Monitoring of benzodiazepines (clobazam, diazepam and their main active metabolites) in human plasma by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic method for the simultaneous determination of clobazam, diazepam and their main metabolites in human plasma is described. A 200-μl plasma sample was directly injected into a precolumn filled with TSK-gel PW. After a washing step with potassium phosphate buffer, the retained substances were backflushed into a reversed-phase column with a mobile phase of acetonitrile—phosphate buffer—diethylamine. Various drugs frequently co-administered with clobazam or diazepam do not interfere with the determination.     

Determination of glimepiride in human plasma using semi-microbore high performance liquid chromatography with column-switching

A fully automated semi-microbore high performance liquid chromatographic (HPLC) method with column-switching using UV detection was developed for the determination of glimepiride from human plasma samples. Plasma sample (900 microl) was deproteinated and extracted with ethanol and acetonitrile. The extract (70 microl) was directly injected into a Capcell Pak MF Ph-1 pre-column where the primary separation occurred to remove proteins and retain drugs using a mixture of acetonitrile and 10mM phosphate buffer (pH 2.18) (20:80, v/v). The analytes were transferred from the pre-column to an intermediate column using a switching valve and then subsequently separated on an analytical column and monitored with UV detection at 228 nm. Glimepiride was eluted with retention time 34.9 min without interference of endogenous substance from plasma. The limit of quantification (LOQ) was 10 ng/ml for glimepiride. The calibration curves were linear over the concentration range of 10-400 ng/ml (r(2) = 0.9997). Moreover, inter- and intra-day precisions of the method were less than 15% and accuracies were higher than 99%. The developed method was successfully applied for the quantification of glimepiride in human plasma and was used to support a human pharmacokinetic study following a single oral administration of 2 mg glimepiride.     

Determination of three main antileprosy drugs and their main metabolites in serum by high-performance liquid chromatography

The simultaneous analysis of main antileprosy drugs such as 4,4′-diaminodiphenyl sulfone (DDS), clofazimine, rifampicin and their main metabolites in serum was examined by high-performance liquid chromatography using a μBondapak C_1a column. When the drugs dissoluted from serum were developed by tetrahydrofuran—0.5% acetic acid (40:60), clofazimine and rifampicins could be analyzed separately. Apart from the mutual separation of water-soluble conjugates of DDS, the individual analysis of DDS, its main liposoluble metabolite and a few related sulfone compounds is possible when the drugs are first developed by acetonitrile—water (20:80). By the use of tetrahydrofuran—water (50:50) containing PIC B-5, the rapid measurement of clofazimine isolated from the other compounds is also possible.     

Analysis of monoamines and their metabolites by high performance liquid chromatography with coulometric electrochemical detection

High performance liquid chromatography with coulometric electrochemical detection has been used to achieve simultaneous determination of norepinephrine, epinephrine, 5-hydroxytryptophan, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylacetic acid, N-acetyldopamine, tyramine, tryptophan, 5-hydroxyindoleacetic acid, 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, homovanillic acid, tyrosine, p-octopamine, N-acetyl-p-octopamine, and p-synephrine. The procedure has been applied to study monoamine degradation in the insect brain and to demonstrate that N-acetylation rather than oxidative deamination is the primary route of monoamine catabolism in insects.     

Bio-medical applications of high performance liquid chromatography (HPLC) with amperometric detection

Electrochemical detection of eluted solutes in HPLC is now well established as a selective and highly sensitive technique. Measurement of catecholamines and metabolites has been the most popular application with an exponential increase in literature. Recent improvements include the use of diphenylborate for extractions, the introduction of smaller diameter HPLC materials which improve resolution and sensitivity, and the replacement of carbon paste with glassy carbon which now gives a more stable electrode with similar sensitivity. Applications of HPLC with amperometric detection to the determination of drugs, amino acids and peptides are discussed.     

Determination of the antidepressant agent citalopram and metabolites in plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of citalopram [1-(3-(dimethylaminopropyl)-1-(4-fluorophenyl)-5-phthalancarbonitrile] and its two main metabolites (the methylamino and amino derivatives). The compounds were extracted from alkaline plasma with diethyl ether. The combined ether layers were evaporated after addition of 50 μl of 0.1 N HCl. The residual extracts were purified with diethyl ether and 20 μl were injected into a Spherisorb ODS 5-μm column with acetonitrile–0.6% phosphate buffer pH 3 (55:45, v/v) as the mobile phase. Using a fluorescence detector the detection limits are 1 ng/ml of plasma for citalopram and the methylamino metabolite and 0.5 ng/ml for the amino metabolite.     

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.     

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.     

Determination of metformin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the hypoglycemic agent metformin is described. Acidified samples of plasma were deproteinated with acetonitrile, washed with dichloromethane and the resulting supernatant injected. Chromatography was performed at 40°C by pumping a mobile phase of acetonitrile (250 ml) in pH 7, 0.03 M diammonium hydrogen phosphate buffer (750 ml) at a flow-rate of 1 ml/min through a silica column. Metformin and the internal standard (atenolol) were detected at 240 nm and were eluted 7.8 and 6.8 min, respectively, after injection. No endogenous substances were found to interfere. Calibration curves were linear (r>0.999) from 10 to 2000 ng/ml. The absolute recovery of both metformin and atenolol was greater than 76%. The detection limit and limit of quantitation were 2.5 and 10 ng/ml, respectively. The intra- and inter-day precision (C.V.) was 12%, or less, and the accuracy was within 6.2% of the nominal concentration. This method is suitable for clinical investigation and monitoring metformin concentration.     

Determination of oseltamivir carboxylic acid in human serum by solid phase extraction and high performance liquid chromatography with UV detection

A new straightforward method based on cloud-point extraction (CPE) has been developed, optimized and validated for the determination of venlafaxine in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection. The non-ionic surfactant Triton X-114 (polyethylene glycol tert-octylphenyl ether) was chosen as the extract solvent. Separation was obtained using a reversed-phase Diamonsil column (C(18), 250mmx4.6mm I.D., 5mum) and a mobile phase composed of acetonitrile-phosphate buffer solution (pH 3.0)-triethylamine (33.5:66.5:0.4). Fluorescence detection was used (lambda(ex) 276nm, lambda(em) 598nm). Maprotiline was used as the internal standard. Under the optimum conditions, the linear range of venlafaxine in human plasma was 10-800ngmL(-1) (r(2)=0.9995). The limit of detection (LOD) was less than 2ngmL(-1) (S/N=3) and the limit of quantification (LOQ) was less than 10ngmL(-1) (S/N=10). The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine capsules in nine healthy volunteers.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Automated analysis of quetiapine and other antipsychotic drugs in human blood by high performance-liquid chromatography with column-switching and spectrophotometric detection

An automated HPLC method with column switching is described for the determination of quetiapine, clozapine, perazine, olanzapine and metabolites in blood serum. After clean-up on silica C8 material (20 microm particle size) drugs were separated on ODS Hypersil C18 material (5 microm; column size 250 mm x 4.6 mm i.d.) within 25 min and quantified by ultraviolet (UV) detection at 254 nm. The limit of quantification ranged between 10 and 50 ng/ml. At therapeutic concentrations of the drugs, the inter-assay reproducibility was below 10%. Analyses of drug concentrations in serum of 75-295 patients treated with therapeutic doses of the antipsychotic drugs revealed mean+/-S.D. steady state concentrations of 139+/-136 ng/ml for quetiapine, 328+/-195 ng/ml for clozapine, 48+/-27 ng/ml for olanzapine and 71+/-52 ng/ml for perazine. The method was thus suitable for routine therapeutic drug monitoring and may be extended to other drugs.     

Automated determination of clomipramine and its major metabolites in human and rat serum by high-performance liquid chromatography with on-line column-switching

A fully automated method including column-switching and isocratic high-performance liquid chromatography (HPLC) was developed for simultaneous determination of the tricyclic antidepressant clomipramine and its metabolites demethylclomipramine, 2-, 8-, and 10-hydroxyclomipramine, 2-, and 8-hydroxydemethylclomipramine and didemethylclomipramine in serum. After serum injection into the HPLC system and on-line sample clean-up on a clean-up column (Hypersil CN; 10×4.6 mm) by an eluent consisting of 35% acetonitrile and 65% deionized water, the chromatographic separation was performed on an analytical column (LiChrospher CN; 250×4.6 mm I.D.) by an eluent consisting of 38% acetonitrile and 62% aqueous sodium perchlorate (0.02 M, pH 2.5). The UV detector was set at 260 nm. The limit of quantification was about 15 ng/ml for all analytes. The coefficients of variation ranged between 3 and 12% with recovery rates between 64 and 110%. Linear regression analyses revealed coefficients of correlation between 0.98 and 0.99. The method could be applied to therapeutic drug monitoring as well as metabolism studies in man and rat.     

The separation of leukotrienes and hydroxyeicosatetraenoic acid metabolites of arachidonic acid by high performance liquid chromatography (HPLC)

The following high performance liquid chromatography system was found suitable for separating most lipoxygenase metabolites of arachidonic acid: Techsphere 5-C18 column, eluting solvent methanol:water:acetic acid (65:35:0.06 v/v), pH 5.3. Comparisons with other packing materials and solvent systems are described. The method could be used to identify lipoxygenase products released from mouse macrophage cells stimulated with gamma-hexachlorocyclohexane. Detection limits between 1 and 10 ng were obtained.     

Determination of angiotensin metabolites in human plasma by fluorimetric high-performance liquid chromatography using a heart-cut column-switching technique

Fluorimetric column-switching HPLC method with naphthalene-2,3-dialdehyde (NDA) was developed for the determination of endogenous angiotensin (ANG) metabolites in human plasma. After one-step extraction to clean up the ultrafiltered plasma sample on the reversed HPLC system, the zone of the retention time of each ANG analyte was subjected to the NDA-derivatization. After putting into a first Phe-ODS (for ANG (3–4) and (5–8) determinations) or ODS column (for ANG I and II determinations), the heart-cut of the retention time of the NDA-ANG was separated on a second ODS column with a mobile phase containing 5 mM ion-pair reagent. Complete separation and good detection were accomplished within 2 h. Good linearity of the regression equation for all ANG analytes with the correlation coefficient of >0.993 as well as good reproducibility (C.V.<4.0%). Good agreement of the range of ANG II plasma level between the present (25–47 fmol/ml in plasma) and the radioimmunoassay methods (28–52 fmol/ml in plasma) indicated that the column-switching method could be applicable for the determination of endogenous smaller ANGs as well as for ANG I or II in plasma.     

高效液相色谱分析银杏萜内酯的含量

用高效液相色谱法测定银杏叶及提取物中银杏内酯Ａ、Ｂ、Ｃ和白果内酯的含量。采用Ｃ１８分离柱,示差检测器,甲醇：水（３３：６７）为流动相,方法回收率平均达９７％以上,变异系数（ＣＶ）为２．１％。     

Separation of steroidal estrogens and their major unconjugated metabolites by high performance liquid chromatography

A high performance liquid chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedures uses a "Diol" column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone, one of the least polar C18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semi-preparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.