Development of restricted-access media supports and their application to the direct analysis of biological fluid samples via high-performance liquid chromatography.

A quick overview of published methods for analyzing compounds in complex biological samples reveals that the most difficult step is the clean-up or extraction of a required compound from the matrix. The strategy required to analyze exogenous compounds in biological fluids depends greatly upon the nature of the compound and upon the biomatrix. Coupled-column separation using restricted-access media as the first dimension in order to exclude macromolecules and retain micromolecules has been successfully used for a number of biological fluids. This paper presents the history of the development of restricted-access media supports and of their application to the direct injection of biological fluid samples in high-performance liquid chromatography.

Validation of a selective method for determination of paroxetine in human plasma by LC-MS/MS.

PURPOSE: A sensitive, robust, and selective liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) was developed and validated for paroxetine quantification in human EDTA plasma. METHODS: Sample preparation was based on liquid-liquid extraction using a mixture of ethyl acetate/hexane (50/50; v/v) to extract the drug and internal standard from plasma. Chromatography was performed on a C-18 analytical column and the retention times were 1.6 and 1.7 for paroxetine and fluoxetine (IS), respectively. The ionization was optimized using ESI(+) and selectivity was achieved by tandem mass spectrometric analysis using MRM functions, 330.0 --> 70.0 and 310 --> 43.9 for paroxetine and fluoxetine. RESULTS: Analytical curve ranged from 0.2 to 20.0 ng/mL. Inter-day precision and accuracy of the quality control (QC) samples were < 15% relative standard deviation (RSD). Analyte stability during sampling processing and storage were established. CONCLUSION: Validation results on linearity, specificity, accuracy, precision as well as application to the analysis of samples taken up to 120 h after oral administration of 20 mg of paroxetine in 28 healthy volunteers were found to be of good performance in bioequivalence study.

Development and validation of a selective and robust LC-MS/MS method for quantifying amlodipine in human plasma.

A liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for quantifying amlodipine in human plasma was developed and validated. Sample preparation was based on liquid-liquid extraction using NaOH and a mixture of ethyl acetate/hexane (80/20; v/v). Chromatography was performed on a C-18 analytical column and the retention times were 1.9 and 3.0 min for amlodipine and nimodipine (internal standard), respectively. The ionization was optimized using ESI(+) and enhanced selectivity was achieved using tandem mass spectrometric analysis via two MRM functions, 409 --> 238 and 418 --> 343 for amlodipine and nimodipine. The calibration curve ranged from 0.2 to 20.0 ng/mL. The inter-day precision and accuracy and the relative standard deviation (RSD) were <15%. The analyte was shown to be stable over the time-scale of the whole procedure. The robustness of the method was demonstrated by the good reproducibility of the results obtained during the analysis of clinical samples.

Plasma hydroxy-metronidazole/metronidazole ratio in hepatitis C virus-induced liver disease.

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 +/- 0.0044 vs 0.0742 +/- 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 +/- 0.0032 vs 0.0438 +/- 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 +/- 0.0026 (mild chronic hepatitis) and vs 0.0478 +/- 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

Plasma hydroxy-metronidazole/ metronidazole ratio in hepatitis C virus-induced liver disease

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 ± 0.0044 vs 0.0742 ± 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 ± 0.0032 vs 0.0438 ± 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 ± 0.0026 (mild chronic hepatitis) and vs 0.0478 ± 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

Enantiomeric determination of the plasma levels of omeprazole by direct plasma injection using high-performance liquid chromatography with achiral-chiral column-switching.

A direct injection high-performance liquid chromatographic (HPLC) method, with column-switching, for the determination of omeprazole enantiomers in human plasma is described. A restricted access media (RAM) of bovine serum albumin (BSA) octyl column has been used in the first dimension for separation of the analyte from the biological matrix. The omeprazole enantiomers were eluted from the RAM column onto an amylose tris(3,5-dimethylphenylcarbamate) chiral column by the use of a column-switching valve and the enantioseparation was performed using acetonitrile-water (60:40 v/v) as eluent. The analytes were detected by their UV absorbance at 302 nm. The validated method was applied to the analysis of the plasma samples obtained from 10 Brazilian volunteers who received a 40 mg oral dose of racemic omeprazole and was able to quantify the enantiomers of omeprazole in the clinical samples analyzed. The assay was able to determine the cytochrome P450 2C19 phenotype of the subjects participating in this study.

Enantiomeric determination of pantoprazole in human plasma by multidimensional high-performance liquid chromatography.

Multidimensional HPLC is a powerful tool for the analysis of samples of a high degree of complexity. This work reports the use of multidimensional HPLC by coupling a RAM column with a chiral polysaccharide column to the analysis of Pantoprazole in human plasma by direct injection. The enantiomers from the plasma samples were separated with high resolution on a tris(3,5-dimethoxyphenylcarbamate) of amylose phase after clean-up by a RAM BSA octyl column. Water was used as solvent for the first 5 min in a flow-rate of 1.0 ml/min for the elution of the plasmatic proteins and then acetonitrile-water (35:65 v/v) for the transfer and analysis of pantoprazole enantiomers, which were detected by UV at 285 nm. Analysis time was 28 min with no time spent on sample preparation. A good linear relationship was obtained in the concentration range of 0.20 to 1.5 microg/ml for each enantiomer. Inter and intra-day precision and accuracy were determined by one low (0.24 microg/ml), one medium (0.70 microg/ml) and one high (1.3 microg/ml) plasma concentration and gave a C.V. varying from 1.80 to 8.43% and accuracy from 86 to 92%. Recoveries of pantoprazole enantiomers were in the range of 93.7-101.2%. The validated method was applied to the analysis of the plasma samples obtained from ten Brazilian volunteers who received an 80 mg oral dose of racemic pantoprazole and was able to quantify the enantiomers of pantoprazole in all clinical samples analyzed.