Improved liquid chromatography–tandem mass spectrometry method for the analysis of eptifibatide in human plasma

A rapid, selective and highly sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C₁₈ column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6 → m/z 646.4 and m/z 931.6 → m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1–1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within ±7.6% of nominal values. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 μg/kg bolus of eptifibatide to 8 healthy volunteers.     

Determination of 2-hydroxyflutamide in human plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

Flutamide is a potent antiandrogen used for the treatment of prostatic cancer. Flutamide undergoes extensive first-pass metabolism to the pharmacologically active metabolite 2-hydroxyflutamide. A simple, sensitive, precise, accurate and specific HPLC method, using carbamazepine as the internal standard, for the determination of 2-hydroxyflutamide in human plasma was developed and validated. After addition of the internal standard, the analytes were isolated from human plasma by liquid–liquid extraction. The method was linear in the 25 to 1000 ng/ml concentration range (r>0.999). Recovery for 2-hydroxyflutamide was greater than 91.4% and for internal standard was 93.6%. The limit of quantitation was 25 ng/ml. Inter-batch precision, expressed as the relative standard deviation (RSD), ranged from 4.3 to 7.9%, and accuracy was better than 93.9%. Analysis of 2-hydroxyflutamide concentrations in plasma samples from 16 healthy volunteers following oral administration of 250 mg of flutamide provided the following pharmacokinetic data (mean±SD): C_max, 776±400 ng/ml; AUC_0–∞, 5368±2689 ng h/ml; AUC_0–t, 5005±2605 ng h/ml; T_max, 2.6±1.6 h; elimination half-life, 5.2±2.0 h.     

Determination of rhoifolin and daidzin in human plasma by high-performance liquid chromatography

A method for determining flavonoids in human plasma is presented for application to pharmacokinetic studies of two flavonoids, rhoifolin and daidzin. Isocratic reversed-phase high-performance liquid chromatography (HPLC) was used with genistin as an internal standard and solid-phase extraction using a Sep-Pak C₁₈ cartridge. The mobile phases were acetonitrile—0.1 M ammonium acetate solution (20:80, v/v) for rhoifolin and methanol—0.1 M ammonium acetate solution (33:67, v/v) for daidzin. The detection limits on-column were 2 ng for rhoifolin and 0.5 ng for daidzin.     

Liquid chromatographic method for the determination of ticlopidine in human plasma

A simple high-performance liquid chromatographic method for determination of ticlopidine in human plasma using ultra violet detection was developed. The separation of the investigated compound and internal standard was achieved on a C₁₈ BD column with a 0.01 M potassium dihydrogen phosphate buffer (pH 4)–acetonitrile–methanol (20:40:40, v/v) mobile phase. The detection was performed at 215 nm. The compounds were isolated from plasma by Bond Elut C₁₈ solid-phase extraction, the mean absolute recovery was 84.9%. The limit of quantitation was 10 ng ml⁻¹, the limit of detection was 5 ng ml⁻¹. The bioanalytical method was validated with respect to linearity, within- and between-day accuracy and precision, system suitability and stability. All validated parameters were found to be within the internationally required limits. The developed analytical method for ticlopidine was found to be suitable for application in pharmacokinetic studies and human drug monitoring.     

High-performance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease inhibitor, in human plasma

Nelfinavir mesylate, a potent and orally bioavailable inhibitor of HIV-1 protease (K_i=2 nM), has undergone Phase III clinical evaluation in a large population of HIV-positive patients. A high-performance liquid chromatography analytical method was developed to determine the pharmacokinetic parameters of the free base, nelfinavir, in these human subjects. The method involved the extraction of nelfinavir and an internal standard, 6,7-dimethyl-2,3-di-(2-pyridyl)quinoxaline, from 250 μl of human plasma with a mixture of ethyl acetate–acetonitrile (90:10, v/v). The analysis was via ultraviolet detection at 220 nm using a reversed-phase C₁₈ analytical column and a mobile phase consisting of 25 mM monobasic sodium phosphate buffer (adjusted to pH 3.4 with phosphoric acid)–acetonitrile (58:42, v/v) that resolved the drug and internal standard peaks from non-specific substances in human plasma. The method was validated under Good Laboratory Practice (GLP) conditions for specificity, inter- and intra-assay precision and accuracy, absolute recovery and stability. The mean recovery ranged from 92.4 to 83.0% for nelfinavir and was 95.7% for the internal standard. The method was linear over a concentration range of 0.0300 μg/ml to 10 μg/ml, with a minimum quantifiable level of 0.0500 μg/ml for nelfinavir.     

Sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its N-desethyl metabolite in human saliva or cerebrospinal fluid using solid-phase extraction

A sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its primary metabolite in human saliva or cerebrospinal fluid using solid-phase extraction is described. Samples mixed with internal standard and sodium phosphate buffer were applied to an activated C₁₈ solid-phase extraction column. The reconstituted eluate was injected onto a Zorbax RX C₈ column utilizing a mobile phase of 100 mM ammonium acetate (pH 4.0)–isopropyl alcohol–acetonitrile (55:20:25, v/v/v). Fluorescence detection was employed with excitation at 295 nm and emission at 456 nm. Quantitation was achieved using peak-height ratios. The detection response curve was linear from 2 to 850 nM for atevirdine in both human saliva and cerebrospinal fluid and from 2 to 250 nM for the metabolite in human saliva. The method was utilized to analyze cerebrospinal fluid and saliva samples from clinical studies.     

Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography

A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite phosphoramide mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70°C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C₈ column with acetonitrile–0.025 M potassium phosphate buffer, pH 8.0, (32:68, v/v) as the mobile phase. The entire sample handling procedure, from collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10 000 ng/ml. The lower limit of quantification and the limit of detection (using a signal-to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 μl of plasma. Within-day and between-day precisions were below 11% over the entire concentration range and the accuracies were between 100 and 106%. PM was found to be stable at −30°C for at least 10 weeks both in plasma and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1000 mg/m² CP in a 1-h infusion demonstrated the applicability of the assay.     

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).     

Determination of picamilon concentration in human plasma by liquid chromatography–tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of picamilon concentration in human plasma. Picamilon was extracted from human plasma by protein precipitation. High performance liquid chromatography separation was performed on a Venusil ASB C₁₈ column with a mobile phase consisting of methanol ?10 mM ammonium acetate–formic acid (55:45:01, v/v/v) at a flow rate of 0.65 ml/min. Acquisition of mass spectrometric data was performed in selected reaction monitoring mode, using the transitions of m/z 209.0 → m/z (78.0 + 106.0) for picamilon and m/z 152.0 → m/z (93.0 + 110.0) for paracetamol (internal standard). The method was linear in the concentration range of 1.00–5000 ng/ml for the analyte. The lower limit of quantification was 1.00 ng/ml. The intra- and inter-assay precision were below 13.5%, and the accuracy was between 99.6% and 101.6%. The method was successfully applied to characterize the pharmacokinetic profiles of picamilon in healthy volunteers. This validated LC–MS/MS method was selective and rapid, and is suitable for the pharmacokinetic study of picamilon in humans.     

Liquid chromatography–tandem mass spectrometry quantification of levosulpiride in human plasma and its application to bioequivalence study

An improved method for determining levels of levosulpiride in human plasma using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and validated. The protein precipitation method was used for plasma sample preparation. Levosulpiride and an internal standard (IS) were isocratically separated on a UPLC BEH C₁₈ column with a mobile phase of ammonium formate buffer (1 mM, adjusted to pH 3 with formic acid) and acetonitrile (60:40, v/v). MS/MS detection was performed by monitoring the parent → daughter pair of levosulpiride and the IS at m/z 342 → 112 and 329 → 256, respectively. The method was linear from 2.5 to 200 ng/mL and exhibited acceptable precision and percent recovery. The method was successfully demonstrated in pharmacokinetic and bioequivalence studies of two levosulpiride oral formulations administered to healthy volunteers. When compared to the previous LC–MS methods, the proposed method is faster, well-validated, and uses lesser plasma volume and a similar sensitivity. The use of UPLC allowed rapid and sensitive quantification of levosulpiride, making this method suitable for high-throughput clinical applications.     

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.     

Stereospecific determination of tiaprofenic acid in plasma: problems with drug degradation

A sensitive stereospecific high-performance liquid chromatographic assay for the quantification of tiaprofenic acid in human plasma was developed. The procedure involved extraction of tiaprofenic acid from acidified plasma into hexane-diethyl ether (8:2, v/v). Stereospecific separation was achieved with a prepacked ga₁-acid glycoprotein column without derivatization. The mobile phase consisted of 2% 2-propanol in 0.01 M phosphate buffer, pH 6.5. Tiaprofenic acid was detected at 317 nm. The limit of quantification was found to be 25 ng/ml for each enantiomer using a 0.5 ml plasma sample. The assay was reproducible and accurate to be applied to the stereoselective pharmacokinetic analysis of tiaprofenic acid in plasma. Because of photoinstability of tiaprofenic acid plasma sampling and sample extraction should be performed under light protection.     

Characterization of human lymphocyte N-acetyltransferase and its relationship to the isoniazid acetylator polymorphism

Characterization of human lymphocyte N-acetyltransferase (NAT) for specific activity, substrate specificity, inhibition, pH optimum, apparent K_m, kinetic mechanism, trypsin stability, freezing stability, and heat stability was carried out in rapid and slow isoniazid (INH) acetylators. There is a statistically significant difference in the heat stability of lymphocyte NAT from rapid and slow INH phenotypes. The lymphocyte enzyme from rapid INH acetylators is less heat stable than the lymphocyte enzyme from slow INH acetylators. This is an indication of a structural, possibly polymorphic, difference in lymphocyte NAT from the two acetylator phenotypes.     

High-performance liquid chromatographic determination of tianeptine in plasma applied to pharmacokinetic studies

An improved analytical method for the quantitative measurement of tianeptine and its main metabolite MC₅ in human plasma was designed. Extraction involved ion-paired liquid–liquid extraction of the compounds from 1.0 ml of human plasma adjusted to pH 7.0. HPLC separation was performed using a Nucleosil C₁₈, 5 μm column (150×4.6 mm I.D.) and a mixture of acetonitrile and pH 3, 2.7 g l⁻¹ solution of sodium heptanesulfonate in distilled water (40:60, v/v) as mobile phase. UV detection was performed using a diode array detector in the 200–400 nm passband, and quantification of the analytes was made at 220 nm. For both tianeptine and MC₅ metabolite, the limit of quantitation was 5 μg l⁻¹ and the calibration curves were linear from 5 to 500 μg l⁻¹. Intra- and inter-assay precision and accuracy fulfilled the international requirements. The recovery of tianeptine and its metabolite from plasma was, respectively, 71.5 and 74.3% at 20 μg l⁻¹, 71.2 and 70.8% at 400 μg l⁻¹. The selectivity of the method was checked by verifying the absence of chromatographic interference from pure solutions of the most commonly associated therapeutic drugs. This method, validated according to the criteria established by the Journal of Chromatography B, was applied to the determination of tianeptine and MC₅-metabolite in human plasma in pharmacokinetic studies.     

Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of acetylsalicylic acid (ASA) and its main metabolite salicylic acid (SA) in human plasma. Acidified plasma is deproteinized with acetonitrile which is separated from the aqueous layer by adding sodium chloride. ASA and SA are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: Novapak C₁₈ 4 μm silica,150×4mm I.D.; eluent: 740 ml water, 900 μl 85% orthophosphoric acid, 180 ml acetonitrile) and photometric detection (237 nm). 2-Methylbenzoic acid is used as internal standard. The method allows the determination of ASA and SA in human plasma as low as 100 ng/ml with good precision (better than 10%). The assay was used to determine the pharmacokinetic parameters of ASA and SA following oral administration of 100–500 mg ASA in healthy volunteers.     

A preliminary pharmacokinetic study of the enantiomers of the terfenadine acid metabolite in humans

A stereoselective and sensitive achiral/chiral method for the determination of terfenadine acid metabolite in human plasma was developed. The metabolite was separated and quantitated using an achiral chromatographic procedure with a cyano column. The mobile phase was 1 mM sodium acetate buffer (pH 4.0) and acetonitrile (25:75% v/v) at a flow rate of 2 ml/min, at ambient temperature. The stereospecific resolution was accomplished using a chiral-AGP column and a mobile phase consisting of sodium acetate (0.01 M): methanol (98.7:1.3% v/v), and 20 mM di-n-butylamine at a flow rate of 1.2 ml/min. The column temperature was maintained at 32°C. The eluent was monitored at 230 nm (excitation) and 300 nm (emission) with a cut-off filter at 270 nm. This assay was used for a pharmacokinetic study in five subjects after administration of a single dose of 60 mg of terfenadine. The t_½ values of the two enantiomers were similar, but the AUC values of the (+)-enantiomer were 2.05–2.35 times higher than those of (?)-enantiomer. © 1994 Wiley-Liss, Inc.     

Liquid chromatography/tandem mass spectrometry method for the simultaneous determination of vardenafil and its major metabolite,N-desethylvardenafil,in human plasma: Application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method for the determination of vardenafil and its major metabolite, N-desethylvardenafil, in human plasma using sildenafil as an internal standard was developed and validated. The analytes were extracted from 0.25-mL aliquots of human plasma by liquid–liquid extraction, using 1 mL of ethyl acetate. Chromatographic separation was carried on a Luna C₁₈ column (50 mm × 2.0 mm, 3 μm) at 40 °C, with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 5.0) and acetonitrile (10:90, v/v), a flow rate of 0.2 mL/min, and a total run time of 2 min. Detection and quantification were performed using a mass spectrometer in the selected reaction-monitoring mode with positive electrospray ionization at m/z 489.1 → 151.2 for vardenafil, m/z 460.9 → 151.2 for N-desethylvardenafil, and m/z 475.3 → 100.1 for the internal standard (IS), respectively. This assay was linear over a concentration range of 0.5–200 ng/mL with a lower limit of quantification of 0.5 ng/mL for both vardenafil and N-desethylvardenafil. The coefficient of variation for the assay precision was <13.6%, and the accuracy was >93.1%. This method was successfully applied to a pharmacokinetic study after oral administration of vardenafil 20 mg tablet in Korean healthy male volunteers.     

Determination of rifampin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifampin in human plasma. Rifampin and sulindac (internal standard) are extracted from human plasma using a C₂ Bond Elut extraction column. A 100-μl volume of 0.1 M HCl is added to the plasma before extraction to increase the retenction of the compounds on the extraction column. Methanol (1 ml) is used to elute the compounds and 0.5 ml of 3 mg/ml ascorbic acid in water is added to the final eluate to reduce the oxidation of rifampin. Separation is achieved by reversed-phase chromatography on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate-acetonitrile (55:45, v/v). Detection is by ultraviolet absorbance at 340 nm. The retention times of rifampin and internal standard are approximately 4.4 and 7.8 min, respectively. The assay is linear in concentration ranges of 50 to 35 000 ng/ml. The quantitation limit is 50 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 μl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

Determination of omeprazole in human plasma by high-performance liquid chromatography

A new method for the determination of omeprazole in human plasma was developed. Omeprazole was extracted from plasma with toluene-isoamylalcohol (95:5, v/v), the organic phase was evaporated, dissolved in the mobile phase and injected into a reversed-phase C₁₈ column. Flunitrazepam was used as an internal standard. The mobile phase consisted of 47% methanol and 53% of 0.1 M dipotassium hydrogenphosphate, pH 7.8. The spectrophotometric detection was performed at 302 nm. Limit of quantitation was 9.7 ng/ml and the calibration curve was linear up to 1240 ng/ml.