A simple high performance liquid chromatographic method for the quantification of total cotinine, total 3'-hydroxycotinine and caffeine in the plasma of smokers

A simple isocratic HPLC procedure has been developed for the quantification of caffeine and the nicotine metabolites cotinine, 3'-hydroxycotinine, cotinine glucuronide and 3'-hydroxycotinine glucuronide in the plasma of smokers. The glucuronide conjugates were determined indirectly via initial basic hydrolysis of the analyte sample followed by quantification of the resulting deconjugation product. Plasma was basified, extracted with dichloromethane, evaporated, the residue dissolved water and an aliquot part was analyzed by HPLC. The method utilized a Partisil-10 SCX cation-exchange column and an isocratic mobile phase of sodium phosphate buffer: methanol (92:8 v/v, 0.1 M, adjusted to pH 4.8 with triethylamine) at a flow rate of 1.5 ml/min. UV detection was at 254 nm. All solutes were separated with good resolution, and quantification was determined using an internal standard of N,N-diethylnicotinamide. The retention times were: caffeine 5.1 min, 3'-hydroxycotinine 7.2 min, N,N-diethylnicotinamide 9.5 min, and cotinine 15.5 min. Detection limits for caffeine, 3'-hydroxycotinine, cotinine, and total cotinine were 10 ng/ml; the detection limit for total 3'-hydroxycotinine was 20 ng/ml. The inter-day and intra-day variations for all analytes were between 1 and 8%. This analytical method is suitable for the determination of caffeine and nicotine metabolite levels in large numbers of clinical samples.     

High performance liquid chromatographic determination of nicotine and cotinine in plasma and nicotine and cotinine, simultaneously, in urine

Three analytical procedures were developed to determine nicotine in plasma, cotinine in plasma and, simultaneously, nicotine and cotinine in urine. After liquid or solid-phase extraction, the purified aqueous phase is injected into a high performance liquid chromatograph equipped with an ultra-violet detector using a CN Spheri-5 micron cartridge-column with an inner diameter of 4.6 mm and a length of 10 or 22 cm. The limit of quantitation for nicotine in plasma was around 8 to 15 ng/ml, that of cotinine in plasma around 50 ng/ml and that of nicotine and cotinine in urine around 170 ng/ml and 70 ng/ml, respectively. The limit of detection of nicotine in plasma was around 1 ng/ml and that of nicotine and cotinine in urine around 20 ng/ml and 10 ng/ml, respectively. The passive exposure to cigarette smoke by non-smokers and the "resting levels" of nicotine in plasma and urine of smokers were studied. The analytical methods were set up to study the pharmacokinetics and bioavailability of nicotine in healthy volunteers following single and repeated administrations of different doses of transdermal nicotine systems.     

反相HPLC法同时测定人尿中茶碱及其两种代谢产物

目的:建立一种同时测定人尿中茶碱及其1,3-二甲基尿酸(1,3-DMU)和3-甲基噻嗪(3-MX)代谢产物的HPLC方法.方法:尿样用异丙醇/二氯甲烷(2/8)混合液提取,有机相在空气吹干,用流动相复溶后进行HPLC分析.色谱柱为Diamonsil ODS C_(18)5 μm,150 mm×4.6 mm I.D),流动相由0.1%甲酸液和乙腈(95:5)组成,流速1.0 mL/min,测定波长280 nm.测定12名受试者单剂量和多剂量口服茶碱后24 h内尿中茶碱及其代谢物累计排泄量.结果:尿中茶碱及其代谢物1,3 DMU和3-MX的线性范围分别为0.312～40.0、0.156～20.0、0.078～10.μg/mL,最低可定量浓度分别为0.312、0.156、0.078μg/mL.批间和批内的变异小于15%,回收率大于70%.结论:该方法的特异性、灵敏度能够满足临床上对人尿中茶碱及其代谢产物同时测定的要求.     

A validated high performance liquid chromatographic method for the analysis of Goldenseal

Goldenseal (Hydrastis canadensis L.) has emerged as one of the top ten herbal supplements on the worldwide market. A rapid, simple and validated high performance liquid chromatographic method, with photodiode array detection, has been developed for the analysis of commercial Goldenseal products. Samples were treated by sonication with acidified methanol/water. The method was validated for LOD, LOQ, linearity, reproducibility and recovery with good results.     

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbamyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 microm ODS (C18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min-1 and the column temperature was maintained at 30 degrees C. Galactosamine hydrochloride (Gal-HCl) was used as an internal standard. Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32+/-1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15+/-0.1 cm2. The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1% v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 microg ml-1. The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) <12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was 相似文献   

Specific high performance liquid chromatographic determination of quinidine in serum, blood, and urine

A reversed-phase high performance liquid chromatographic method for determination of quinidine in serum, blood, and urine has been developed. An alkylnitrile column is used with a mobile phase of acetonitrile in an acetate buffer. The method was rigorously tested and shown to be specific for quinidine using the following methods: comparison of capacity factors among methanolic reference standards of quinidine, known metabolites, and 36 other drugs; comparison of the quinidine capacity factor with the capacity factors from components in patient sera and urines, from which quinidine was selectively removed by thin-layer chromatography; and, correlation of quinidine concentrations in patient sera using ultraviolet absorbance versus fluorescence detection. Application of the method to a single-dose pharmacokinetic study, including serum protein binding and blood/serum concentration ratio measurements.     

Simplified high performance liquid chromatographic method for the determination of clonazepam and other benzodiazepines in serum

A convenient and sensitive high performance liquid chromatographic method was developed for determination of clonazepam in serum using a C-18 reverse-phase column, and mobile phase consisting of a 50:35:15 mixture by volume of pH 6.0 phosphate buffer:methanol:acetonitrile. Quantitation was performed at 313 nm with flunitrazepam as the internal standard. Using 1 ml of serum for extraction, the assay is linear for clonazepam concentrations between 10 and 250 ng/ml. The relative recovery averaged 100.3%, and the coefficient of variation for between-day and within-day assays was less than 7%. A simple modification permits analysis of 200 microliter of serum, with little loss of precision and with a detection limit of 20 ng/ml. Only three drugs tested (nitrazepam, methaqualone, and norchlordiazepoxide) interfered with the assay, and none are likely to be used therapeutically with clonazepam. Importantly, carbamazepine and carbamazepine-10,11-epoxide do not interfere under the conditions of the assay. The method is equally suitable for the determination of nitrazepam. By adjusting the mobile phase so that volume ratios of phosphate buffer, methanol, and acetonitrile are 45:35:20, and using nitrazepam as the internal standard, seven other benzodiazepines (demoxepam, oxazepam, chlordiazepoxide, norchlordiazepoxide, temazepam, diazepam, and nordiazepam) can be resolved at 254 nm.     

Acidic degredation of cephaloglycin and high performance liquid chromatographic determination of deacetylcephaloglycin in human urine.

In order to provide fundamental knowledge about the determination of deacetylcephaloglycin excreted in human urine as an active metabolite of cephaloglycin, the degradation of cephaloglycin in acidic media has been investigated with varying reaction temperature between 30 degrees and 50 degrees C and pH between 1.2 and 2.8. The degradation pathway observed under these conditions was the elimination of the 3-acetyl group yielding deacetylcephaloglycin followed by formation of deacetylcephaloglycin lactone. Estimation of first order rate constants revealed that deacetylation is the rate-determining step for the degradation of cephaloglycin to the lactone. It is found from the kinetic results that reproducible assays of deacetylcephaloglycin excreted in urine can be achieved by a quantitative conversion to deacetylcephaloglycin lactone in a medium of pH 1.4 at 37 degrees C for 2 hours, followed by a reversed-phase ion-pair high-performance liquid chromatography. The utility of the present method is demonstrated by determining the time course of urinary excretion of deacetylcephaloglycin after oral administration of cephaloglycin capsule.     

A validated ultra high pressure liquid chromatographic method for qualification and quantification of folic acid in pharmaceutical preparations

A fully validated UHPLC method for the identification and quantification of folic acid in pharmaceutical preparations was developed. The starting conditions for the development were calculated starting from the HPLC conditions of a validated method. These start conditions were tested on four different UHPLC columns: Grace Vision HT? C18-P, C18, C18-HL and C18-B (2 mm × 100 mm, 1.5 μm). After selection of the stationary phase, the method was further optimised by testing two aqueous and two organic phases and by adapting to a gradient method. The obtained method was fully validated based on its measurement uncertainty (accuracy profile) and robustness tests. A UHPLC method was obtained for the identification and quantification of folic acid in pharmaceutical preparations, which will cut analysis times and solvent consumption.     

Narrow-bore high performance liquid chromatographic method for the determination of cetirizine in human plasma using column switching

An improved column switching high performance liquid chromatographic (HPLC) method was developed for determination of cetirizine in human plasma. Plasma samples were prepared by liquid-liquid extraction using methylene chloride. The samples extracted were initially injected into a clean-up Capcell Pak MF C8 column and the peaks of cetirizine and internal standard were separated to an analytical C18 micro-column via column switching device. This analysis showed highly sensitive and selective results. Also, it was successfully applied to evaluate the pharmacokinetics of cetirizine in human volunteers after single oral administration.     

High performance liquid chromatographic analysis of hydrochlorothiazide in serum and urine

We report a high performance liquid chromatographic method for the analysis of hydrochlorothiazide in 500microliter of serum of 25 microliter of urine. The between-day precision of the analysis (CV 5.0-7.0%) is acceptable, as is the recovery of hydrochlorothiazide (90-96%) and trichloromethiazide (96%) added to serum. No drug or drug metabolite interferences have been noted. The method represents a considerable improvement over existing techniques described in the literature.     

Development of a high performance liquid chromatography method for quantification of PAC-1 in rat plasma

A sensitive and specific high performance liquid chromatography method with UV detection was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on a Diamonsil C₁₈ column (150 mm × 4.6 mm i.d., 5 μm particle size, Zhonghuida) protected by a ODS guard column (10 mm × 4.6 mm i.d., 5 μm particle size), using acetonitrile–methanol–phosphate buffer (pH 3.0, 30 mM) (31:3:66, v/v/v) as mobile phase at a flow rate of 1.0 mL/min, and wavelength of the UV detector was set at 281 nm. No interference from any endogenous substances was observed during the elution of PAC-1 and internal standard (IS, indapamide). The calibration curve was linear over the range of 0.05–20 μg/mL (r > 0.99). The lower limit of quantification was evaluated to be 50 ng/mL. The method was successfully applied to the pharmacokinetic study of PAC-1 after intravenous and oral administration in rats, respectively.     

HPLC法测定人血清及尿中美洛培南浓度

目的　建立测定人血清样品及尿样品中美洛培南的定量分析方法。方法　采用高效液相色谱法 ,以 μ- Bondapak C1 8柱 (3.9mm× 30 0 mm,10 μm)为色谱柱 ;甲醇∶ 5 mmol/ L 磷酸二氢钾缓冲液 (17∶ 83,v/ v)为流动相 ,p H2 .5 ,检测波长 30 8nm。结果　血清中美洛培南的回收率平均为 97.5 4 % (n=6 ) ;日内 ,日间RSD≤ 3.80 % ,在 1～ 10 0 mg/ L 血药浓度范围内线性关系良好 ,r=0 .9997。尿中美洛培南的平均回收率为96 .90 % (n=6 ) ;日内、日间 RSD<3.5 0 % ,在 5～ 10 0 mg/ L 尿药浓度范围内线性关系良好 ,r=0 .9994。结论该方法灵敏准确 ,适用于临床药代动力学的研究。     

用高效液相色谱法测定儿童氨苄青霉素的药动学参数

本文报道用高效液相色谱法研究儿童时期氨苄青霉素的药物动力学。本法以β-羟乙基茶碱为内标物,C_(18)反相色谱柱,0.1mol/L 磷酸二氢钾/乙腈(92/8)作流动相,214nm 检测,方法学考查表明该方法灵敏度、精密度高,特异性强,可用于药动学研究和治疗药物监测。8名儿童静滴氨苄青霉素100mg/kg,药物动力学符合二室开放式模型。其消除半衰期t_(1/2)β1.07h。     

A validated high performance liquid chromatographic method for analysis of nicotine in pure form and from formulations

A reverse phase HPLC method using C18 column has been developed for the quantitative estimation of nicotine in the bulk material and formulations (extended release and immediate release dosage forms). The method is specific to nicotine (RT approximately 4.64 min, asymmetry approximately 1.75), and can resolve analyte peak from excipient interferences. It is linear (coefficient of variation approximately 0.9993), accurate (average recovery approximately 100%), and passed all the system suitability requirements. Applicability of the method was evaluated in analysis of drug-excipient compatibility samples, commercial dosage form (such as nicotine gum) and two novel in-house formulations.     

Development and validation of a specific stability indicating high performance liquid chromatographic method for rizatriptan benzoate

A gradient, reversed-phase liquid chromatographic (RP-LC) method was developed for the quantitative determination of rizatriptan benzoate, used to treat relieves migraine headache symptoms. The developed method can be also employed for the related substance determination in bulk samples. Forced degradation studies were performed on bulk sample of rizatriptan benzoate using acid (0.5 N hydrochloric acid), base (0.1 N sodium hydroxide), oxidation (3.0% hydrogen peroxide), water hydrolysis, heat (60 degrees C) and photolytic degradation. Mild degradation of the drug substance was observed in base hydrolysis and considerable degradation observed during oxidative stress. The chromatographic method was fine tuned using the samples generated from forced degradation studies. Good resolution between the peaks corresponds to degradation products and the analyte was achieved on Agilent Zorbax SB-CN (250 mm x 4.6 mm, 5 microm) column. The mobile phase consists of a mixture of aqueous potassium di hydrogen ortho phosphate (pH 3.4), acetonitrile and methanol. The stress sample solutions were assayed against the qualified reference standard of rizatriptan benzoate and the mass balance in each case was close to 99.7% indicating that the developed method is stability indicating. Validation of the developed method was carried out as per ICH requirements.     

Sensitive high performance liquid chromatographic analysis of ethmozin in plasma

A sensitive high performance liquid chromatographic procedure for the quantitative determination of ethmozin is described. Following a one-step extraction of the drug and the internal standard, protriptyline, by diethylether at pH 9.0, the organic solvent is evaporated and the residue is reconstituted in the mobile phase and injected into the chromatograph. The separation is obtained using a CN-bonded column with a methanol-propanol-2-perchloric acid 1.16 M mobile phase. Ethmozin is detected at 268 nm. Under these conditions, the lower limit of detection is 11 nM, and the lower limit of quantification is 22 nM (10 ng/ml) for ethmozin. The procedure is linear between 0 and 8,620 nM.