Quantification of zolpidem in human plasma by high-performance liquid chromatography with fluorescence detection

A simple, reliable HPLC method with fluorescence detection (excitation 320 and emission 388 nm) was developed and validated for quantitation of zolpidem in human plasma. Following a single-step liquid-liquid extraction, the analyte and internal standard (quinine) were separated using an isocratic mobile phase on a reversed-phase C(18) column. The lower limit of quantitation was 1.8 ng/mL, with a relative standard deviation of less than 5%. A linear dynamic range of 1.8-288 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.7-4.8 and 1.2-2.3%, respectively. The between-batch and within-batch accuracy was 95.3-100.4 and 95.5-102.7%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of zolpidem in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is simple and repeatable enough to be used in pharmacokinetic studies.     

Determination of cisapride in human plasma by high-performance liquid chromatography with fluorescence detection

Summary A new sensitive HPLC-FLD method has been developed and validated for the determination of cisapride in human plasma for a bioequivalence study. A gradient method was used to remove late-eluting plasma components of no interest. The separation was performed on a Li-ChroCART 250-4 Purospher RP-18 (5 μm particle) analytical column fitted with a LiChroCART 4-4 Purospher RP-18 endcapped (5 μm particle) guard column. The excitation and emission wavelengths were 295 and 350 nm during fluorescence detection. The calibration plot was linear in the range of 5–200 ng mL⁻¹. A demethoxy analogue of cisapride was used as internal standard.     

Quantitation of plasma and biliary cefpiramide concentrations in human samples using high-performance liquid chromatography

Cefpiramide is frequently used to treat biliary infections. However, no bioanalytical method has been validated to quantitate cefpiramide in human samples, particularly in bile. Therefore, this study was conducted to develop a simple, selective and validated high-performance liquid chromatographic method to determine cefpiramide in human plasma and bile. A protein precipitation procedure was used to extract cefpiramide and cefoperazone (internal standard, IS) from 200 μl of plasma and bile. Utilizing a Capcell Pak C₁₈ column (4.6 × 250 mm), cefpiramide and IS were separated using the timed-gradient mobile phase consisting of 0.1 m sodium acetate (pH 5.2) and acetonitrile at a flow rate of 1 ml/min with photodiode array detector (wavelength set at 273 nm). The calibration curves showed linearity at concentrations ranging from 1 to 150 μg/ml in both plasma and bile (r² > 0.999). The within- and between-run coefficients of variation (CVs) for plasma samples were 0.570–4.43 and 1.10–2.76%, respectively; for bile samples, the within- and between-day precision (CV) was 0.814–6.34 and 2.05–4.00%, respectively. Our newly developed bioanalytical method was successfully employed to quantify cefpiramide concentrations in both plasma and bile at multiple time points in patients with acute cholangitis.     

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r² values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.     

Quantification of pramipexole in human plasma by liquid chromatography tandem mass spectrometry using tamsulosin as internal standard

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of pramipexole in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 212/152 for pramipexole and m/z 409/228 for the IS. The method exhibited a linear dynamic range of 200-8000 pg/mL for pramipexole in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 200 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Convenient and rapid determination of cimetidine in human plasma using perchloric acid-mediated plasma protein precipitation and high-performance liquid chromatography

This study demonstrates the analysis of cimetidine in human plasma with HPLC using a simplified sample preparation by protein precipitation with perchloric acid. Plasma cimetidine concentration was determined by plotting peak height ratio of cimetidine to ranitidine (internal standard, IS) against cimetidine concentrations in plasma. The cimetidine and ranitidine peaks were completely separated and no interference from plasma was observed. The lower limit of quantification (LLOQ) of the method was established at 0.1 microg/mL with a precision of 4.3% and a relative error of 1.9%. The average analytical recovery was >90% over the range of cimetidine concentrations (0.1-15.0 microg/mL). The linearity of calibration curve was excellent (r(2) > 0.999). The within- and between-day precision and accuracy, expressed as the coefficients of variation and relative error, were found to be less than 5%. Compared with previously reported methods, the analytical technique for cimetidine determination in human plasma presented here demonstrates comparable accuracy and precision, an acceptable analysis time, shorter and simpler sample preparation, and a reduced need for complicated equipment. The method presented here is simple and rapid, and the precision and sensitivity are appropriate for the determination of cimetidine in plasma in pharmacokinetic studies.     

Quantification of fexofenadine in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry using mosapride as internal standard

A rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of fexofenadine in human plasma using mosapride as internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 1-500 ng/mL for fexofenadine in human plasma. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 5% for fexofenadine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Simple method for the assay of clindamycin in human plasma by reversed-phase high-performance liquid chromatography with UV detector

A rapid and simple high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of clindamycin in human plasma. After precipitation with 50% trichloroacetic acid (TCA) containing the internal standard, propranolol, the analysis of the clindamycin level in the plasma samples was carried out using a reverse-phase cyano (CN) column with ultraviolet detection (204 nm). The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-distilled water-7.6 mm tetramethylammonium chloride (TMA) (60:40:0.075, v/v/v), adjusted to pH 3.2. The proposed method was specific and sensitive with a lower limit of quantitation (LLOQ) of 0.2 microg/mL. This HPLC method was validated by examining the precision and accuracy for inter- and intraday analysis in the concentration range 0.2-20.0 microg/mL. The relative standard deviations (RSD) in the inter- and intraday validation were 6.1-14.9 and 6.0-16.1%, respectively. In the stability test, clindamycin was found to be stable in human plasma during the storage and assay procedure. The present HPLC method was applied to the analysis of samples taken up to 12 h after a single oral administration of clindamycin in healthy volunteers.     

Clonazepam quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method for quantifying clonazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a hexane/diethylether (20 : 80, v/v) solution. The extracts were analysed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on a Jones Genesis C8 4 microm analytical column (100 x 2.1 mm i.d.). The method had a chromatographic run time of 3.0 min and a linear calibration curve over the range 0.5-50 ng/ml (r2 > 0.9965). The limit of quantification was 0.5 ng/ml. This HPLC/MS/MS procedure was used to assess the bioequivalence of two clonazepam 2 mg tablet formulations (clonazepam test formulation from Ranbaxy Laboratories Ltd and Rivotril from Roche Laboratórios Ltda as standard reference formulation).     

Quantitative determination of lisinopril in human plasma by high performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine lisinopril in human plasma. Sample pretreatment involved a one-step protein precipitation with methanol of 0.1 mL plasma. Analysis was performed on an Inertsil ODS-3 column (2.1 × 50 mm i.d., 3 μm) with mobile phase consisting of methanol-water (containing 0.2% formic acid; 55:45, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via an electrospray ionization source. Each plasma sample was chromatographed within 2.5 min. The linear calibration curves for lisinopril were obtained in the concentration range of 1.03-206 ng/mL (r(2) ≥ 0.99) with a lower limit of quantification of 1.03 ng/mL. The intra- and inter-day precisions (relative standard deviation) were not higher than 11%, and accuracy (relative error) was within ±6.8%, determined from quality control samples for lisinopril, which corresponded to the guidance of the Food and Drug Administration. The method described herein was fully validated and successfully applied to the pharmacokinetic study of lisinopril tablets in healthy male volunteers after oral administration.     

4-Methylamino antipyrine determination in human plasma by high-performance liquid chromatography tandem mass spectrometry

In the present study, a simple and rapid method for metamizole metabolite 4-methylamino antipyrine (MAA) determination in human plasma was developed, validated and successfully applied to a clinical trial. Chromatographic separation was achieved in HILIC mode on a YMC-Pack SIL column (100 × 2.0 mm; S-5 μm, 30 nm), with a mobile phase consisting of acetonitrile, water and formic acid. Protein precipitation of a small plasma volume using acetonitrile was selected for sample preparation. The multiple reaction monitoring transitions in the positive ionization mode were m/z 218.2 → 56.2 for MAA and m/z 221.2 → 56.2 for MAA-d3 (IS, internal standard). Concentration levels of MAA calibration standards were in the range of 0.100–20 μg/ml. Metamizole conversion into MAA in both water and organic media was investigated, and the level of the conversion in commercially available injection solutions was estimated.     

Quantitation of clobazam in human plasma using high-performance liquid chromatography.

A rapid and simple reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of clobazam concentrations in human blood samples is developed and validated. Solid-phase column extraction is performed to clean up blood samples before running the analytical HPLC system. The chromatography is isocratic with a mobile phase consisting of acetonitrile (20%, v/v), methanol (23%, v/v), and 0.1 M potassium hydrogen phosphate buffer (pH 3.6; 57%, v/v) at a constant flow rate of 2 mL/min. Clobazam is detected at 226 nm. Chromatography is completed within less than 25 min. The recovery rate is greater than 95% and linear over a wide range of drug concentrations. The intra-assay coefficient of variation percentage varies between 4.3 and 12. This method is used for therapeutic drug monitoring in patients undergoing antiepileptic therapy with clobazam. Plasma levels of clobazam ranged from 21 to 663 ng/mL. Other antiepileptic compounds, such as clonazepam and phenobarbital, did not interfere with the detection of clobazam.     

Determination of vancomycin in human plasma using high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the quantification of vancomycin in human plasma was developed and validated. The method includes an extraction of vancomycin by deproteinization with acetonitrile. The analyses were carried out at 258 nm as the emission wavelength while exciting at 225 nm on a reversed-phase column (30 cm × 4 mm i.d. × 10 μm Waters Associates μBondapak C18) using a mobile phase composed of methanol and phosphate buffer at pH 6.3. Vancomycin was quantitatively recovered from human plasma samples (>96%) with high values of precision. The separation was completed within 27 min. The calibration curve was linear over the range from 5 to 1,000 ng/mL with the detection and quantification limits of 2 ng/mL and 5 ng/mL, respectively. This method is suitable for the routine assay of plasma samples. Figure The effect of the deproteinization solvent on the signal of the interference peak at retention time of 15.0 min. The peak which interferes with the peaks of Erythromycin and Vancomycin has been disappeared by using 2 mL acetonitrile as the deproteinization solvent.     

Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

Improvement of doxazosin determination in human plasma using high-performance liquid chromatography with fluorescence detection

A simple, sensitive, rapid, and reproducible high-performance liquid chromatographic method is developed and validated for the determination of doxazosin in human plasma without a solvent extraction procedure. This method involves plasma protein precipitation using methanol. The structurally related compound prazosin is used as an internal standard. Doxazosin is detected with high sensitivity using spectrofluorimetry. Over the concentration range 0.5-20 ng/mL, the absolute recovery values are all greater than 98%. The method has a quantitation limit of 0.5 ng/mL. The intra- and interday coefficient of variation and inaccuracy values are all less than 8% and 7%, respectively. Therefore, the method has been applied in pharmacokinetic studies of doxazosin.