Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid–liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78–46.95 μmol/l, with a mean coefficient of correlation (r)≥0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 μmol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.     

Determination of naringin and naringenin in human urine by high-performance liquid chromatography utilizing solid-phase extraction

An HPLC method for determining a flavonoid naringin and its metabolite, naringenin, in human urine is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using hesperidin for naringin or hesperetin for naringenin as internal standard and solid-phase extraction using a strong anion exchanger, Sep-Pak Accell QMA cartridge. The HPLC assay was carried out using an Inertsil ODS-2 column (250×4.6 mm I.D., 5 μm particle size). The mobile phases were acetonitrile–0.1 M ammonium acetate–acetic acid (18:81:1, v/v; pH 4.7) for naringin and acetonitrile–0.1 M ammonium acetate–triethylamine (25:75:0.05; v/v; pH 8.0) for naringenin. The flow-rate was 1.0 ml min⁻¹. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 282 nm for naringin and at 324 nm for naringenin. The lower limits of quantification were ca. 25 ng/ml for naringin and naringenin with R.S.D. less than 10%. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 5 ng for naringin and 1 ng for naringenin. A preliminary experiment to investigate the urinary excretion of naringin, naringenin and naringenin glucuronides after oral administration of 500 mg of naringin to a healthy volunteer demonstrated that the present method was suitable for determining naringin and naringenin in human urine.     

Validated liquid chromatographic method for the determination of bexarotene in human plasma

A new liquid chromatographic method is described for the determination of the anti-tumour agent bexarotene in human plasma over the range 0.500-1500 ng/ml, using 1 ml of sample. Sample preparation consists of liberating the analyte from plasma lipids by adding acetonitrile, followed by acidification of the plasma and liquid extraction using a mixture of isoamyl alcohol and pentane or hexane. Separation and quantitation are performed by reversed-phase column liquid chromatography with fluorescence detection. Parameters affecting the performance of these steps are discussed. Validation results on linearity, selectivity, accuracy, precision, recovery and stability are shown, as well as the application of the method to samples from clinical trials.     

Solid-phase extraction and determination of ranitidine in human plasma by a high-performance liquid chromatographic method utilizing midbore chromatography

An improved high-performance liquid chromatographic (HPLC) method utilizing solid-phase extraction (SPE) and midbore chromatography was developed for the determination of ranitidine in human plasma. A mobile phase of 20 mM K₂HPO₄-acetonitrile-triethylamine (87.9:12.0:0.1, v/v) pH 6.0 was used with a phenyl analytical column and ultraviolet detection (UV). The method demonstrated linearity from 25 to 1000 ng/ml in 500 μl of plasma with a detection limit of 10 ng/ml. The method was utilized in a pharmacokinetic study evaluating the effects of pancreatico-biliary secretions on ranitidine absorption.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

High-performance liquid chromatographic determination of diclofenac in human plasma after solid-phase extraction

A novel high-performance liquid chromatographic (HPLC) method for the quantification of diclofenac in human plasma was set up. Samples, added with ibuprofen (used as internal standard) were purified by solid-phase extraction using Abselut Nexus cartridges (Varian) not requiring pre-conditioning. Drugs of interest were eluted directly into the autosampler vials and injected. The recovery of diclofenac was 92%, the analysis lasted 7 min with a sensitivity of 5 ng/ml and intra- and inter-day RSDs of 3 and 8%, respectively. The pharmacokinetics of diclofenac after oral and rectal administration in 10 healthy volunteers are reported.     

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Reliable and specific high-performance liquid chromatographic method for simultaneous determination of loratadine and its metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid-liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol-acetonitrile-phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5-16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.     

Simple high-performance liquid chromatographic method for the determination of tocotrienols in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of vitamin E especially δ-, γ- and α-tocotrienols in human plasma. The method entailed direct injection of plasma sample after deproteinization using a 3:2 mixture of acetonitrile–tetrahydrofuran. The mobile phase comprised 0.5% (v/v) of distilled water in methanol. Analyses were run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 296 nm and emission wavelength of 330 nm. This method is specific and sensitive, with a quantification limit of approximately 40, 34 and 16 ng/ml for α-, γ- and δ-tocotrienol, respectively. The mean absolute recovery values were about 98% while the within-day and between-day relative standard deviation and percent error values of the assay method were all less than 12.0% for α-, γ- and δ-tocotrienol. The calibration curve was linear over a concentration range of 40–2500, 30–4000 and 16–1000 ng/ml for α-, γ- and δ-tocotrienol, respectively. Application of the method in a bioavailability study for determination of the above compounds was also demonstrated.     

Simple high-performance liquid chromatographic method for the determination of metformin in human plasma

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5–4000 ng/ml.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Improved high-performance liquid chromatographic method for the routine determination of unconjugated 3-methoxy-4-hydroxyphenylethyleneglycol in human plasma using solid-phase extraction and electrochemical detection

An improved semi-automated high-performance liquid chromatographic method is described for the routine determination of unconjugated 3-methoxy-4-hydroxyphenylethyleneglycol in plasma. The 3-ethoxy analogue of the compound is used as an internal standard. The method is based on purification of 0.5-ml plasma samples with phenyl-type reversed-phase extraction columns, reversed-phase separation with an acetate—citrate—methanol mobile phase with an octadecyl-bonded column, and dual-electrode coulometric detection with oxidation at +0.44 V and reduction at −0.25 V. The precision and accuracy of the assay are satisfactory: the lower limit of reliable detection corresponds to a plasma concentration of 1.5 nM. The validity of the determination is demonstrated by an 18% mean increase in plasma levels of 3-methoxy-4-hydroxyphenylethyleneglycol during physical exercise (duration 16 min, n = 13) and a 50% mean reduction in plasma levels induced by a single dose of the monoamine oxidase inhibitor, moclobemide (n = 8). The method is suitable for routine use in pharmacological and physiological experiments.     

High-performance liquid chromatographic analysis for the determination of miconazole in human plasma using solid-phase extraction

A high-performance liquid chromatographic method for the determination of miconazole in human plasma is described. A solid-phase extraction was performed on an octadecyl (C₁₈) cartridge. Miconazole was eluted with methanol, separated on a reversed-phase column and was measured by ultraviolet detection at 230 nm. The absolute extraction recovery from plasma samples was 85%. The limit of detection was established as 5 ng/ml. The coefficient of variation of the determination of plasma levels by this method over the standard curve concentration range was less than 10%, except with the concentration of 10 ng/ml. The plasma levels of miconazole in twelve healthy volunteers given a 250-mg oral dose of two tablet forms were determined by this method.     

On-line solid-phase extraction and high-performance liquid chromatographic determination of chlorthalidone in urine

A simple and rapid on-line method for the determination of chlorthalidone in urine is proposed. The sample containing the internal standard is injected in a CN precolumn. After a 2-ml water rinsing, the precolumn is coupled for 30 s to the HPLC column via a switching valve, allowing the on-line elution of the compounds of interest. Analysis is carried out by reversed-phase chromatography with an acetonitrile-0.01 M phosphate buffer pH 7 (20:80, v/v) eluent, using UV detection at 214 nm. While the LC separation is performed, the precolumn is regenerated and conditioned, and is ready to receive the next sample at the end of the run. Accurate (>95%) and precise (<10%) analyses, in the range of 0.1–20 μg/ml of chlorthalidone in urine, have been achieved using this method.     

Validated liquid chromatographic determination of 5-fluorouracil in human plasma

A sensitive, reproducible, selective and accurate high performance liquid chromatographic (HPLC) method for the quantitative determination of 5-flurorouracil in plasma has been developed and validated using isocratic elution and UV detection. The method provides a selective quantifications of 5-flurorouracil without any interference of the endogenous uracil. The assay is performed after a double extraction of 5-flurorouracil and thymine (internal standard) from human plasma using ethyl acetate. The drug and the internal standard were eluted from a Genesis C(18) analytical column at ambient temperature with mobile phase consisting of methanol:water (10:90, v/v) adjusted to pH 3.2 with perchloric acid at a flow rate of 1.0 ml/min. The effluent was monitored with an ultraviolet detector at 260 nm. Quantification was achieved by the measurement of the peak-height ratios and the limit of quantification for 5-flurorouracil in plasma was 30 ng/ml. The retention times for 5-flurorouracil, uracil, and thymine were 4.5, 6.0, and 9.0, respectively. The intra-day coefficient of variation (CV) ranged from 1.35 to 4.53% at three different concentrations and the inter-day CVs varied from 1.29 to 4.98%. The relative and absolute recoveries varied from 96 to 101%. Stability tests showed that 5-flurorouracil is stable for at least 72 h in plasma after freezing. The simple method may permit the assessment of 5-flurorouracil plasma concentrations for pharmacokinetic studies in combination with clinical trials.     

Validated liquid chromatographic ultraviolet method for the quantitation of Etoricoxib in human plasma using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (284 nm) was developed and validated for quantitation of Etoricoxib in human plasma, the newest addition to the group of nonsteroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. Following a single-step liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v), the analyte and internal standard (Zaleplon) were separated using an isocratic mobile phase of water/acetonitrile (58/42, v/v) on reverse phase Waters symmetry C(18) column. The lower limit of quantitation was 5 ng/mL, with a relative standard deviation of less than 20%. A linear range of 5-2500 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 4.1-5.1% and 1.1-2.4%, respectively. The between- and within-batch bias was -3.8-4.7% and -0.6-9.4%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Etoricoxib in plasma was >90%, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive and simple with between-batch precision of <6% and was used in pharmacokinetic studies.     

Efficient high-performance liquid chromatographic assay for the simultaneous determination of metoprolol and two main metabolites in human urine by solid-phase extraction and fluorescence detection

An improved, more efficient method for the determination of metoprolol and its two metabolites in human urine is reported. The simultaneous analysis of the zwitterionic metoprolol acidic metabolite (III, H117/04) with the basic metabolites α-hydroxymetoprolol (II, H119/66), metoprolol (I) and guanoxan (IV, internal standard) was achieved employing solid-phase extraction and isocratic reversed-phase HPLC. The analytes were extracted from urine (100 μl) using C₁₈ solid-phase extraction cartridges (100 mg), and eluted with aqueous acetic acid (0.1%, v/v)–methanol mixture (40:60, v/v, 1.2 ml). The eluents were concentrated (250 μl) under vacuum, and aliquots (100 μl) were analysed by HPLC with fluorescence detection at 229 nm (excitation) and 309 nm (emission) using simple isocratic reversed-phase HPLC (Novapak C₁₈ radial compression cartridge, 4 μm, 100×5 mm I.D.). Acetonitrile–methanol–TEA/phosphate buffer pH 3.0 (9:1:90, v/v) was employed as the eluent (1.4 ml/min). All components were fully resolved within 18 min, and the calibration curves for the individual analytes were linear (r²≥0.996) within the concentration range of 0.25–40.0 mg/ml. Recoveries for all four analytes were greater than 76% (n=4). The assay method was validated with intra-day and inter-day variations less than 2.5%.     

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

Sensitive high-performance liquid chromatographic method for the determination of coumarin in plasma

A high-performance liquid chromatographic method was developed for the determination of coumarin in plasma at low concentrations. The method involves a single-step extraction of the alkalinized sample with hexane and subsequent evaporation of the organic phase in the presence of hydrochloric acid to collect and concentrate the coumarin. Analysis of the acidic phase was performed on a C₈ column and coumarin was detected by measuring the UV absorbance at 275 nm. The limit of detection was 0.3 μg l⁻¹. The assay was used to study the evolution of concentrations of coumarin in one volunteer after oral administration of a single 10-mg dose.