A Reversed-Phase High-Performance Liquid Chromatography Assay Procedure for Progabide and Its Related Metabolic Derivatives

A reversed-phase high-performance liquid chromatography (HPLC) assay procedure for Progabide, its active acid metabolite (PGA), and its hydrolytic degradation product (SL79.182) has been developed. This highly specific technique has allowed the simultaneous determination of these drugs in aqueous samples, and when coupled with a single and easy extraction step, spiked plasma samples could also be analyzed. The method had a sensitivity of about 30, 45, and 100 ng/ml for Progabide, SL79.182, and PGA, respectively.     

High Performance Liquid Chromatographic (HPLC) Determination of Centchroman in Human Serum and Application to Single-Dose Pharmacokinetics

A simple and sensitive (2 ng/ml) HPLC method with fluorescence detection has been developed to measure serum concentrations of centchroman, a new nonsteroidal antifertility agent. The method was sufficiently sensitive to follow the drug over 21 days in human volunteers. Pharmacokinetic parameters of centchroman were determined after a single oral dose of 60 mg (2 × 30-mg tablets) in two healthy female volunteers. Centchroman is slowly eliminated from serum, showing a biexponential disappearance curve from serum. The terminal half-life of centchroman in the two volunteers was 168 and 175 hr, respectively.     

Pharmacokinetics of Sulfasalazine Metabolites in Rats Following Concomitant Oral Administration of Riboflavin

Sulfasalazine, 60 mg/kg, was administered orally to groups of rats (n = 4) along with 1, 5, or 10 mg/kg of riboflavin. Plasma and urine were assayed for 5-aminosalicylic acid, acetyl-5-aminosalicylic acid, sulfapyridine, and acetyl-sulfapyridine using an HPLC method. The mean percent of dose recovered as total metabolites in urine was significantly greater ( = 0.01) for the group receiving 10 mg/kg riboflavin compared to the controls or the group receiving 1 mg/kg riboflavin. Plasma AUC and C _max values were also significantly greater ( = 0.05) for the 10 mg/kg riboflavin group. These results suggest that at higher doses, a significant fraction of riboflavin reaches the colon intact and stimulates more efficient reduction of the azo bond in sulfasalazine. Since the concentrations of 5-ASA achieved in the colon may be directly related to the efficacy of sulfasalazine in treating inflammatory bowel disease, concomitant administration of riboflavin may enhance sulfasalazine's efficacy in humans.     

The Pharmacokinetics of Butorphanol and its Metabolites at Steady State Following Nasal Administration in Humans

The single-dose and steady state pharmacokinetics of butorphanol and its metabolites, hydroxybutorphanol (HO-B) and norbutorphanol (NOR-B), were studied in nine healthy male volunteers. Each subject received a single 1 mg dose of butorphanol on days 1 and 6, and a 1 mg dose every 6 h (q6h) on days 2–5, via nasal administration. Serial blood and urine samples were collected for 24 h after the first dose on day 1 and for 72 h at steady state on day 6. Plasma and urine samples were analyzed for free and conjugated butorphanol, HO-B, and NOR-B. The plasma samples were analyzed using validated gas chromatography–electron capture negative chemical ionization–mass spectrometric methods and the urine samples were analyzed using a validated HPLC procedure. In the plasma, conjugated metabolites were not detected and only trace amounts of NOR-B were present. Therefore, pharmacokinetic parameters could not be estimated for NOR-B and conjugated metabolites. AUC_0→τ of butorphanol after the first dose and AUC_0→τ at steady state were not statistically different, indicating that the kinetics of butorphanol were not significantly altered after repeated dosing. Steady state levels of butorphanol were attained within 3 days (d) of q6h dosing and the accumulation index was 1·2 for butorphanol. Due to a relatively long t_1/2 of 15 h of HO-B compared to the dosing interval (q6h), the accumulation index was 6·0 for this metabolite. The evaluation of the molar plasma concentration ratio of HO-B to butorphanol as a function of time revealed that HO-B exhibits elimination-rate-limited kinetics. Similarly to butorphanol, steady state levels of HO-B were attained within 3 d of q6h dosing. © 1997 John Wiley & Sons, Ltd.     

The Pharmacokinetics of Antipyrine and Three of Its Metabolites in the Rabbit: Intravenous Administration of Pure Metabolites

Antipyrine (AP) is a commonly used probe of oxidative metabolism. Indirect evidence demonstrates formation rate limited disposition of its metabolites. Kinetic studies using antipyrine and its major metabolites 3-hydroxymethylantipyrine (HMA), norantipyrine (NORA), and 4-hydroxyantipyrine (OHA) were completed to investigate the metabolic fate of preformed antipyrine metabolite and to demonstrate directly formation rate-limited metabolite disposition in vivo. Bolus injections of antipyrine and preformed metabolites (40-50 mg/kg) were administered to male, New Zealand white rabbits. Plasma and urine were analyzed using HPLC. These studies demonstrate that HMA, NORA, and OHA are formation rate limited in the rabbit. NORA appears to undergo further extensive oxidative and conjugative metabolism. Unknown additional peaks were detected in urine after NORA dosing but not after HMA or OHA administration. Mass spectroscopy of the unknown HPLC eluents identified potential structures of these NORA metabolites.     

A High-Performance Liquid Chromatographic Microassay Employing a Liquid–Solid Extraction Technique for Etintidine in Plasma

This paper describes a new, rapid solid extraction method for the determination of etintidine in plasma. The method employs a semiautomatic sample preparation system. Plasma samples and the internal standard (cimetidine) were applied onto octyl-bonded silica extraction columns. The extraction columns were then subjected to Tris buffer and water wash and were subsequently loaded onto an automatic sample injection system. The contents of the extraction columns were eluted on-line with a mobile phase of acetonitrile:methanol:0.1% ammonium hydroxide (85:10:5, by volume) onto a silica analytical column and detected by UV absorption at 229 nm. The chromatographic condition separates etintidine from some of its metabolites and other endogenous components in plasma. The detection limit for etintidine was 0.02–0.05 µg/ml when 0.2 ml of plasma was used. This method has been used for the determination of plasma etintidine levels in humans and mice after oral administration of etintidine and was found to be suitable for pharmacokinetic/bioavailability studies of etintidine in humans and animals. The method can also be used for the quantitative determination of cimetidine and certain metabolites of etintidine.     

A High-Performance Liquid Chromatographic Assay for the Enantiomers of Bevantolol in Human Plasma

A method was developed and validated for the simultaneous analysis of (+)- and (–)-bevantolol in human plasma. The assay involves plasma protein precipitation, derivatization of racemic bevantolol to its diastereomeric thioureas with 2,3,4,5-tetra-o-acetyl--D-gluco-pyranosyl isothiocyanate, and solid-phase extraction of the diastereomers from 0.5 ml human plasma. Chromatographic separation was accomplished under isocratic conditions using a reversed-phase C-18 analytical column and mobile phase consisting of equal parts of 75 mM dibasic ammonium phosphate buffer (adjusted to pH 3.5 with phosphoric acid) and acetonitrile, with a detection wavelength of 220 nm. The absolute peak-height method was employed for quantitation. Retention times for the diastereomers of ( + )- and (–)-bevantolol were 7.4 and 6.4 min, respectively. The method is suitable for the quantification of the enantiomers over a concentration range of 40 to 800 ng/ml per enantiomer.     

去铁酮在大鼠体内的药代动力学与组织分布

目的研究去铁酮(DFP)在大鼠体内的药代动力学和组织分布。方法雄性Wistar大鼠ig给予DFP35,70和140mg.kg-1后,于不同时间点收集血液和组织样本。采用高效液相色谱法测定大鼠血浆及组织中的DFP的含量,运用DAS2.0药代动力学智能分析软件拟合房室模型,并进行药代动力学参数计算。结果大鼠ig给予DFP35,70和140mg.kg-1后,体内药代动力学过程符合二室模型,t1/2α分别为23.3,22.2和20.9min,t1/2β分别为53.3,50.9和46.3min,Cl分别为0.017,0.021和0.016L.min-1.kg-1。大鼠ig给予DFP70mg.kg-1后,DFP在胃和肝中浓度较高,60min时肝中DFP含量可达(359.22±31.16)μg.g-1,其他组织含量较低。结论DFP在大鼠体内吸收和消除较迅速,在体内组织分布广。     

反相高效液相色谱法测定人血浆中甲磺丁脲的含量

目的建立反相高效液相色谱法用于测定人血浆中甲磺丁脲的含量。方法采用Eclipse XDB-C_(18)色谱柱,以乙腈-25 mmol·L~(-1)乙酸钠缓冲液(pH=3.3,32:68)为流动相,流速1.0 mL·min~(-1),检测波长为229 nm,柱温35℃。血样经等体积比的0.6 mol·L~(-1)三氯乙酸处理后,离心,取上清液20μL,进样检测。结果甲磺丁脲血浆药物浓度在0.5～100 mg·L~(-1)内,线性关系良好(r=0.999 6);回收率为93.0%～105.0%;日内RSD≤3.80%,日间RSD≤6.31%。结论本方法简单快速、准确灵敏、回收率高、重现性好,适用于甲磺丁脲的血药浓度测定。     

High-Performance Liquid Chromatography Determination of Acitretin in Plasma and Its Application to a Pharmacokinetic Study in Human Subjects

Pharmaceutical Research -     

High-Performance Liquid Chromatographic Assay of a Central Nervous System (CNS)-Directed Estradiol Chemical Delivery System and Its Application After Intravenous Administration to Rats

A redox-based chemical delivery system for estradiol (E₂-CDS) has been shown capable of sustained and brain-selective delivery of estradiol (E₂). A re versed-phase high-performance liquid chromatographic (HPLC) method is presented for the analysis of E₂-CDS and its oxidized quaternary metabolite (E₂-Quat) in biological fluids or tissues. The assay utilizes a precolumn enrichment technique and detects plasma levels down to 10 ng/ml E₂-Quat and 20 ng/ml E₂-CDS. Sample preparation is rapid and simple. Samples are homogenized with acetonitrile, then centrifuged, and the supernatant is directly injected into the HPLC system. A water delivering pump injects the sample on a precolumn where the drug is concentrated. The mobile phase backflushes the retained compound onto the analytical column. At the same time, another sample can be injected onto a second precolumn. This alternating precolumn sample enrichment technique allows the injection of large volumes, up to 1800 µl. Plasma and tissue samples of rats collected after i.v. administration of a single 15-mg/kg E₂-CDS dose were analyzed for E₂-CDS and E₂-Quat by this procedure. The results show sustained brain levels of E₂-Quat and prolonged half-life in brain compared to six peripheral tissues measured. These data support the concept of brain-targeted delivery using redox carrier systems of this type.     

Column-Switching High-Performance Liquid Chromatography for On-Line Simultaneous Determination and Resolution of Enantiomers of Verapamil and Its Metabolites in Plasma

An on-line simultaneous assay for the enantiomers of verapamil (VA) and its three metabolites in plasma was developed with column-switching HPLC. This system consists of an ovomucoid protein chiral stationary phase coupled to an achiral reversed-phase column via a dilution tube and a trapping column. The reversed-phase column was used to separate and quantitate VA, its metabolites, and internal standard, without interference from plasma components. Then each of the eluates containing VA and its metabolites was selectively switched into a sampling loop, and the samples were transferred successively to the trapping column after dilution with a new mobile phase. After concentration on the trapping column, each sample was passed to the ovomucoid column, where the enantiomers were resolved and quantitated. The mobile phases for both HPLC columns were independently optimized and the diffusion of the sample during column switching was minimized. This method was shown to be efficient and reliable.     

Bioavailability of Propranolol After Oral,Sublingual, and Intranasal Administration

The bioavailability of propranolol was compared after oral, sublingual, and intranasal administration in eight healthy male volunteers. Relative to the bioavailability after intranasal (in) administration, which was previously shown to be nearly complete (F _relin = 100%), the sublingual (sl) administration of a standard 10-mg tablet gave a bioavailability of F _relsl = 63 ± 22%, while the oral (or) administration yielded only F _relor = 25 ± 8%. The serum concentration–time curves of propranolol after sublingual administration resembled those of a sustained-release preparation. This sustained-release phenomenon after the sublingual route is reflected in the mean residence times (MRTs) of propranolol in the body (MRT_or = 5.7 ± 1.3 hr, MRT_sl - 6.4 ± 1.3 hr, MRT_in = 4.6 ± 1.0 hr; mean ± SD; N = 8). MRTs after sublingual administration were significantly longer than after the oral and the intranasal doses (P < 0.05 and P < 0.002, respectively).     

用高效液相色谱法测定人血浆中黄芩苷与血浆蛋白的结合率

用高效液相色谱法对人血浆中黄芩苷进行了测定 .在 5、15、2 5 μg/mL范围的回收率为95 .0 %、88.7%、94.2 % .人血浆中黄芩苷与血浆蛋白的结合率测定 ,为设计合理的给药方案提供了实验依据 .     

Pharmacokinetics of oral fumarates in healthy subjects

AIMS: To characterize the pharmacokinetics of fumarates in healthy subjects. METHODS: Ten subjects received a single fumarate tablet (containing 120 mg of dimethylfumarate and 95 mg of calcium-monoethylfumarate) in the fasted state and after a standardized breakfast in randomized order. Prior to and at fixed intervals after the dose, blood samples were drawn and the concentrations of monomethylfumarate, the biologically active metabolite, as well as dimethylfumarate and fumaric acid were measured using high-performance liquid chromatography. RESULTS: After a lag time, a transient increase in serum monomethylfumarate concentrations in the blood was observed, whereas dimethylfumarate and fumaric acid concentrations remained below the detection limit. The tlag was 240 min [range 60-603 min; 95% confidence interval (CI) 139, 471] shorter when the tablet was taken after an overnight fast (90 min; range 60-120 min; 95% CI 66, 107) than when taken with breakfast (300 min; range 180-723 min; 95% CI 0, 1002). The tmax was 241 min (range 60-1189 min, 95% CI 53, 781) shorter when the tablet was taken after an overnight fast (182 min; range 120-240 min; 95% CI 146, 211) than when taken with breakfast (361 min; range 240-1429 min; 95% CI 0, 1062). The mean Cmax for monomethylfumarate in the blood of fasting subjects was to 0.84 mg l(-1) (range 0.37-1.29 mg l(-1); 95% CI 0.52, 1.07) and did not differ from that in fed subjects (0.48 mg l(-1); range 0-1.22 mg l(-1); 95% CI 0, 5.55). CONCLUSIONS: The pharmacokinetics of monomethylfumarate in healthy subjects after a single tablet of fumarate are highly variable, particularly after food intake. Further experiments exploring the pharmacokinetics of oral fumarates are warranted in order to elucidate the mechanisms underlying variability in response in patients.     

Disposition of synthetic glucocorticoids I. Pharmacokinetics of dexamethasone in healthy adults

The pharmacokinetics of dexamethasone alcohol is described in six male and six female healthy adult volunteers who each received 8 mg of dexamethasone phosphate by bolus intravenous injection. Quantitation of the alcohol was done using a high-performance liquid Chromatographic method with improved specificity. Statistical evaluation of the results generated by nonlinear least-squares regression analysis of the plasma concentration-time data shows that the phosphate ester is very rapidly hydrolyzed to the alcohol and a biexponential equation is the simplest poly exponential equation that is consistent with the data. The terminal phase half-lifet _1/2 was significantly greater (p<0.05) in males (mean 201.5 min) than in females (mean 142.3 min). The prolongedt _1/2 in males did not appear to be caused by an impaired capacity to eliminate dexamethasone since the total plasma clearance did not differ between males (mean 247.5ml/min) and females (mean 242.9 ml/min). There was, however, a high positive correlation betweent _1/2 and among the 12 adults (r=0.92, p<0.001). There were also significant correlations between and body weight (r=0.67, p<0.05) andt _1/2 (r=0.80, p<0.01).The difference in body weight between the sexes seems to be the main factor contributing to the difference observed in t _1/2. An average of only 2.6% of the dose was found unchanged in a 24-hr urine sample, and hence it appears that dexamethasone is primarily eliminated by extrarenal, probably hepatic, mechanisms.     

Assay of antipyrine and its main metabolites 3-hydroxymethylantipyrine, norantipyrine and 4-hydroxyantipyrine in urine

Two assay methods for antipyrine (AP) and its main metabolites 3-hydroxymethylantipyrine (3-HMA), norantipyrine (NORA) and 4-hydroxyantipyrine (4-HA) in urine samples have been compared. Method I involved a 3 h incubation at 37°C with β-glucuronidase, whereas method II used acid hydrolysis with 3 M hydrochloric acid to convert NORA glucuronide to the aglycone and 24 h incubation at 37°C with β-glucuronidase for hydrolysis of 3-HMA and 4-HA glucuronides. The precision of both sample preparation procedures was evaluated by means of HPLC with UV detection. The relative standard deviation (RSD) for the metabolites were considerably greater than 10% with method I. Application of method II, however, led to intra-assay and inter-assay RSD of less than 10%.     

反相高效液相色谱法测定心肺转流术患者血浆丙泊酚浓度

目的 建立反相高效液相色谱法（RP-HPLC）测定患者血浆中丙泊酚浓度.方法 使用Phenomenex Gemini C18柱,（4.6 mm×250 mm,5 μm）,以甲醇-水（80：20）为流动相,流速1.0 mL·min-1,柱温30℃,荧光激发波长（Ex）274 mm,发射波长（Em）312 nm.以甲醇沉淀法处理血浆,测定患者血浆中药物浓度.结果 丙泊酚总浓度在0.108～8.64μg·mL-1与峰面积线性关系良好,低、中、高3个浓度方法回收率在95.5%～102.9%,日内RSD为2.5%～3.9%,日间RSD为3.9%～5.8%.结论 本方法简便、快速,灵敏度高,适用于临床上丙泊酚血药浓度监测.     

Stereospecific High-Performance Liquid Chromatographic (HPLC) Assay of Fenoprofen Enantiomers in Plasma and Urine

A new high-performance liquid chromatographic (HPLC) assay suitable for pharmacokinetic studies of enantiomers of fenoprofen (FEN) was developed. Following the addition of internal standard (IS; racemic ketoprofen), the plasma or urine constituents are extracted into a mixture of isooctane:isopropanol (95:5), back extracted into water, and finally, extracted into chloroform. After evaporation of the organic layer, the drug and IS are derivatized with l-leucinamide hydrochloride via ethyl chloroformate intermediate. The formed diastereoisomers are chromatographed on a reversed-phase HPLC with a mobile phase consisting of monopotassium phosphate solution:acetonitrile:triethylamine (65:35:0.02) at a flow rate of 1 ml/min. The detection UV wavelengths are 232 and 275 for the drug and IS, respectively. The suitability of the assay for pharmacokinetic analysis of FEN enantiomers was examined by analysis of the plasma and urine samples taken from a healthy subject, following peroral administration of a single 300-mg dose of racemic FEN.