansen; Ulrik Sidenius; Kjeld Schmiegelow (149-155).
A HPLC method for determination of 6-thioguanine nucleotide in DNA was developed. Leukocyte DNA was isolated from peripheral blood, derivatized with chloroacetaldehyde and the formed etheno derivatives N2,3-etheno 6-thioguanine (ɛ6TG), 1,N6-etheno adenine (ɛA) and N2,3-etheno guanine (ɛG) were released from the DNA backbone by hydrolysis at pH 6.0 and 80 °C for 60 min. After extraction of ɛ6TG by immobilized metal ion affinity chromatography (IMAC) the sample was analysed by ion-pair reversed-phase HPLC with fluorescence detection. The limit of quantification was 9.0 nM and the intra- and interday precision ranged from 2.8 to 15.5%. In a small cohort of eight children with acute lymphoblastic leukaemia (ALL), a median of one 6-thioguanine base was found for each 3000 normal bases (range 1:2000–1:11000).
Keywords: 6-Thioguanine nucleotide; Etheno adenine; High-performance liquid chromatography; DNA; Acute lymphoblastic leukaemia; Chloroacetaldehyde; Immobilized metal ion affinity chromatography;

An LC–ESI–MS–MS method for the analysis of metabolites of four nitrofurans (furazolidone, furaltadone, nitrofurazone and nitrofurantoin) in raw milk has been developed. The samples were achieved by hydrolysis of the protein-bound drug metabolites, derivatization with 2-nitrobenzaldehyd (2-NBA) and clean-up extraction liquid–liquid with ethyl acetate. LC separation was achieved by using a Phenomenex Luna C-18 column. The mass spectrometer operated in multiple reaction monitoring mode (MRM) with positive electro-spray interface (ESI). The method validation was done according to the criteria laid down in Commission Decision No. 2002/657 EC. The validation includes the determination of linearity, repeatability, within-laboratory reproducibility, accuracy, decision limit (CCα) and detection capability (CCβ). The calibration curves were linear, with typical (R 2) values higher than 0.991. The coefficient of variation (CV, %) was lower than 9.3% and the accuracy (RE, %) ranged from −9.0% to 7.0%. CV within-laboratory reproducibility was lower than 13%. The limits of decision (CCα) and detection capability (CCβ) were 0.12–0.29 μg/kg and 0.15–0.37 μg/kg, thus below the minimum required performance limit (MRPL) set at 1 μg/kg by the UE. This validated method was successfully applied for the determination of nitrofuran metabolites in a large number of milk samples.
Keywords: Milk; Nitrofuran metabolites; Residue; LC–ESI–MS–MS;

Monitoring of plasma antibiotic drugs is useful for better clinical management in infected patients, particularly in intensive care units. A simple and sensitive high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection has been developed and validated for simultaneous quantification of five β-lactam antibiotics in human plasma: cefepim, ceftazidim, cefuroxim, meropenem and piperacillin. The plasma sample, after spiked with ceforanid as an internal standard (IS), was submitted to a solid-phase extraction (SPE) prior to HPLC analysis. A chromatographic separation was achieved on a C8 symmetry column with a mobile phase consisting of an acetonitrile and phosphate buffer (pH 7.4) mixture in a gradient mode. Detection was carried out at a wavelength between 200 and 400 nm. The method developed was linear over the concentration range of 2.5–60 μg/mL for each antibiotic in the plasma samples. Accuracy ranged from 93.2 to 107.1% and precision was between 0.9 and 12.2%. The method has been applied to plasma samples obtained from patients treated with β-lactam antibiotics and is appropriated for easy determination of plasma concentrations for therapeutic monitoring applications.
Keywords: β-Lactam antibiotics; Plasma; HPLC–UV;

A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of anti platelet agent, clopidogrel, in human serum. The analytical procedure involves liquid–liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. A mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 mL/L; pH 5.7) and acetonitrile (56:44 v/v) was used and chromatographic separation was achieved using C18 analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05–10 μg/mL of CCA in human serum. The total run time of analysis was 5.5 min and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 μg/mL, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.
Keywords: Chromatography; HPLC; Clopidogrel; Bioequivalence study;

Bromide–sulfur interchange: Ion chromatographic determination of total reduced thiol levels in plasma by Sneha A. Chitre; Grace-Ann M. Lobo; Spandan M. Rathod; Robert B. Smith; Ray Leslie; Callum Livingstone; James Davis (173-177).
Plasma thiol concentration has long been recognised as a potential indicator for assessing the severity of oxidative stress processes within physiological systems. While such measurements are normally restricted to research studies, this communication has sought to develop and characterise a novel approach through which this parameter could be exploited within routine clinical settings. The protocol is based on the rapid derivatisation of reduced thiol functionalities (protein and monomolecular moieties) through the homogenous reaction of a naphthoquinone bromide derivative. Bromide released in the reaction can be easily quantified through ion chromatography (Isocractic Dionex DX-120 incorporating an IonPac® AS14 anion exchange column and a 25 μL sample loop with conductivity detector. Mobile phase consisted sodium carbonate/bicarbonate (3.5 mM/1 mM) at a flow rate of 1.5 mL/min). Method selectivity and sensitivity has been critically evaluated. The technique covers the range 15 μM–3.5 mM PSH with a detection limit of 9 μM PSH and analysis time of 5 min. The efficacy of the approach for the analysis of human plasma from five volunteers was assessed (ranging from 49 to 72 μM with an intra assay variation of less than 5% in all cases). The responses were validated through comparison with the standard Ellman colorimetric technique.
Keywords: Albumin; Plasma; Thiol; Bromide; Ion chromatography; Oxidative stress;