Column-switching techniques for high-performance liquid chromatography of ibuprofen and mefenamic acid in human serum with short-wavelength ultraviolet detection

Column-switching techniques for high-performance liquid chromatography of two acidic drugs, ibuprofen and mefenamic acid, in human serum with short-wavelength ultraviolet detection are described. The method involved extraction of the analyte from acidified serum followed by the chromatographic analysis using column switching. Three ODS columns were used each with different mobile phase, utilizing the difference of ion-pair formation or of ionization caused by pH change. The method offered high sensitivity and selectivity, with short-wavelength ultraviolet detection at 221 nm for ibuprofen and at 219 nm for mefenamic acid. The detection limits were 0.5 ng/ml (2.4 pmol/ml) for ibuprofen and 0.1 ng/ml (0.4 pmol/ml) for mefenamic acid using 1 ml of serum, both at a signal-to-noise ratio of 3. With some modifications, the principle of the method would be applicable to other acidic compounds in biological fluids.     

Determination of free serum didanosine by ultrafiltration and high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid chromatographic method was developed to determine free didanosine concentrations in human serum. An ultrafiltration technique was used to recover didanosine from the samples. Didanosine was analyzed using a 150 mm × 3.9 mm I.D. Nova-Pak phenyl column and a mobile phase of 0.02 M sodium citrate (pH 5)-isopropanol (97.5:2.5, v/v) with detection set at 250 nm. Linearity was verified from 25 to 3000 ng/ml. The limit of detection at a signal-to-noise ratio of 3 was 25 ng/ml. The mean recovery of didanosine added to serum at 50, 100, 250 and 750 ng/ml was 97.4%, 97.3%, 92.9% and 95.4%, respectively. A within-day variation of 3.6% at 50 ng/ml and 1.7% at 250 ng/ml, and a day-to-day variation of 9.3% at 50 ng/ml and 3.6% at 230 ng/ml were found. Stability studies indicated that didanosine is stable in serum for at least 8.5 months at 20°C, 4°C and −20°C.     

Reversed-phase liquid chromatography method to determine COL-3, a matrix metalloproteinase inhibitor, in biological samples

A reversed-phase high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of 6-deoxy-6-demethyl-4-dedimethylamino-tetracycline (COL-3), a matrix metalloproteinase (MMPs) inhibitor, in rat serum. This simple, sensitive, rapid and reproducible assay involved a preliminary serum deproteinization by adding a mixture of acetonitrile-methanol-0.5 M oxalic acid (70:20:10 (v/v)), as the combined precipitant and metal blocking agent, into serum samples (2:1 (v/v)). An aliquot (20 microl) of the supernatant was injected into the HPLC system linked to a Waters XTerra RP(18) column (150 mm x 4.6 mm i.d., particle size 5 microm). The compound was eluted by a mixture of acetonitrile-methanol-0.01 M oxalic acid (40:10:50 (v/v), pH 2.00), as the mobile phase, and detected at the wavelength of 350 nm. The total running time was 10 min. The low and high concentration calibration curves were linear in the range of 50-1200 ng/ml and 1200-12,000 ng/ml, respectively. The intra- and inter-day coefficients of variation at three quality control concentrations of 100, 1200, and 12,000 ng/ml were all less than 6%, while the percent error ranged from -2.5 to 6.6%. The limit of quantitation (LOQ) for COL-3 in serum was 50 ng/ml. This assay was successfully employed to study the serum concentration-time profiles of COL-3 after its intravenous and oral administration in rats. The method with some minor modifications in sample pretreatment was also applicable to the determination of the concentrations of COL-3 in rat bile, urine and feces.     

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C₁₈ column (250 × 4.6 mm I.D.) using 10 mM phosphoric acid—acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 μg/ml and the detection limits were 0.06 μg/ml for indomethacin and 0.08 μg/ml for mefenamic acid, for 50μl plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

Albendazole sulphoxide concentrations in plasma of endemic normals from a lymphatic filariasis endemic region using liquid chromatography

A simple and sensitive reversed-phase isocratic HPLC method for the determination of albendazole and its metabolites has been developed. The mobile phase consisting of acetonitrile-water-perchloric acid (70%) (30:110:0.06 (v/v/v)) was pumped at a flow rate of 0.80 ml/min on a 5 microm, reverse phase, Discovery RPamide C16 column with UV detection at 290 nm. The calibration graphs were linear in the range of 0.05- 1 microg/ml for albendazole, albendazole sulphoxide and albendazole sulphone. The limit of quantification was 50 ng/ml for albendazole, 25 ng/ml for albendazole sulphoxide and 30 ng/ml for albendazole sulphone. The within-day and day-to-day coefficient of variation averaged 4.98 and 6.95% for albendazole, 3.83 and 6.83% for albendazole sulphoxide and 3.44 and 5.51% for albendazole sulphone, respectively. The mean extraction recoveries of albendazole, albendazole sulphoxide and albendazole sulphone were 79.25, 93.03 and 88.78%, respectively. The method was applied to determine the plasma levels of albendazole sulphoxide in endemic normals administered with albendazole during pharmacokinetic studies.     

A simple and sensitive HPLC method for determination of gliclazide in human serum

A simple, rapid and specific method for analysis of gliclazide in serum by a sensitive high-performance liquid chromatographic method is described. Only 100 microl of serum and a little sample work-up is required. A simple procedure of extraction by toluene followed by evaporation to dryness under a gentle stream of air and dissolving the dried residue in mobile was used. The gliclazide peak was separated from endogenous peaks on a C(8) column by a mobile phase of acetonitrile-water (45:55, v/v), pH 3. Gliclazide and internal standard (phenytoin) were eluted at 6.8 and 3.8 min, respectively. The limit of quantitation (LOQ) for gliclazide in serum was 75 ng/ml at 230 nm. The method was linear over the range of 75-10,000 ng/ml with r(2) of 0.999. Mean recovery for gliclazide and internal standard was 84.5 and 87%, respectively.     

An HPLC method for the determination of ng mifepristone in human plasma

An HPLC method was developed and validated for the determination of mifepristone in human plasma. C(18) solid-phase extraction cartridges were used to extract plasma samples. Separation was by C(18) column; mobile phase, methanol-acetonitrile-water (50:25:25, v/v/v); flow rate, 0.8 ml/min; UV detection at 302 nm. The calibration curve was linear in the concentration range of 10 ng/ml to 20 microg/ml (r=0.9991). Within- and between-day variability were acceptable. The limit of detection for the assay was 6 ng/ml. Plasma samples were stable for at least 7 days in the state of plasma or residue treated at -20 degrees C. The method was simple, sensitive and accurate, and allowed to determine ng mifepristone in human plasma. It could be applied to assess the plasma level of mifepristone in women receiving low oral doses of mifepristone.     

Simple high-performance liquid chromatography method for the simultaneous determination of serum caffeine and paraxanthine following rapid sample preparation

A simple reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of caffeine and paraxanthine in human serum is described. Serum proteins are precipitated with perchloric acid and the resulting supernatant neutralized for direct injection onto an HPLC column. The method uses a phosphate–methanol mobile phase (85:15, v/v) at pH 4.9 with a flow-rate of 1.75 ml/min and quantitation is by UV absorbance at 274 nm. Elution times are approximately 18 min for caffeine and 8 min for paraxanthine. Theobromine and theophylline have elution times of 5.4 and 9.4 min and do not interfere in the assay. The intra-assay and between-assay means for precision and accuracy for both drugs are: 4.5% C.V. and 3.3% deviation. The sensitivity of the method is 50 ng/ml for each drug.     

Optimization of a solid-phase extraction method for determination of indapamide in biological fluids using high-performance liquid chromatography

A new simple and rapid high-performance liquid chromatographic (HPLC) method with UV detection for the determination of indapamide in biological fluids has been developed. Indapamide and internal standard were isolated from serum and whole blood samples by solid-phase extraction with RP select B cartridges. The chromatographic separation was accomplished on a reversed-phase C(8) column with a mobile phase composed of 0.1% (v/v) triethylamine in water (pH 3.5) and acetonitrile (63:37, v/v). UV detection was set at 240 nm. The calibration curves were linear in the concentration range of 10.0-100.0 ng/ml for serum, and 50.0-500.0 ng/ml for whole blood, and the limits of quantification were 10.0 and 50.0 ng/ml, respectively.     

Liquid chromatographic method for the determination of rizatriptan in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the determination of rizatriptan in human plasma. Following a single-step liquid-liquid extraction with methyl tertiarybutyl ether, the analytes were separated using a mobile phase consisting of 0.05% (v/v) triethylamine in water (adjusting to pH 2.75 with 85% phosphoric acid) and acetonitrile (92:8, v/v). Fluorescence detection was performed at an excitation wavelength of 225nm and an emission wavelength of 360nm. The linearity for rizatriptan was within the concentration range of 0.5-50ng/ml. The intra- and inter-day precisions of the method were not more than 8.0%. The lower limit of quantification (LLOQ) was 0.5ng/ml for rizatriptan. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Determination of 6-hydroxychlorzoxazone and chlorzoxazone in porcine microsome samples

A simple, accurate and sensitive HPLC method for the in vitro determination of 6-hydroxychlorzoxazone and chlorzoxazone in porcine microsome samples is described. Chromatography was performed on a YMC-Pack ODS-AQ column using a mobile phase of 0.05% phosphoric acid pH 3-methanol (60:40, v/v). UV detection was carried out at 287 nm. The detector response was linear over the concentration range 25-2000 ng/ml. This assay produced quick, accurate, and repeatable results.     

Simultaneous determination of digoxin and permeability markers in rat in situ intestinal perfusion samples by RP-HPLC

A simple, sensitive and specific reversed-phase high performance liquid chromatographic (RP-HPLC) assay for simultaneous determination of digoxin and permeability markers, in samples obtained from intestinal in situ single-pass perfusion studies, was developed and validated. Chromatography was carried on C-18 column with mobile phase comprising of acetate buffer (pH 3.0), acetonitrile and methanol in the ratio of 50:25:25 (v/v/v), was pumped at a flow rate of 0.5 ml/min and UV detection was employed at 220 nm. The average retention times for phenolred, propranolol, frusemide and digoxin were 9.1, 10.7, 12.9 and 15.3 min, respectively. The calibration curves were linear (R(2) > 0.998) in the range for each analyte. The method is specific and sensitive with limit of quantification of 25 ng/ml for digoxin and frusemide and 10 ng/ml for phenolred and propranolol. The method is accurate and precise with recoveries of digoxin in the range of 95.2 and 103.2% and relative standard deviation (R.S.D.) <5%. We found that this method was simple and reliable in permeability determination and to estimate the contribution of P-glycoprotein in limiting intestinal absorption.     

Determination of cimetidine in human plasma by high-performance liquid chromatography following liquid-liquid extraction

A new method is described for the determination of cimetidine in human plasma. The drug and internal standard (ranitidine) were separated on a Nucleosil C₁₈ 5 μm (25 × 4.6 mm I.D.) column using a mobile phase of acetonitrile-phosphate buffer, pH 6.2 (25:75, v/v) containing 2.5 g/l heptane sulphonic acid. The mobile phase was delivered at a flow-rate of 0.9 ml/min, detection was by ultraviolet absorption at 228 nm and concentrations were calculated on the basis of peak areas. The drugs were extracted from alkaline plasma into ethyl acetate using a salting out procedure which involved the addition of 100 ml of a saturated solution of K₂CO₃ to each 250-μl plasma aliquot. The method was validated over the concentration ranges 50–3000 ng/ml and 100–7000 ng/ml for two separate studies. Mean coefficients of variation were less than 6% for both intra- and inter-assay in both studies and recoveries varied between 71 and 81%. The method was successfully applied to the determination of cimetidine in plasma for a pharmacokinetic study.     

Simultaneous determination of all-trans-, 13-cis-, 9-cis-retinoic acid and their 4-oxo-metabolites in plasma by high-performance liquid chromatography

A gradient reversed-phase high-performance liquid chromatographic technique is described for the easy separation and quantification of some retinoids; all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and their corresponding 4-oxometabolites, in plasma. The method involved a diethyl ether-ethyl acetate (50:50, v/v) mixture extraction at pH 7 with acitretin and 13-cis-acitretin as internal standards. A Nova-Pak C₁₈ steel cartridge column was used. The mobile phase was methanol-acetonitrile (65:35, v/v) and 5% tetrahydrofuran (solvent A) and 2% aqueous acetic acid (solvent B) at 1 ml/min. The gradient composition was (only the percentages of solvent B are mentioned): I, 25% solvent B at the time of injection; II, 12% solvent B at 11 min until 30 min; III, 25% solvent B and maintenance of 25% solvent B for 10 min until a new injection. Total time between injections was 40 min. Detection was by absorbance at 350 nm. The precision calculated for plasma concentrations ranging from 2 to 250 ng/ml was better than 15% and the accuracy was less than 12%. The linearity of the method was in the range of 2 to 400 ng/ml of plasma. The limit of quantification was 2 ng/ml for each of the compounds. The HPLC method was applied to plasma specimens collected from animals receiving single dose administrations of all-trans-retinoic acid, 13-cis-retinoic acid and 9-cis-retinoic acid.     

Simultaneous determination of thienorphine and its active metabolite thienorphine glucuronide in rat plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies

A simple, sensitive and reliable method was developed to determine simultaneously the concentrations of thienorphine and its metabolite thienorphine glucuronide conjugate in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The metabolite was identified by MS: thienorphine glucuronide conjugate. Sample preparation involved protein precipitation with methanol. Analytes were separated on Finnigan BetaBasic-18 column (150 mm x 2.1mm i.d., 5 microm) using methanol: water: formic acid (56:44:0.1, v/v/v) as mobile phase at a flow rate of 0.2 ml/min. The method had a linear calibration curve over the concentration range of 0.1-50 ng/ml for thienorphine and 2-1000 ng/ml for thienorphine glucuronide conjugate, respectively. LOQ of thienorphine and thienorphine glucuronide conjugate was 0.1 and 2 ng/ml, respectively. The intra- and inter-batch precisions were less than 12% and their recoveries were greater than 80%. Pharmacokinetic data of thienorphine and its metabolite thienorphine glucuronide conjugate obtained with this method following a single oral dose of 3mg/kg thienorphine to rats were also reported for the first time.     

Selective method for the determination of cefdinir in human plasma using liquid chromatography electrospray ionization tandam mass spectrometry

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of cefdinir in human plasma. After a simple protein precipitation using trichloracetic acid, the post-treatment samples were applied to a prepacked RP18 Waters SymmetryShield column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of methanol-water-formic acid (25:75:0.075, v/v/v). The analyte and I.S. cefaclor were both detected by the use of selected reaction monitoring mode. The method was linear in the concentration range of 5-2,000 ng/ml. The lower limit of quantification was 5 ng/ml. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 4.3%. The accuracy determined at three concentrations (36, 360 and 1,800 ng/ml for cefdinir) ranged from 99.6 to 106.7% in terms of recovery. The chromatographic run time for each plasma sample was less than 3 min. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of cefdinir capsule in 12 healthy volunteers.     

Simple and sensitive method for the determination of celecoxib in human serum by high-performance liquid chromatography with fluorescence detection

A simple method is described for the determination of the cyclooxygenase-2 specific inhibitor celecoxib in human serum by HPLC using the demethylated analogue as internal standard. After protein precipitation with acetonitrile, samples were extracted with chloroform. Separation was achieved on a Prontosil C18 AQ column (150x3 mm I.D., 3-microm particle size) at a flow-rate of 0.35 ml/min using water-acetonitrile (40:60, v/v) as the mobile phase. Using fluorescence detection with excitation at 240 nm and emission at 380 nm, the limit of quantification was 12.5 ng/ml for a sample size of 0.5 ml of serum. The assay was linear in the concentration range of 12.5-1500 ng/ml and showed good accuracy and reproducibility. At all concentrations intra- and inter-assay variabilities were below 11% with less than 9% error. The method was applied to the determination of celecoxib for pharmacokinetic studies in man.     

Ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of strychnine and brucine in mice plasma

A selective, simple and efficient method-ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for determination of two toxic alkaloids, namely strychnine and brucine in mice plasma. The UPLC separation was carried out using a 1.7 μm BEH C(18) column (50 mm × 2.1 mm) with a mobile phase consisting of methanol:0.1% formic acid (25:75, v/v), hence providing high efficiency, high resolution and excellent peak shape for the analytes and internal standard. The method was validated over the range of 2.48-496.4 ng/ml for strychnine and 2.64-528 ng/ml for brucine, respectively. Intra- and inter-day accuracy ranged from 95.0% to 107.9% for strychnine, 93.4% to 103.3% for brucine, and the precisions were within 13.8%. The extraction recoveries of both the two alkaloids exceed 81.9%. With a simple and minor sample preparation procedure and short run-time (<3 min), the proposed method was applicable for the pharmacokinetic and toxicological analysis of strychnine and brucine in vivo.     

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.