Automated high-performance liquid chromatography method for the determination of rosiglitazone in human plasma

A robust, fully automated assay procedure for the determination of rosiglitazone (I, BRL-49653) in human plasma has been developed. Plasma concentrations of I were determined using automated sequential trace enrichment of dialysates (ASTED) coupled to reversed-phase high-performance liquid chromatography. Sequential automated dialysis of human plasma samples was followed by concentration of the dialysate by trace enrichment on a C₁₈ cartridge. Drug and internal standard, SB-204882 (II) were eluted from the trace enrichment cartridge by mobile phase (0.01 M ammonium acetate, pH 8–acetonitrile, 65:35, v/v) onto the HPLC column (a Novapak C₁₈, 4 μm, 100×5 mm radial compression cartridge) protected by a Guard-Pak C₁₈ cartridge. The compounds were detected by fluorescence detection, using an excitation wavelength of 247 nm, and emission wavelength of 367 nm. The lower limit of quantitation of the method was 3 ng/ml (200 μl aliquot) with linearity demonstrated up to 100 ng/ml. Within- and between-run precision and accuracy of determination were better than 10% across the calibration range. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can be safely stored for at least 7 months at −20°C. This method has been successfully utilised to provide pharmacokinetic data throughout the clinical development of rosiglitazone.     

Analysis of clozapine and norclozapine by high-performance liquid chromatography

A simple and reliable method for analyzing the concentrations of clozapine and its biologically active metabolite, norclozapine, in human serum or plasma has been developed. This method is based on reversed-phase high-performance liquid chromatography (HPLC) with automated solid-phase extraction (SPE). For HPLC analysis, samples and standards are prepared with an ASPEC automatic sample preparator using 100 mg Bond-Elut C₁₈ SPE columns. The HPLC assay is an isocratic method with a mobile phase of acetonitrile-methanol-10 mM dipotassium hydrogenphosphate, pH 3.7 (30:2:100, v/v/v) at a flow-rate of 1.5 ml/min with a C₁₈ reversed-phase column. Detection is performed with a diode array detector set at 220 nm and with peak purity analyses at 210–365 nm. The absolute recovery varied from 85 and 95%. The intra-assay coefficients of variation (C.V.s) were from 4.2 and 8.0% and the inter-assay C.V.s were from 1.1. to 9.3% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method has been developed for use in a clinical laboratory for therapeutic drug monitoring.     

Quantitation of itraconazole in rat heparinized plasma by liquid chromatography–mass spectrometry

A liquid chromatographic–mass spectrometric (LC–MS) assay was developed and validated for the determination of itraconazole (ITZ) in rat heparinized plasma using reversed-phase HPLC combined with positive atmospheric pressure ionization (API) mass spectrometry. After protein precipitation of plasma samples (0.1 ml) with acetonitrile containing nefazodone as an internal standard (I.S.), a 50-μl aliquot of the supernatant was mixed with 100 μl of 10 mM ammonium formate (pH 4.0). An aliquot of 25 μl of the mixture was injected onto a BDS Hypersil C₁₈ column (50×2 mm; 3 μm) at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ was detected in single ion monitoring (SIM) mode. Standard curves were linear (r²≥0.994) over the concentration range of 4–1000 ng/ml. The mean predicted concentrations of the quality control (QC) samples deviated by less than 10% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8% relative standard deviation. Both ITZ and I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of ITZ was 96%. The validated assay was applied to a pharmacokinetic study of ITZ in rats following administration of a single dose of itraconazole (15 mg/kg).     

New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard

A simple and reproducible HPLC method for the analysis of amphotericin B (AmB) in serum, lung and liver using natamycin as the internal standard was developed. AmB and natamycin were extracted from serum, lung and liver and were separated using an isocratic elution from a C₁₈ reversed-phase column. The mobile phase consisted of acetonitrile-10 mM acetate buffer pH 4.0 (37:63, v/v). The HPLC system had two detectors in series. One was set at 303 nm and the other at 383 nm for the detection of natamycin and AmB, respectively. The retention times of AmB and natamycin were 15 and 6 min, respectively. The recovery efficiency was 96-70%. The limit of quantification was 0.1 μg/ml. The assay was reproducible, the within-day coefficient of variation (n=6) was <8% for serum, lungs and liver. The between-day variability (n=6) was <7.7% for serum, liver and lungs at 1 μg/ml or 1 μg/g tissue concentration. The assay was linear within the range 1–40 μg/ml (r²=0.999).     

Sensitive assay for measuring amoxicillin in human plasma and middle ear fluid using solid-phase extraction and reversed-phase high-performance liquid chromatography

We developed a sensitive assay to measure amoxicillin in human plasma and midle ear fluid (MEF) using solid-phase extraction and reversed-phase HPLC. Amoxicillin and cefadroxil, the internal standard, were extracted from 50–200 μl of sample with Bond Elut C₁₈ cartridges. The exact was analyzed on a 15 cm × 2 mm, 5μm Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer (pH = 6.5) and 5 mM tetrabutylammonium. The average absolute recovery of amoxicillin and cefadroxil were 91.2 ± 16.6% and 91.0 ± 6.8%, respectively. The limit of quantitation was 0.125 μg/ml with 200 μl sample size. The linear range was from 0.125 to 35.0 μg/ml with correlation coefficients greater than 0.999. These analytic conditions produced a highly sensitive amoxicillin assay in human body fluids without derivatization.     

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 μl of the mixture was injected onto a Spherex C₁₈ column (150×4.6 mm; 5 μm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 mM ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 μM in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 μM. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.     

Simultaneous determination of selegiline-N-oxide,a new indicator for selegiline administration,and other metabolites in urine by high-performance liquid chromatography–electrospray ionization mass spectrometry

In order to discriminate selegiline (SG) use from methamphetamine (MA) use, the urinary metabolites of SG users have been investigated using high-performance liquid chromatography (HPLC)–electrospray ionization mass spectrometry (HPLC–ESI–MS). Selegiline-N-oxide (SGO), a specific metabolite of SG, was for the first time detected in the urine, in addition to other metabolites MA, amphetamine (AP) and desmethylselegiline (DM-SG). A combination of a Sep-pak C₁₈ cartridge for the solid-phase extraction, a semi-micro SCX column (1.5 mm I.D.×150 mm) for HPLC separation and ESI–MS for detection provided a simple and sensitive procedure for the simultaneous determination of these analytes. Acetonitrile–10 mM ammonium formate buffer adjusted to pH 3.0 (70:30, v/v) at a flow-rate of 0.1 ml/min was found to be the most effective mobile phase. Linear calibration curves were obtained over the concentration range from 0.5 to 100 ng/ml for all the analytes by monitoring each protonated molecular ion in the selected ion monitoring (SIM) mode. The detection limits ranged from 0.1 to 0.5 ng/ml. Upon applying the scan mode, 10–20 ng/ml were the detection limits. Quantitative investigation utilizing this revealed that SGO was about three times more abundant (47 ng/ml, 79 ng/ml) than DM-SG in two SG users’ urine samples tested here. This newly-detected, specific metabolite SGO was found to be an effective indicator for SG administration.     

Rapid,highly sensitive method for the determination of morphine and its metabolites in body fluids by liquid chromatography–mass spectrometry

A rapid, highly sensitive method for the determination of morphine and its metabolites morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and normorphine has been developed using high-performance liquid chromatography–electrospray mass spectrometry, with the deuterated analogues as internal standards. The analytes were extracted automatically using end-capped C₂ solid-phase extraction cartridges. Baseline separation of morphine, M3G and M6G was achieved on a LiChrospher 100 RP-18 end-capped analytical column (125×3 mm I.D., 5 μm particle size) with water–acetonitrile–tetrahydrofuran–formic acid (100:1:1:0.1, v/v) as the mobile phase. Morphine and normorphine coeluate and were separated mass spectrometrically. The mass spectrometer was operated in the selected-ion monitoring mode using m/z 272 for normorphine, m/z 286 for morphine, m/z 462 for morphine-6-glucuronide. Due to an interfering peak, M3G was measured by tandem mass spectrometry in the daughter-ion mode. The limits of quantitation achieved with this method were 1.3 pmol/ml for morphine, 1.5 pmol/ml for normorphine, 1.0 pmol/ml for M6G and 5.4 pmol/ml for M3G in serum or cerebrospinal fluid. The limits of quantitation achieved in urine were 10 pmol/ml for morphine, 20 pmol/ml for normorphine and M6G and 50 pmol/ml for M3G using a sample size of 100 μl. The method described was successfully applied to the determination of morphine and its metabolites in human serum, cerebrospinal fluid and urine in pharmacokinetic and drug interaction studies.     

Determination of midazolam and its metabolites in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating midazolam and its two hydroxy metabolites in rat serum microsamples (50 μl). The separation used a 2 mm I.D. reversed-phase Symmetry C₁₈ column with an isocratic mobile phase consisting of methanol-acetonitrile-14.9 mM sodium acetate in water at pH 3.0 (10:23:67, v/v). The detection limit was 10 ng/ml for all the compounds using an ultraviolet detector operated at 230 nm. The method was used to study the pharmacokinetics of midazolam after an intravenous bolus dose (0.75 mg/kg).     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

High-performance liquid chromatography determination of praziquantel enantiomers in human serum using a reversed-phase cellulose-based chiral stationary phase and disc solid-phase extraction

A sensitive HPLC method for the quantification of praziquantel enantiomers in human serum is described. The method involves the use of a novel disc solid-phase extraction for sample clean-up prior to HPLC analysis and is also free of interference from trans-4-hydroxypraziquantel, the major metabolite of praziquantel. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OJ-R) under isocratic conditions using a mobile phase consisting of 0.1 M sodium perchlorate–acetonitrile (66:34, v/v) at a flow-rate of 0.5 ml/min. Recoveries for R-(−)- and S-(+)-praziquantel enantiomers were in the range of 84–89% at 50–500 ng/ml levels. Intra-day and inter-day precisions calculated as R.S.D. were in the ranges of 3–8% and 1–8% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percent error were in the 0.2–5% and 0.3–8% ranges for both enantiomers, respectively. Linear calibration curves were in the concentration range 10–600 ng/ml for each enantiomer in serum. The limit of quantification of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using a UV detector set at 210 nm was 5 ng/ml (S/N=2).     

High-performance liquid chromatographic method for the determination of AG-331, a novel anti-cancer agent,in human serum and urine using solid-phase extraction and photodiode-array detection

A reversed-phase isocratic high-performance liquid chromatographic method has been developed for the determination of AG-331, a novel thymidylate synthase inhibitor, in human serum and urine. The method involves a solid-phase extraction from C₁₈ cartridges without addition of an internal standard. The methanol eluent is evaporated under nitrogen at 40°C, and reconstituted in mobile phase, acetonitrile-water (35:65, v/v) containing 25 mM ammonium phosphate. Separation of AG-331 was obtained on a C₁₈ column at a flow-rate of 1 ml/min. Chromatographic signals were monitored by a photodiode-array detector at a primary wavelength of 457 nm with a bandwidth of 4.8 nm. Standard curves are linear in the range of 22–2175 ng/ml in plasma and 44–2175 ng/ml in urine, respectively. The extraction recovery ranged from 92.9–102.4%. Intra-day coefficient of variation was less than 9.5%, and inter-day coefficient of variation was less than 14.3% for an AG-331 concentration of 44 ng/ml. This method has been used to characterize the pharmacokinetics of AG-331 in cancer patients as part of ongoing Phase I trials.     

Simultaneous determination of all-trans and 13-cis retinoic acids and their 4-oxo metabolites by adsorption liquid chromatography after solid-phase extraction

All-trans retinoic acid (all-trans RA), the active metabolite of vitamin A, has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL), the AML₃ subtype of the FAB cytological classification. Complete remission is obtained by inducing terminal granulocytic differentiation of the leukemic cells. To study all-trans RA pharmacokinetics in patients with APL, a rapid, precise and selective high-performance liquid chromatographis (HPLC) assay was developed. This method is easy and shows good repeatability (C.V. = 8.41–12.44%), reproducibility (C.V. = 9.19–14.73%), accuracy (C.V. = 3.5–11%) and sensitivity with a detection limit of 5 pmol/ml. The analysis is performed using normal-phase HPLC in an isocratic mode with UV detection after solid-phase extraction on octadecyl (C₁₈) columns. The mobile phase is hexanedichloromethane-dioxane (78:18:4, v/v) containing 1% acetic acid.     

Rapid determination of the anti-cancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C₁₈ sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4–800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 μl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran™ following single doses of oral tablet formulations.     

Solid-phase extraction and high-performance liquid chromatographic determination of articaine and its metabolite articainic acid in human serum

A new method is described using solid-phase extraction (SPE) for preconcentration of articaine and the metabolite articainic acid and high-performance liquid chromatography (HPLC) for the determination of both compounds in human serum. Articaine and articainic acid were extracted in one step with SDB-RPS disk cartridges after precipitation of the serum proteins by perchloric acid. The HPLC separation was then performed on a reversed-phase C₈ column using phosphate buffer–acetonitrile (88:12, v/v). UV absorption at 274 nm was used for measuring the analytes with a low limit of quantitation of about 10 ng/ml, which is appropriate for pharmacokinetic studies of low dose submucosal injections of the local anaesthetic agent articaine hydrochloride in dentistry.     

Sensitive high-performance liquid chromatographic method for the determination of 2-phenylethylamine in human urine

A new sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of 2-phenylethylamine (PEA) in human urine. The analytical procedure involved a simple extraction of the analyte from urine, followed by precolumn derivatisation of the sample with o-phthalaldehyde. The HPLC separation was performed under isocratic conditions using an Erbasil S C₁₈ (250 × 4.0 mm I.D., particle size 3 μm) reversed-phase column. The limit of quantification was 0.5 ng of PEA/ml of urine. The method showed good linearity, accuracy and precision data in the concentration range 0.5–200 ng/ml of urine. The method was successfully applied to the determination of PEA urinary excretion in Parkinsonian patients after oral administration of the monoamine oxidase B (MAO-B) inhibitor, selegiline.     

An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-Retinol (Vitamin A) and α-Tocopherol (Vitamin E) in human serum: Comparison of different particulate reversed-phase HPLC columns

A novel, simple and fast reversed-phase HPLC/UV method was developed, optimized for various chromatographic conditions, and validated according to international guidelines for simultaneous determination of all-trans-retinol and α-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5 μg/ml. A liquid-phase extraction was applied to the 250 μl of serum with n-hexane–dichloromethane mixture (70:30, v/v), in two steps, using ethanol–methanol mixture (95:5, v/v) for protein precipitation and BHT (butylated hydroxy toluene) as stabilizer for sample preparation. Both analytes were analyzed on Kromasil 100 C₁₈ column (150 mm × 4.6 mm, 5 μm), Brownlee analytical (Perkin Elmer) C₁₈ column (150 mm × 4.6 mm, 5 μm), and Supelco (Supelcosil) LC-18 column (150 mm × 3 mm, 3 μm), protected by a Perkin Elmer C₁₈ (30 mm × 4.6 mm, 10 μm; Norwalk, USA) pre-column guard cartridge, at 292 nm wavelength, using methanol–water (99:1, v/v), in isocratic mode as mobile phase applied at flow rate of 1.5 ml/min and 1 ml/min for both 5 μm and 3 μm columns, respectively. Complete separation of all the analytes was achieved in 3 and 6 min on 3 μm and 5 μm columns, respectively by injecting 20 μl of sample into the HPLC system by autosampler, keeping column oven temperature at 25 °C. Different particulate reversed-phase chromatographic columns were evaluated in order to select the best column in terms of sensitivity, selectivity, resolution and short run time of both the analytes and it was concluded that 3 μm columns are better to be used in clinical set up as well as in laboratories for the separation of these analytes in a shorter time as compared with 5 μm columns. The method was validated and applied for the analysis of all-trans-retinol and α-tocopherol in the serum of human volunteers.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

Gradient ion-pair high-performance liquid chromatographic method for analysis of 3-hydroxypyridin-4-one iron chelators

A gradient ion-pair HPLC separation of highly hydrophilic 3-hydroxypyridin-4-one (HPO) iron chelators is described. The separation of HPOs was performed using a reversed-phase polymer HPLC column (PLRP-S 100 Å, 15×0.46 cm ID, 5 μm). The ion-pair buffer contained 1-heptanesulfonic acid (sodium salt) (5 mM) and the pH was adjusted to 2.0 using HCl. The gradient was 2%–35% CH₃CN in 20 min and post-run was followed for 5 min using 2% CH₃CN and 98% buffer. The flow-rate was 1 ml/min and the analytes were monitored at 280 nm. The retention times of 30 hydrophilic HPOs fell in the range of 10–18 min with sharp peak shapes, although these iron chelators possess various functional groups and distribution coefficients. The application of this HPLC method in the analysis of HPO chelators and their metabolites in rat bile and urine is described.     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.