Analysis of ritonavir in plasma/serum and tissues by high-performance liquid chromatography

A method has been developed to quantify ritonavir concentrations in human plasma and in mouse serum, liver, and brain using high-performance liquid chromatography. Extraction recoveries for ritonavir and its internal standard averaged greater than 95%. Within-day variability, expressed as a coefficient of variation, averaged 6% over the concentration range 0.5 μg/mL to 15 μg/mL ritonavir, and between-day variability averaged 5.6% over 5 μg/mL to 15 μg/mL ritonavir. The method was applied to quantitation of ritonavir in mouse serum and tissue. Measured values deviated less than 5% from the actual values in mouse serum, liver, and brain samples containing 5 μg/mL ritonavir. The slopes of calibration curves for extracted calf serum, mouse serum, mouse liver and mouse brain standards were nearly identical to the calibration slope of standards which were not extracted. All curves were linear through zero, and r² was no less than 0.998 for any form of calibration. In addition, there was no chromatographic interference from commonly prescribed medications.     

Potent mechanism-based inhibition of human CYP3A in vitro by amprenavir and ritonavir: comparison with ketoconazole

Objective: Biotransformation of triazolam to its α-hydroxy and 4-hydroxy metabolites by human liver microsomes in vitro was used as an index of human cytochrome P ₄₅₀ 3A (CYP3A) activity. Results: The reaction was strongly inhibited by co-incubation with the viral protease inhibitors ritonavir (IC₅₀=0.14 μM) and amprenavir (IC₅₀=2.5–2.9 μM), and by the azole derivative ketoconazole (IC₅₀ = 0.07 μM). Pre-incubation of microsomes with ritonavir or amprenavir increased inhibitory potency (IC₅₀ reduced to 0.07 μM and 1.4 μM, respectively). This was not the case with ketoconazole. Conclusions: Thus, ritonavir and amprenavir are highly potent mechanism-based inhibitors of human CYP3A isoforms. Received: 11 January 2000 / Accepted in revised form: 9 March 2000     

Validated stability indicating chromatographic method for determination of baricitinib and its degradation products in their tablet dosage form: Implementation to content uniformity and in vitro dissolution studies

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Scheme 1: Graphical Abstract for the proposed method.Schéma 1 : Résumé graphique de la méthode proposée.

     

Evaluation of pharmacokinetic interaction between cetirizine and ritonavir, an HIV-1 protease inhibitor, in healthy male volunteers

Background Serious adverse effects have been observed with some non-sedative H₁-antihistamines (terfenadine and astemizole) when they were associated with drugs known to inhibit their metabolism. However, this is not a class effect, and this interaction should be considered on a case-by-case basis. The aim of this study was to evaluate the potential of pharmacokinetic interaction between cetirizine and ritonavir, the most potent cytochrome P ₄₅₀ (CYP) inhibitor.Methods An open-label, single-center, one-sequence crossover pharmacokinetic study was conducted in three running periods: cetirizine (CTZ) alone, ritonavir (RTV) alone and then CTZ plus RTV. For each period, steady-state pharmacokinetics were obtained. RTV and CTZ plasma concentrations were determined using validated liquid chromatography methods. The statistical method was based on a 90% confidence interval (CI) for the ratio of population geometric means (combination/drug alone) for each drug and for each parameter [area under the plasma concentration versus time curve (AUC_0-,ss), value of maximum plasma concentration (C_max,ss)] and compared to bioequivalence ranges 80–125% and 70–143% for AUC_0-,ss and C_max,ss, respectively.Results Among the 17 male subjects enrolled (26.4±8.6 years), 16 completed the study (1 withdrawal after the first period). The RTV pharmacokinetic parameter values were not affected by CTZ co-treatment. With RTV, a 42% increase in the CTZ AUC_0-,ss (3406 versus 4840 gh/l, 90% CI of 128–158%), a 53% increase in the CTZ elimination half-life (7.8 h versus 11.9 h, P = 0.001), a slight increase (15%) in the CTZ apparent volume of distribution (V_d,ss/ f) (34.7 l versus 39.8 l, P = 0.035), a 29% decrease in the CTZ apparent total body clearance (49.9 ml/min versus 35.3 ml/min, P<0.001) and bioequivalent C_max,ss (374 g/l versus 408 g/l) were observed. No serious drug related adverse effects were notified.Conclusions CTZ does not significantly affect the pharmacokinetic parameters of RTV, and the association does not, thus, require a modification of the dosage of the protease inhibitor. The increased extent of exposure to CTZ in healthy subjects, in the presence of RTV administered at high doses, remained in the same range as previously observed in the elderly or in mildly renally impaired subjects.Presented in part at the 42nd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, California, 27–30 September 2002, Poster H-1718.     

Safety and Efficacy in HIV-1-Infected Patients Treated with Ritonavir-Boosted Saquinavir Mesylate

Objective.  To evaluate the safety, tolerability, and efficacy of ritonavir-boosted saquinavir 1000/100 mg twice daily administered as a 500 mg film-coated tablet in HIV-1-infected patients.
Methods.  In this open-label, observational, 24-week survey conducted in 8 European countries, eligible HIV-infected participants had been prescribed saquinavir/ritonavir in combination with other nonprotease inhibitor (PI) antiretroviral agents as part of their HIV treatment regimen. The safety (grade 3 or 4 adverse events [AEs]), tolerability (by an investigator-reported subjective rating system), and efficacy (the percentage of participants with <50 and <400 copies/mL HIV RNA and change from baseline in mean CD4+ cell count) were analyzed for the overall study population and 7 subpopulations.
Results.  The enrolled population included 2122 participants with 1908 completing the study; 44 (2.1%) withdrew prematurely because of AEs, including 7 nontreatment-related deaths. There were 33 grade 3 or 4 AEs in 29 (1.4%) participants; 7 AEs in 7 (0.3%) participants were considered treatment-related. Tolerability was reported to be "very good" or "good" in 42% and 25% of participants, respectively. From baseline to week 24, the proportion of participants with HIV RNA <50 copies/mL increased from 31.2% to 47.6% and the proportion with <400 copies/mL increased from 42.5% to 61.4%; the mean CD4+ cell count increased by 75 cells/µL. In the subpopulation analysis, the greatest efficacy benefits occurred in participants who were treatment-naïve and in those not having received prior PI therapy.
Conclusions.  Treatment with the saquinavir 500 mg film-coated tablet resulted in few grade 3 or 4 AEs and was well tolerated and effective in a broad population of patients.     

RP-HPLC法检查复方氨酚葡锌片中盐酸二氧丙嗪含量均匀度及含量测定

目的采用HPLC法同时测定复方氨酚葡锌片中盐酸二氧丙嗪的含量均匀度及含量。方法以C18化学键合硅胶为固定相,以水-乙腈-三乙胺（90∶10∶0.2）为流动相,检测波长为226nm。结果平均加样回收率盐酸二氧丙嗪为100.5%,RSD=1.48%（n=9）。盐酸二氧丙嗪在0.08~0.8μg.mL-1范围内,浓度与峰面积值呈良好的线性关系。结论方法简便、快速、结果准确。     

RP-HPLC法和液质联用技术检查雌三醇中的有关物质

目的用反相高效液相色谱法和液质联用技术检查雌三醇中的有关物质,并对未知杂质进行定性。方法采用Phenomenex Hypersil C8柱5μm岛津VP-ODS C18柱,流动相为A相：水;B相：乙腈;梯度洗脱;检测波长：280nm;并用UPLC-MS技术对未知杂质进行了鉴定。结果雌三醇和未知杂质及16-表雌三醇、雌二醇和雌酮分离度良好,未知杂质初步鉴定为9,11-二脱氢雌三醇,其含量为0.73%,16-表雌三醇含量为0.42%,雌二醇和雌酮均未检出,总杂质为2.6%。结论本法可简便、准确的检查雌三醇中的有关物质。     

RP-HPLC法测定普鲁卡因肾上腺素注射液中盐酸普鲁卡因含量及其杂质对氨基苯甲酸

目的：采用RP—HPLC法同时测定普鲁卡因肾上腺素注射液中盐酸普鲁卡因含量及对氨基苯甲酸的方法。方法：色谱柱为Agilent—C18，流动相为水-乙腈-三乙胺（75：25：0．05），用冰醋酸调节pH3、3，流速为1．0mL·min^-1，检测波长为286nm。结果：盐酸普鲁卡因检测浓度平均加样回收率为101．5％（RSD 0．5，n=5），线性范围为0．8～3．2μg（r=0．9999）。结论：本法专属性强，操作简便，结果准确可靠。     

RP-HPLC法测定百乐眠胶囊中大黄素和大黄素甲醚的含量

目的建立RP-HPLC法测定百乐眠胶囊中大黄素和大黄素甲醚的含量。方法采用lichrospher C18色谱柱,流动相为甲醇：0.1%磷酸水溶液（82∶18）,流速为：1.0mL·min^-1,检测波长：254nm,柱温35℃。结果大黄素在0.0392～0.2741μg,r=0.9999,大黄素甲醚在0.0506～0.3544μg线性关系良好,r=0.9999。平均回收率大黄素为100.54%（RSD=2.18%）、大黄素甲醚为102.63%（RSD=1.63%）。结论本方法操作可靠、准确,适用于百乐眠胶囊中大黄素和大黄素甲醚的含量测定。     

RP-HPLC法测定冰蟾乳膏中马钱子碱和士的宁的含量

目的：建立冰蟾乳膏中马钱子碱和士的宁的含量测定方法。方法：采用DiamonsilTMC18色谱柱（250 mm×4.6 mm,5μm）,流动相为乙腈-0.015 mol/L庚烷磺酸钠与0.02 mol/L磷酸二氢钾等量混合溶液（含0.1%三乙胺,用10%磷酸调节pH值至3.0）（20∶80）,流速：1.0 mL/min,检测波长：256 nm。结果：马钱子碱在2.4-24 mg/L范围内（r=0.999 7）、士的宁在2.95-29.5 mg/L范围内（r=0.999 9）线性关系良好,其平均加样回收率分别为98.3%和97.9%,RSD分别为1.54%和2.20%（n=5）。结论：本法快速、准确、重复性好,可作为冰蟾乳膏的质量控制方法。     

Comparison of the efficacy,safety and predictive value of HIV genotyping using distinct ritonavir-boosted protease inhibitors

The pharmacokinetic profile of protease inhibitors (PI) is improved significantly by adding low doses of ritonavir (rit). The use of rit-boosted PI allows the reduction of pill burden, improves dosing schedules and enhances drug exposure, all factors that have been associated with a greater benefit of antiretroviral therapy. All patients receiving rit 100 mg bid together with saquinavir (SQV) soft gel 1000 mg bid, indinavir (IDV) 800 mg bid or lopinavir (LPV) 400 mg bid, as part of a salvage regimen, were retrospectively analyzed in a reference institution. Only subjects who had failed all three antiretroviral drug families in the past were included. A total of 299 patients were included in the study (121 on SQV, 62 on IDV and 116 on LPV). Mean plasma HIV-RNA and CD4 lymphocyte counts at baseline were less favourable in the LPV group (4.4 logs, 275 cells/μl) with respect to SQV (4.3 logs, 355 cells/μl) and IDV (3.7 logs, 409 cells/μl) groups (P<0.05). The proportion of subjects experiencing virological success (attainment of either <500 HIV-RNA copies/ml or >1 log reduction) in an intent-to-treat analysis at 24 weeks was 61% with LPV, 49% with SQV and 48% with IDV. The CD4 count increased 48% with SQV, 45% with IDV and 37% with LPV. The proportion of subjects discontinuing therapy due to adverse events was higher using IDV (27%) than using either SQV (11%) or LPV (6%) (P<0.05). The presence of <5 or >5 PI resistance mutations at baseline discriminated significantly better who would respond to therapy in all instances: 90 vs. 48% with LPV, 95 vs. 21% with SQV and 90 vs. 23% with IDV. The efficacy of rit-boosted PI combinations is greatly influenced by the extent of baseline PI resistance. Differences, not only in potency, but mainly in tolerance may favour the selection of one dual PI combination over others.     

RP-HPLC法测定强筋健骨片中马钱子碱和士的宁的含量

目的：建立强筋健骨片中马钱子碱和士的宁的含量测定方法。方法：采用反相高效液相色谱法。色谱柱为ODSC18（250mm×4．6mm,5μm）;流动相为0．01mol·L-1庚烷磺酸钠与0．02mol·L-1磷酸二氢钾等量混合（用10％磷酸调节pH值为2．8）一乙腈（79：21）;流速1．0ml·min-1,检测波长260nm。结果：马钱子碱和士的宁分别在0．0126—0．1260,0．0155～0．1550mg·ml-1的进样浓度范围内与其峰面积呈良好的线性关系;相关系数分别为0．9997,0．9999,平均回收率分别为98．43％（RSD为0．58％,n=6）、99．32％（RSD为0．32％,n=6）。结论：本方法简便可行,灵敏度高,重复性好,可用于强筋健骨片的质量控制。     

RP-HPLC法测定阿魏酸钠胶囊的含量和有关物质

目的建立阿魏酸钠胶囊剂含量及有关物质的RP-HPLC测定方法。方法采用Agilent ZORBAX SB-C18色谱柱,以水-甲醇-冰醋酸（64∶35∶1）为流动相,流速为0.9mL·min^-1,检测波长为322nm。结果阿魏酸钠胶囊中有关物质能完全分离,在15.6～67.5μg·mL^-1范围内线性关系良好（r=0.9999）;平均回收率为99.9%（n=9,RSD=0.5%）。结论本法专属性强,准确度高,能满足制剂质量控制的要求。     

RP-HPLC法测定PAC-1盐酸盐的含量及有关物质

目的建立RP-HPLC方法测定PAC-1盐酸盐含量并进行有关物质检查。方法采用KromasilC18色谱柱(150 mm×4.6 mm,5μm),流动相:乙腈-甲醇-磷酸盐缓冲液(30 mmo.lL-1磷酸二氢钾,体积分数为0.06%的磷酸溶液)(体积比为35∶5∶60),流速:1.0 mL.min-1,UV检测波长:281 nm,柱温:35℃。结果PAC-1盐酸盐在2.00~50.10 mg.L-1内线性关系良好(r=0.999 9),平均回收率为100.3%(RSD=0.8%)。PAC-1盐酸盐与其有关物质得到良好分离,检测限为0.2 ng。结论本方法可用作PAC-1盐酸盐含量测定和有关物质检查。     

RP-HPLC法同时测定合剂中麻黄碱、苯海拉明的含量

目的: 用RP-HPLC法同时测定小儿麻黄碱苯海拉明合剂中盐酸麻黄碱、盐酸苯海拉明的含量.方法: 采用C18色谱柱,流动相为乙腈-0.02 mol·L-1十二烷基硫酸钠溶液-三乙胺(52∶48∶0.1),用磷酸调pH3.5,检测波长254 nm.结果: 盐酸麻黄碱、盐酸苯海拉明的线性范围分别为(0.500 8～5.008μg和0.503 2～5.032)μg,平均回收率分别为99.6%(RSD=0.8%)和100.2%(RSD=1.0%)(n=6).结论: 该法可同时测定合剂中盐酸麻黄碱、盐酸苯海拉明的含量.     

RP-HPLC法同时测定乙肝舒康胶囊中没食子酸和丹参素含量

目的建立同时测定乙肝舒康胶囊中没食子酸和丹参素的高效液相色谱法。方法采用Hy-persil C18色谱柱(200.0 mm×4.6 mm,5μm),以甲醇-体积分数为0.5%的冰醋酸溶液(体积比为4∶96)为流动相,流速:1.0 mL.min-1,检测波长:280 nm。结果没食子酸和丹参素的线性分别为4.5~90.0 mg.L-1(r=0.999 9)和7.5~150.0 mg.L-1(r=0.9994),平均回收率分别为99.1%和100.2%,RSD分别为2.2%和2.4%。     

RP-HPLC法测定复方氧氟沙星鱼肝油乳剂中氧氟沙星和甲硝唑的含量

目的:建立反相高效液相色谱法测定复方氧氟沙星鱼肝油乳剂中甲硝唑和氧氟沙星的含量.方法:采用反相高效液相色谱法,色谱柱为Diamonsii C18柱(250mm×4.6mm,5μm), 流动相为甲醇-1%醋酸铵(40∶60),流速1.0mL·min-1, 检测波长293nm.结果:甲硝唑在3.0～30.0μg·mL-1范围内线性关系良好,回归方程:Y=0.123 30 1.833 75X,r=0.999 9(n=5),平均回收率100.33%,RSD=1.49%(n=9);氧氟沙星在2.0～20.0μg·mL-1范围内线性关系良好,回归方程:Y=0.157 53 0.623 53X,r=0.999 9(n=5),平均回收率100.31%,RSD=1.19%(n=9).结论:该方法灵敏度高;专属性好;操作简便;重现性好.     

RP-HPLC法测定益肾灵颗粒中补骨脂素和异补骨脂素的含量

采用RP-HPLC法测定益肾灵颗粒中补骨脂素和异补骨脂素的含量。采用Dikma C18柱,流动相：甲醇-0.01 mol.L^-1磷酸氢二钾溶液（用磷酸调pH=6.5）（48∶52）,流速：1.0 mL.min^-1,检测波长：245 nm。补骨脂素线性范围：0.012-0.100μg,r=0.9999;异补骨脂素线性范围：0.016-0.124μg,r=0.9999。平均回收率：补骨脂素99.1%（RSD=1.54%）,异补骨脂素100.3%（RSD=1.36%）。本法简便快捷,结果准确,重复性好。     

RP-HPLC法同时测定知柏地黄丸中芒果苷和丹皮酚含量

目的：建立知柏地黄丸中芒果苷和丹皮酚RP—HPLC含量测定方法。方法：采用Agilcnt ZORBAX Eclipse XDB C18色谱柱（4．6×150mm,5μm）,流速1ml／min,以乙腈-0．04％磷酸（22：88）为流动相,梯度洗脱,检测波长为258nm;进样量10μl。结果：芒果苷、丹皮酚的线性范围分别为在0．00504～0．2344mg／ml,r=0．9999;0．003872-0．04259mg／ml,r=0．9997,平均回收率分别为200．8％（RSD为1．2％,n=6）、98．26％（RSD为1．5％,n=6）。结论：该方法操作简便准确,重现性好,能有效控制知柏地黄丸的质量。