Evaluation of drug-free plasma profiles by high-performance liquid chromatography following on-line solid-phase extraction

The effect of varying the type of column and eluent composition on drug-free plasma profiles was investigated. The study was based on a C18 and a CN column; methanol and acetonitrile were the organic modifiers used. The plasma profiles were evaluated quantitatively by measuring the number of interfering peaks greater than 8 . 10(-4) absorbance units in the area of interest along the chromatogram. Results were subjected to statistical treatment using a three-factor analysis of variance design. The three factors were the column, the type of organic modifier and either the percentage organic modifier, the pH or the ionic strength. Analysis of the data revealed that significant effects were seen with changing eluent composition, particularly with regard to the percentage of organic modifier, and that the observed effects were strongly dependent on the type of column and the type of organic modifier under consideration.     

Quantification of trovafloxacin in serum by high-performance liquid chromatography with on-line solid-phase extraction

Summary A rapid, simple, accurate and sensitive liquid chromatographic assay with on-line solid-phase extraction is described for determination of trovafloxacin in human serum. Samples were deproteinized with acetonitrile and injected on to an NH₂ extraction column for sample clean-up. Thereafter, an on-line column-switching system was used for quantitative transfer of the drug to a C₁₈ analytical column. Separation was performed by ion-pair chromatography and detection was by ultraviolet absorbance at 275 nm. Recovery was 98.5%. The linear range was from 0.25 to 20μg mL⁻¹, with a correlation coefficient of 0.999. Detection limit was 0.1 μg mL⁻¹ from extraction of 25 μL serum.     

Determination of tetracyclines residues in honey by on-line solid-phase extraction high-performance liquid chromatography

An automated system using on-line solid-phase extraction (SPE) high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of tetracyclines (TCs), such as tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), metacycline (MC), and doxycycline (DC) in honey. One milliliter diluted honey sample was injected into a conditioned C18 SPE column and the matrix was washed out with water for 3 min. By rotation of the switching valve, TCs were eluted and transferred to the analytical column by the chromatographic mobile phase. Chromatographic conditions were optimized. TCs were separated in less than 8 min with a gradient elution using a mixture of 0.8% formic acid and acetonitrile. The UV detection was performed at 365 nm. The conditions for on-line SPE, including solvent and total time for loading sample and washing matrix were also optimized. Time for extraction and separation decreased greatly. For the five kinds of TCs, the limits of detection (LODs) at a signal-to-noise of 3 ranged from 5 to 12 ng g⁻¹. The relative standard deviations (R.S.D.) for the determination of TCs ranged from 3.4 to 7.1% within a day and ranged from 3.2 to 8.9% in 3 days, respectively.     

Piroxicam quantitation in human plasma by high-performance liquid chromatography with on- and off-line solid-phase extraction.

A comparative study of two analytical methodologies for piroxicam quantitation in plasma by off-line and on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) is described. The SPE cartridges contained C8 for both extraction methods. The analytes piroxicam and tenoxican (internal standard) were separated on a C18 column with a mobile phase consisting of acetonitrile:20 mM phosphate buffer pH 3.1 (50:50, v/v) followed by UV detection at 360 nm. The validation of the methods demonstrated good recoveries (over 90%), sensitivity (limits of quantification of 0.05 microgram/ml with on-line SPE and 0.1 microgram/ml with off-line SPE, based on a 100 microliters and 200 microliters sample volume, respectively), accuracy and precision (better than 9.5%). Both methodologies have been used for bioequivalence studies.     

Determination of estazolam in plasma by high-performance liquid chromatography with solid-phase extraction.

A high-performance liquid chromatography (HPLC) assay was developed for the determination of estazolam in human plasma. Estazolam and alprazolam as an internal standard were detected by ultraviolet absorbance at 240 nm. Estazolam in plasma was extracted by a rapid and simple procedure based on cyanopropyl bonded-phase extraction. Chromatographic separation was achieved with a reversed-phase C8-5 column using a mobile phase of 0.5% potassium dihydrogenphosphate(pH 4.5)-acetonitrile (70:30, v/v). The determination of estazolam was possible in the concentration range of 1.0 - 200.0 ng/mL. The mean recovery of estazolam added to plasma was 96.1 +/- 1.5% with coefficients of variation of less than 5.5%. This method is applicable for accurately monitoring the plasma level of estazolam in healthy subjects participating in scientific research.     

Quantification of myrislignan in rat plasma by solid-phase extraction and reversed-phase high-performance liquid chromatography

A rapid and simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for determination of myrislignan in rat plasma after intravenous administration. The analytes extracted from plasma samples by solid-phase extraction were successfully carried out on a Diamonsiltrade mark ODS C(18) column (250 x 4.6 mm i.d., 5 microm) with an RP(18) guard column (8 x 4.6 mm i.d., 5 microm) and a mobile phase of MeOH-H(2)O (4:1, v/v). The UV detector was set at a single wavelength of 270 nm. The linear ranges of the standard curves were 0.5-30.0 microg/mL with the correlation coefficients greater than 0.9992. The lower limits of detection and quantification were 0.1 and 0.3 microg/mL for myrislignan. Intra- and inter-day precisions were 2.4-7.5 and 1.3-5.7%, respectively. The extraction recovery from plasma was more than 90%. This assay method has been successfully used to study the pharmacokinetics of myrislignan in rats.     

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.     

Efficient purification and metabolite analysis of radiotracers using high-performance liquid chromatography and on-line solid-phase extraction

This study describes an efficient method using on-line solid-phase extraction (SPE) (Oasis HLB) for preparative HPLC purification of short-lived radiotracers for positron emission tomography (PET) and for HPLC analysis of radiotracers and their metabolites in cell homogenates, plasma and urine samples. The radiochemical purity of tracers (fluorine-18 labeled) purified using this method (Oasis column) was >99% compared to 90% when no Oasis column was used. Radiometabolites of several fluorine-18 and carbon-11-labeled tracers and one technetium-99m tracer were quantified in cell homogenates, plasma and urine samples. Samples were analyzed using Oasis column and analytical HPLC system without prior precipitation of proteins or removal of other biological matrices. The metabolites observed for the evaluated tracers were all polar relative to the unchanged tracer. The extraction repeatability was found to be good (RSD 2.2%) and recoveries of Oasis column/HPLC-injected radioactivity (plasma) were found to be high (mean recovery >91%). The same Oasis column was used for several times without back pressure build-up or decrease of the HPLC separation characteristics.     

Determination of amineptine and its main metabolite in plasma by high-performance liquid chromatography after solid-phase extraction

Amineptine and its main metabolite were determined simultaneously in plasma by high-performance liquid chromatography using quinupramine as internal standard. The method comprised adsorption on Extrelut column from alkaline plasma, elution with diethyl ether-methylene chloride, evaporation in the presence of 0.01 M hydrochloric acid and injection of the acid solution onto a mu Bondapak C18 column, using acetonitrile-0.025 M potassium dihydrogenphosphate as mobile phase and ultraviolet detection at 210 nm. Average steady-state concentrations of the two compounds were determined in four patients under treatment regimen (two 100-mg doses of amineptine per day, at 8.00 and 12.00 h). The concentrations determined 20 h after the last dose were undetectable in all cases, whereas the concentrations determined 1 h after the second dose were found to be 780 +/- 96 ng ml-1 for amineptine and 690 +/- 137 ng ml-1 for its metabolite. The technique can also be applied to whole blood with, if necessary, identification on the basis of the ultraviolet spectrum obtained by photodiode-array detection.     

Quantitation of zopiclone and desmethylzopiclone in human plasma by high-performance liquid chromatography using fluorescence detection

A simple, reliable HPLC method using fluorescence detection (excitation 307 and emission 483 nm) was developed and validated for simultaneous quantitation of zopiclone and its metabolite desmethylzopiclone in human plasma. Following a single-step liquid-liquid extraction, the analytes and internal standard (zaleplon) were separated using an isocratic mobile phase on a reversed-phase C18 column. The lower limit of quantitation was 3 ng/mL for zopiclone and 6 ng/mL for desmethylzopiclone with a relative standard deviation of less than 5%. A linear dynamic range of 3-300 ng/mL for zopiclone and of 6-500 ng/mL for desmethylzopiclone was established. This HPLC method was validated with between-batch precision of 1.7-4.2% and 3.2-7.5% for zopiclone and desmethylzopiclone respectively. The between-batch accuracy was 99.4-111.5% and 101.6-104.8% for zopiclone and desmethylzopiclone, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of zopiclone and desmethylzopiclone 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 atorvastatin and its metabolite ortho-hydroxyatorvastatin in human plasma by on-line anion-exchange solid-phase extraction and liquid chromatography tandem mass spectrometry

A rapid, sensitive, and specific method was developed and validated using liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of atorvastatin (ATV) and its major metabolite ortho-hydroxyatorvastatin (o-HATV) in human plasma. The sample preparation involved a liquid–liquid extraction without chlorinated solvents and an on-line solid-phase extraction exploring the possibilities that anion exchange offers. The analytical method presented intraday and day-to-day variation below 10%; intraday and day-to-day accuracy stood between 94% and 105%; the limit of quantification was 0.1 ng/mL for ATV and 0.5 ng/mL for o-HATV; and the recovery was above 75% for both molecules. This method was applied successfully to quantitate ATV and o-HATV concentrations in an unstudied renal transplant recipient cohort treated with an immunosuppressive regime of tacrolimus and mycophenolic acid and a cohort of hypercholesterolemic patients included in the study as a control group. It can be used to evaluate patient adherence, drug–drug interactions, and pharmacokinetic/pharmacodynamic relationships. The results in our study showed that ATV and o-HATV levels in the renal transplant group were significantly increased (p < 0.001), compared to the hypercholesterolemic group.     

Determination of andrographolide and dehydroandrographolide in rabbit plasma by on-line solid phase extraction of high-performance liquid chromatography

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining andrographolide and dehydroandrographolide in rabbit plasma. Plasma samples (100 μL) were injected directly into a C18 SPE column and the biological matrix was washed out for 6 min using 15% aqueous methanol. By rotation of the switching valve, andrographolide and dehydroandrographolide were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase consisted of methanol:acetonitrile (ACN):water (50:10:40; v/v). The UV detection was performed at 225 nm. The calibration curves showed excellent linear relationship (R ≥ 0.9993) over the concentration range of 0.05-5.0 μg mL⁻¹. The within- and between-day precisions (R.S.D.) of two analytes were in the range of 1.2-6.5% and the accuracies were between 92.0% and 102.1%. Their recoveries were all greater than 94%. The limits of detection were 0.019 μg mL⁻¹ for andrographolide and 0.022 μg mL⁻¹ for dehydroandrographolide. This method was successfully applied to the plasma concentration-time curve study after oral administration of Andrographis paniculata Nees extract in rabbit.