Simultaneous determination of NK-104 and its lactone in biological samples by column-switching high-performance liquid chromatography with ultraviolet detection

A simple and sensitive column-switching HPLC method has been developed for the simultaneous determination of NK-104 (HMG–CoA reductase inhibitor) and its lactone in human and dog plasma. Plasma sample was extracted with methyl tert-butyl ether and then the extract was subjected to methylation with diazomethane to prevent the mutual conversion between NK-104 and its lactone. The extract was injected into the column-switching HPLC system. The calibration curves of NK-104 and NK-104 lactone were linear over the ranges 0.5 to 100 ng/ml for human plasma samples and 0.5 to 500 ng/ml for dog plasma, respectively. The intra-day and inter-day C.V. values of these analytes were less than 13.3%. The intra-day and inter-day accuracies of these analytes were between −14.0 and 6.5%. The proposed method has been applied to plasma samples obtained after oral administration of a single 2 mg dose of NK-104 to volunteers.     

Simultaneous determination of estriol and estriol 3-sulfate in serum by column-switching semi-micro high-performance liquid chromatography with ultraviolet and electrochemical detection

A column-switching HPLC with semi-microcolumn enabled us a direct and simultaneous analysis of estriol (E3) and estriol 3-sulfate (E3 S) in human serum in combination with ultraviolet (for E3 S) and electrochemical (for E3) detectors. The mobile phases (phosphate buffer pH 7.0) contained 5 mM tetra-n-butylammonium ion (TBA) as a counter ion for E3 S. Serum samples were diluted with 200 mM phosphate buffer (pH 7.0) containing 100 mM TBA, then injected to the pre-column. After serum proteins had flowed out from the pre-column, E3 and E3 S were transferred to the enrichment column. Subsequently the analytes were eluted to the analytical column. Detection limits of E3 and E3 S in human serum were 2.5 ng/ml and 295 ng/ml. Serum E3 and E3 S levels (mean±SD) of umbilical artery from 18 full-term healthy neonates were 33±23 ng/ml and 1.26±0.69 μg/ml, respectively.     

Determination of scutellarin in rat plasma by high-performance liquid chromatography with ultraviolet detection

A validated high-performance liquid chromatography method is described for the determination of scutellarin in rat plasma using a liquid-liquid extraction and ultraviolet (UV) absorbance detection. The separation used a Diamonsil ODS column (250 mm x 4.6mm i.d., 5 microm particle size) with an isocratic mobile phase consisting of methanol-acetonitrile-50mM dihydrogen ammonium phosphate buffer (22:15:63 (v/v/v), adjusted to pH 2.5 with 1M phosphoric acid). The ultraviolet detector operated at 335 nm. Plasma samples were extracted with ethyl acetate after acidification. The extraction recovery of scutellarin ranged from 68.1 to 80.5%. High selectivity and a low quantitation limit (0.050 microg/ml) were achieved. The linear range was 0.050-12.5 microg/ml, correlation coefficient r=0.9981. The method has a good reproducibility, R.S.D. values were below 7.9% for within-day and between-day precision. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.     

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.     

Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography–ultraviolet (HPLC–UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0–2000 pg/ml) and midazolam (range 0–400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (< 15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography–mass spectrometry (GC–MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC–UV system.     

Simultaneous determination of ZT-1 and its metabolite Huperzine A in plasma by high-performance liquid chromatography with ultraviolet detection

ZT-1 is a novel acetylcholinesterase (AChE) inhibitor. It is rapidly transformed to Huperzine A (Hup A) in vitro. A simple and rapid HPLC-UV method for the simultaneous determination of ZT-1 and its metabolite Hup A in plasma is described. The chromatographic separations were achieved on a C(18) ODS column (250 mm x 4.6 mm ID) using methanol-1 mmol/L ammonium acetate (70:30,v/v) as mobile phase. The flow rate was 0.7 mL/min, the detection wavelength was 313 nm and the column temperature was kept at 35 degrees C. Plasma samples were prepared as rapidly as possible and extracted immediately with 5 mL of chloroform:iso-propyl alcohol mixture (v/v, 9:1).The retention times of ZT-1 and Huperzine A (Hup A) were 18.7 and 14.4 min, respectively. The mean absolute recoveries of two analytes were >90%. Quantification limits were all 0.02 nmol/mL for ZT-1 and Hup A. This analytical method was reliable and convenient procedure that meets the criteria for the pharmacokinetic evaluation of ZT-1 on experimental animals.     

Simultaneous determination of five antipsychotic drugs in rat plasma by high performance liquid chromatography with ultraviolet detection

A significant percentage of psychiatric patients who are treated with antipsychotics are treated with more than one antipsychotic drug in the clinic. Thus, it is advantageous to use a rapid and reliable assay that is suitable for determination of multiple antipsychotic drugs in plasma in a single run. A simple and sensitive HPLC-UV method was developed and validated for simultaneous quantification of olanzapine, haloperidol, chlorpromazine, ziprasidone, risperidone and its active metabolite 9-hydroxyrisperidone in rat plasma using imipramine as an internal standard (I.S.). The analytes were extracted from rat plasma using a single step liquid-liquid acid solution back extraction technique with wash procedure, which provided the very clear baseline for blank plasma extraction. The compounds were separated on an Agilent Eclipse XDB C8 (150 mm x 4.6 mm i.d., 5 microm) column using a mobile phase of acetonitrile/30 mM ammonium acetate including 0.05% triethylamine (pH 5.86 adjusted with acetic acid) with gradient elution. All of the analytes were monitored using UV detection. The method was validated and the linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries, selectivity and stability were determined. The LLOQ was 2.0 ng/ml and correlation coefficient (R(2)) values for the linear range of 2.0-500.0 ng/ml were 0.998 or greater for all the analytes. The precision and accuracy for intra-day and inter-day were better than 7.44%. The recovery was above 74.8% for all of the analytes. This validated method has been successfully used to quantify the plasma concentration of the analytes for pharmacological and toxicological studies following chronic treatment with antipsychotic drugs in the rat.     

Determination of risedronate in human urine by column-switching ion-pair high-performance liquid chromatography with ultraviolet detection

An HPLC assay for the determination of risedronate in human urine was developed and validated. Risedronate and the internal standard were isolated from 5-ml urine samples in a two-part procedure. First, the analytes were precipitated from urine along with endogenous phosphates as calcium salts by the addition of CaCl(2) at alkaline pH. The precipitate was then dissolved in 0.05 M ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and subjected to ion-pair solid-phase extraction using a Waters HLB cartridge (1 ml, 30 mg) with 1-octyltriethylammonium phosphate as the ion-pair reagent. Following extraction, the analytes were initially separated from the majority of co-extracted endogenous components on a Waters X-Terra RP18 (4.6 x 50 mm, 3.5 microm) column. The effluent from the X-Terra was "heart-cut" onto a Phenomenex Synergi Polar RP (4.6 x 150 mm, 4 microm) column for final separation. UV detection (lambda=262 nm) was used to quantitate risedronate in the concentration range of 7.5-250 ng/ml. Mean recovery was 83.3% for risedronate and 86.5% for the internal standard. The intra-day precision of the assay, as assessed by replicate (n=5) standard curves, was better than 6% RSD for all points on the standard curve. Within-day accuracy for the standards ranged from 96.3 to 106.1% of nominal. Inter-day precision for quality controls assayed over a 3-week period was better than 5%, while inter-day accuracy was within 90% of nominal. The assay was employed to analyze samples collected during a clinical pharmacokinetics study.     

Simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) procedure for the simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma samples is described. A one-step solid-phase extraction (SPE) with C18 cartridges was coupled with a reversed-phase HPLC system. The system requires two mobile phases composed of tetrabutyl ammonium hydrogensulfate (10 mM adjusted to pH 7)-acetonitrile (62:38, v/v) for quinapril, and (25:75, v/v) for quinaprilat elution through a C18 Symmetry column and detection at a wavelength of 215 nm. Calibration curves were linear over the ranges 20 to 1,000 ng/ml for quinaprilat and 10 to 500 for quinapril. The limits of quantification were 20 and 10 ng/ml for quinaprilat and quinapril, respectively. Extraction recoveries were higher than 90% for quinapril and 80% for quinaprilat. This method has been successfully applied to a bioequivalence study of quinapril in healthy subjects.     

Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of ketamine and xylazine in canine plasma is described. Plasma samples (500 microl) are cleaned up via liquid-liquid extraction. The analytes and the internal standard clonidine are separated on a cyano (CN) column using a mobile phase containing acetonitrile-0.005 M phosphate buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. The method was validated according to specificity, sensitivity, accuracy and reproducibility and was used to determine the plasma concentrations of both compounds in dogs after intramuscular injection.     

Simultaneous determination of verapamil and norverapamil in biological samples by high-performance liquid chromatography using ultraviolet detection

In this paper we develop an high-performance liquid chromatographic method with ultraviolet detection for the determination of verapamil and its primary metabolite norverapamil in biological samples. Both compounds, as well as the internal standard, imipramine, were extracted from alkalinised blood, with n-hexane–isobutyl alcohol, back-extracted into 0.01 M phosphoric acid and determined using a reversed-phase column and ultraviolet monitoring at 210 nm. The average coefficient of variation obtained over the concentration range of 1–1000 ng/ml is about 3%. The detection limit is below 5 ng/ml for both compounds, and extraction recoveries close to 80%. The method was applied to a pharmacokinetic study of the drug and its active metabolite and used to analyse blood samples from verapamil treated rabbits.     

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl-cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A₂ and PAF-acetylhydrolase, on single phospholipids or their mixtures.     

Quantitative determination of domperidone in rat plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and selective analytical method for the determination of domperidone in rat plasma is described. The procedure involves liquid–liquid extraction followed by reversed-phase high-performance chromatographic analysis with fluorometric detection at 282 nm for excitation and 328 nm for emission. The detection limit was 1 ng ml⁻¹ using 1 ml of plasma. This assay procedure should be useful for the pharmacokinetic study of domperidone in small animals such as rats.     

Simultaneous determination of guanine nucleotides in rat brain by high-performance liquid chromatography with dual-electrochemical detection

A new, sensitive, and specific assay method for guanine nucleotides using high-performance liquid chromatography with dual-electrochemical detection was developed. GTP, GDP, GMP, and cyclic GMP were separated with reversed-phase "ion-pair" chromatography and detected by a dual-electrochemical detector. Only guanine nucleotides among all purine and pyrimidine nucleotides responded to the electrochemical detector at 0.95 V. The peak heights for these guanine nucleotides were linear at concentrations between 0.5 pmol and 1 nmol. The regional distribution of these guanine nucleotides in the rat brain was studied by this new assay method.     

Determination of montelukast sodium in human plasma by column-switching high-performance liquid chromatography with fluorescence detection

MK-0476 (montelukast sodium) is a potent and selective cysteinyl leukotriene receptor antagonist that is being investigated in the treatment of asthma. A simple and sensitive method for the determination of MK-0476 in human plasma was developed using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. A plasma sample was injected directly onto the HPLC system consisting of a pre-column (Capcell pak MF) and an analytical column (Capcell pak C18) which were connected with a six-port switching valve. The column eluate was monitored with a fluorescence detector (excitation at 350 nm; emission at 400 nm). The calibration curve was linear in a concentration range of 1–500 ng ml⁻¹ for MK-0476 in human plasma. The intra-day coefficients of variation of all concentrations within the range was less than 9.2%, and the intra-day accuracy values were between 97.2 and 114.6%. This method was used to measure the plasma concentration of MK-0476 following oral administration of the drug in humans.     

Simultaneous determination of clofibrate and its active metabolite clofibric acid in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet absorbance detection

A reversed-phase high-performance liquid chromatographic (HPLC) using ultraviolet (UV) absorbance detection method for simultaneous determination of clofibrate (I) and its major metabolite clofibric acid (II) in human plasma has been developed to support a clinical study. I, II and internal standard (I.S., III) are isolated from human plasma by 96-well solid-phase extraction (SPE) C(18)z.ccirf;AR plate and quantified by direct injection of the SPE eluent onto the HPLC with UV detection wavelength at 230 nm. Two chromatographic methods, isocratic and step gradient, have been validated from 1.0 to 100.0 microg/ml and successfully applied to plasma sample analysis for a clinical study. The lower limit of quantitation (LLOQ) is 1.0 microg/ml for both I and II when 500 microl plasma sample is processed. Sample collection and preparation is conducted at 5 degrees C to minimize the hydrolysis of I to II in human plasma.     

Micro determination of gentamicin in serum by high-performance liquid chromatography with ultraviolet detection

A procedure for the high-performance liquid chromatographic determination of gentamicin in serum is described using pre-column derivatisation and UV detection. The serum proteins are precipitated with acetonitrile and the gentamicin components in the supernatant are derivatized with 1-fluoro-2,4-dinitrobenzene. The reaction products are chromatographed on a microparticulate C₁₈ reversed-phase column and detected at 365 nm. Sample volumes of 50 μl are sufficient for the determination of gentamicin concentrations in, and well below, the therapeutic range.     

Determination of rifampin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifampin in human plasma. Rifampin and sulindac (internal standard) are extracted from human plasma using a C₂ Bond Elut extraction column. A 100-μl volume of 0.1 M HCl is added to the plasma before extraction to increase the retenction of the compounds on the extraction column. Methanol (1 ml) is used to elute the compounds and 0.5 ml of 3 mg/ml ascorbic acid in water is added to the final eluate to reduce the oxidation of rifampin. Separation is achieved by reversed-phase chromatography on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate-acetonitrile (55:45, v/v). Detection is by ultraviolet absorbance at 340 nm. The retention times of rifampin and internal standard are approximately 4.4 and 7.8 min, respectively. The assay is linear in concentration ranges of 50 to 35 000 ng/ml. The quantitation limit is 50 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.