Pharmacokinetic monitoring of HIV-1 protease inhibitors in the antiretroviral therapy

Combination therapy with protease inhibitors (PIs) is used for the treatment of patients infected with the human immunodeficiency virus (HIV). To achieve optimal drug concentrations for viral suppression and avoidance of drug toxicity, therapeutic drug monitoring of PIs levels has been considered essential. In this study an analytical procedure for simultaneous monitoring the plasma concentrations of a total four protease inhibitors: indinavir, lopinavir, nelfinavir and saquinavir was presented. The plasma samples were liquid/liquid extracted and subjected to high performance liquid chromatography (HPLC) analysis. The lopinavir and saquinavir in the patient plasma samples were monitored by ultraviolet detection at 215 nm. Extraction procedure using methyl tert-butyl ether and the mobile phase consisted from acetonitrile with phosphate buffer on a Symmetry C18 column were used to monitor these two compounds. Linearity of the method was obtained in the concentration range of 0.1 - 15.0 mg/mL for all four protease inhibitors. Under steady state conditions, the plasma concentrations of protein inhibitors in 23 patients were determined and area under the plasma concentration-time curve was estimated to maintain optimal viral suppression. We developed a simple and specific method for the simultaneous determination of lopinavir and saquinavir.     

Effect of repeated doses of darunavir plus low-dose ritonavir on the pharmacokinetics of sildenafil in healthy male subjects: phase I randomized, open-label, two-way crossover study

BACKGROUND AND OBJECTIVES: Darunavir (DRV, TMC114) is a novel protease inhibitor administered in combination with low-dose ritonavir (DRV/r) and is highly active against both wild-type and multidrug-resistant HIV-1 strains. Sildenafil is an oral therapy for erectile dysfunction. Concomitant administration of protease inhibitors and sildenafil increases sildenafil plasma concentrations. The potential for a pharmacokinetic drug interaction exists when sildenafil and DRV/r are co-administered, as these drugs are primarily metabolized by cytochrome P450 (CYP) 3A, and darunavir and ritonavir are CYP3A inhibitors. The primary objective of this open-label, crossover, phase I study was to assess the effect of multiple doses of DRV/r on the pharmacokinetics of sildenafil and its active metabolite N-desmethyl sildenafil. The secondary objective was to assess the short-term safety and tolerability of co-administration of sildenafil and DRV/r. METHODS: Sixteen HIV-negative healthy male subjects were randomized to one of two sequences. In two sessions each subject received treatments A and B. In treatment A, a single dose of sildenafil 100 mg was administered. In treatment B, the subjects received DRV/r 400/100 mg twice daily for 8 days and on day 7 a single dose of sildenafil 25 mg was co-administered. Full pharmacokinetic profiles of sildenafil, N-desmethyl sildenafil, darunavir and ritonavir were determined. Safety and tolerability were also assessed. RESULTS: Sildenafil exposure (area under the plasma concentration-time curve [AUC]) was comparable between the two treatments despite administration of a lower dose of sildenafil (25 mg) with DRV/r than when sildenafil (100 mg) was administered alone. When sildenafil 25 mg was co-administered with DRV/r, the sildenafil maximum plasma concentration (Cmax) was 38% lower compared with Cmax after administration of sildenafil alone at a dose of 100 mg. N-desmethyl sildenafil Cmax and AUC from the time of administration until the last time point with a measurable concentration after dosing (calculated by linear trapezoidal summation [AUClast]) values decreased by approximately 95% when sildenafil 25 mg was co-administered with DRV/r compared with sildenafil 100 mg alone. Combined treatment with DRV/r and sildenafil was generally safe and well tolerated. CONCLUSION: Sildenafil exposure is increased in the presence of DRV/r. In this setting, a dose adjustment for sildenafil is warranted; no more than 25 mg of sildenafil is recommended over a 48-hour period when co-administered with DRV/r.     

A liquid chromatography tandem mass spectrometry method for the quantification of indocyanine green in dog plasma and bile

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of indocyanine green (ICG) in dog plasma and bile. An ICG analog (IR-820) was used as an internal standard. A protein precipitation method was used with acetonitrile for plasma sample preparation, whereas bile samples were diluted with water (120-fold) prior to analysis. Using MS/MS in the multiple reaction monitoring mode, ICG and IR-820 were detected in both matrices without interference. The lower limit of quantitation for ICG in dog plasma was 3 ng/mL, with an intra- and inter-day accuracy (%Bias) and precision (%CV) of less than 15%, so it was possible to study the pharmacokinetics of ICG in bile duct-cannulated dogs and assess their liver function after surgery. The method described herein is sensitive, selective and faster than other existing methods (e.g., spectrophotometry, HPLC/UV–vis detection, or HPLC/fluorescence detection).     

Lopinavir protein binding in vivo through the 12-hour dosing interval

Most protease inhibitors available for the treatment of human immunodeficiency virus (HIV) infection are highly bound to plasma proteins, mainly alpha-1 acid glycoprotein. Therapeutic drug monitoring (TDM) of total protease inhibitor (PI) concentrations has been increasing in the past few years; however, the pharmacological activity of the PIs is dependent on unbound drug entering cells harboring HIV. There is little information available on unbound drug concentrations of these drugs in vivo. The aim of the study was to measure unbound plasma concentrations of lopinavir (LPV) and to relate them to the total plasma concentrations to establish the unbound percentage in vivo during a full dosage interval. A pharmacokinetic study was performed in HIV-infected subjects (n = 23; median CD4 cell count = 290 x 10(6) cells x L(-1); viral load < 50 copies x mL(-1)) treated with a LPV/ritonavir (RTV)-containing regimen. Ultrafiltration was used to separate unbound LPV in all plasma samples (n = 115). Equilibrium dialysis was also used to compare with ultrafiltration measurements in 10/23 patients at baseline and 2 hours after drug intake. Total and unbound LPV concentrations were measured by a fully validated method using high-performance liquid chromatography-mass spectometry (HPLC-MS/MS). Based on a comparison of AUC(unbound)AUC(total), the mean (+/- SD) unbound percentage of LPV from all the samples studied (n = 115) was 0.92% (+/- 0.22) when measured with ultrafiltration and 1.32% (+/- 0.44) when equilibrium dialysis was used (n = 20), showing a higher drug recovery (P = 0.048). The unbound percentage of LPV was found to be significantly higher after 2 h than at baseline (P < 0.05 with both methods), suggesting a concentration-dependent binding of LPV that has not been observed in vitro. However, the clinical significance of such phenomena is still unclear.     

Determination of efavirenz,a selective non-nucleoside reverse transcriptase inhibitor,in human plasma using HPLC with post-column photochemical derivatization and fluorescence detection

Methods for the quantitative determination of efavirenz in human plasma and the qualitative assessment of the stereochemical integrity of efavirenz in post-dose human plasma samples are described. After the addition of an internal standard, plasma samples were extracted with hexane-methylene chloride (65/35, v/v%). The extracts were evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to form fluorescent products; the major fluorescent product was isolated and identified as a substituted quinoline. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. Reverse phase chromatography was used for the quantitative assay, whereas chromatography with a column containing a chiral stationary phase (dinitrobenzoyl leucine) was used for the stereochemical assessment. The quantitative assay has been validated in the concentration range of 50-1000 ng/ml using 0.5 ml samples. Analyte recovery was better than 89% at all points on the standard curve. Intra-day precision was better than 5% C.V., while accuracy was between 95 and 104% of nominal over the range of the assay. The selective detection method reduces the likelihood of interference by co-administered medications or endogenous species. The stereochemical configuration of efavirenz was confirmed to remain intact in post-dose human plasma samples. The quantitative method has been successfully utilized to support a study in which a possible drug interaction between co-administered HIV protease inhibitors and efavirenz was evaluated.     

Bioanalysis of HIV protease inhibitors in samples from sanctuary sites

The human immunodeficiency virus (HIV) is present in several sites inside the human body, which are hardly accessible to antiretroviral drugs, the so-called sanctuary sites. The most important sanctuary sites are cerebrospinal fluid (CSF), peripheral blood mononuclear cells (PBMCs) and seminal plasma. The determination of drug concentrations in these sanctuary sites may form an important step in treatment optimisation of HIV-infected individuals. However, bioanalysis in these sites is hampered by several factors with regard to sample preparation, chromatography and detection. In this review, we will discuss these issues and give an overview of published methods using high-performance liquid chromatography (HPLC) for the bioanalysis of HIV protease inhibitors in CSF, PBMCs and seminal plasma.     

Simple RP-HPLC method for determination of triple drug combination of valsartan, amlodipine and hydrochlorothiazide in human plasma

A simple RP-HPLC method for the quantification of valsartan (VAL), amlodipine (AML) and hydrochlorothiazide (HCT) in human plasma was developed and validated. VAL, AML and HCT were resolved using a Gemini C18 column and mobile phase gradient starting from 20 % acetonitrile and 80 % 10 mmol L(-1) ammonium formate (V/V, pH 3.5 ± 0.2, by formic acid) to 70 % acetonitrile and 30 % 10 mmol L(-1) ammonium formate, over 20 minutes, with a flow rate of 1 mL min(-1). The samples were purified by protein precipitation and extraction. Telmisartan was used as internal standard. The method was validated according to USFDA and EMEA guidelines with good reproducibility and linear responses R = 0.9985 (VAL), 0.9964 (AML), and 0.9971 (HCT). RSDs of intra- and inter-day precision ranged 2.2-8.1 and 4.6-11.7 %, respectively, for all three drugs. Mean extraction recoveries of three QCs for the triple drug combination were 76.5 (VAL), 72.0 (AML) and 73.0 (HCT) % for human plasma. Although the LC-MS/MS method is more sensitive than HPLC, HPLC is still suitable for preliminary pharmacokinetic study. The experiments performed demostrated that simultaneous determination of all components of the triple drug combination in human plasma can be done by this method. Proposed method can be also used for guidance to the LC-MS/MS method.     

Determination of nelfinavir free drug concentrations in plasma by equilibrium dialysis and liquid chromatography/tandem mass spectrometry: important factors for method optimization.

A method was developed and validated for measuring the free fraction of nelfinavir in plasma employing equilibrium dialysis for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry for quantitation. Nelfinavir, widely used to treat HIV infection, is a highly bound HIV protease inhibitor with the fraction bound in plasma being greater than 98%. Thus variations in the free fraction may be clinically important when interpreting total drug concentrations. Optimization of the method was carried out considering the influence of sample matrix and physicochemical and absorptive properties of nelfinavir. Nelfinavir free fraction averaged 0.41 +/- 0.094, 0.43 +/- 0.087 and 0.41 +/- 0.063% at nelfinavir plasma concentrations of 1000, 2000 and 3000 ng/ml, respectively. Free nelfinavir concentrations were underestimated with this assay by approximately 25% because of unavoidable losses to adsorption. However, the adsorptive loss was reproducible and consistent across the concentration range of the assay. Within-day and between-day precisions ranged from 6.0 to 9.4% and 15.2 to 27.3%, respectively. The lower limit of quantitation of the unbound concentration of nelfinavir was 1.0 ng/ml, permitting analysis of samples with total concentrations of nelfinavir in plasma that are > or = 400 ng/ml. This developed method proves reproducible and sensitive and its application to patient plasma samples is also reported.     

Comparison of solid-phase microextraction and liquid-liquid extraction in 96-well format for the determination of a drug compound in human plasma by liquid chromatography with tandem mass spectrometric detection

Two sensitive and selective methods based on solid phase microextraction (SPME) and liquid-liquid extraction (LLE) in 96-well format, in combination with high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection have been developed to determine a model drug compound in human plasma. Both assays were performed on an Applied Biosystems-Sciex API 4000 tandem mass spectrometer interfaced with a turbo ion-spray probe and operated in the negative ionization mode. A lower limit of quantitation (LLOQ) of 1 ng/mL achieved when 0.25 mL of human plasma was processed. In both methods, a stable isotope labeled internal standard was utilized. The methods were validated in the concentration range of 1-500 ng/mL. The intraday precision (%C.V.) of the method using LLE was 0.8% at LLOQ, and was equal to or lower than 3.3% at all other concentrations, while the intraday precision (%C.V.) of the method using SPME was 6.9% at LLOQ, and was equal to or lower than 5.7% at all other concentrations. Based on the direct comparison of the two methods and their successful applications in clinical sample analysis, it may be concluded that SPME may be considered and used as an alternative approach for quantitative determination of drugs in pharmacokinetic studies.     

Quantification of propiverine by liquid chromatography/electrospray tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy subjects

Here we report on the development and validation of a sensitive and rapid reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of propiverine in human plasma. After adding an internal standard (oxybutynin chloride) to human plasma, samples were extracted using n-hexane/ethylacetate (8:2, v/v). Compounds extracted were analyzed by reversed-phase high-performance liquid chromatography (HPLC) with multiple reaction monitoring (MRM) mode for analyte detection. This method for determination of propiverine proved accurate and reproducible, with a limit of quantitation of 0.5 ng/ml in human plasma. The standard calibration curve for propiverine was linear (r2=0.9988) over the concentration range 0.5-1000.0 ng/ml in human plasma. The intra- and inter-day precision over this concentration range was lower than 8.66% (relative standard deviation, %R.S.D.), and accuracy was between 99.46 and 109.41%, respectively. This method was successfully applied to a bioequivalence study of two propiverine hydrochloride tablet formulations (20 mg) in 24 healthy subjects after a single administration.     

LC-MS/MS法测定犬体内托拉塞米浓度及相对生物利用度研究

目的:建立LC-MS/MS法测定Beagle犬体内托拉塞米的浓度,并研究托拉塞米缓释片的相对生物利用度。方法:色谱柱为Waters Sym-metry-C18(100mm×4.6mm,5μm);流动相为甲醇∶20mmol/L甲酸铵(65∶35,V∶V);流速0.4mL/min;柱温30℃。采用两制剂双周期随机交叉试验设计,分别给予6只Beagle犬受试制剂或参比制剂5mg,采用LC-MS/MS法测定给药后不同时间的血药浓度。结果:托拉塞米线性范围为0.02～5μg/mL,最低定量限为0.02μg/mL,分析方法灵敏、稳定、特异性高。受试制剂和参比制剂的主要药动学参数:峰浓度(Cmax)、达峰时间(tmax)和药-时曲线下面积(AUC0-48h)分别为(2.6±0.5)g/mL和(3.0±0.6)g/mL、(3.3±1.4)h和(1.2±0.8)h、(36.1±11.0)g.h.mL-1和(32.1±13.1)g.h.mL-1。以AUC0-48h计算,受试制剂的相对生物利用度F为(118.0±28.3)%。结论:该方法操作简单、准确、重复性好,并成功地运用于犬体内托拉塞米相对生物利用度的研究。     

A selective and rapid method for the quantification of captopril in human plasma using liquid chromatography/selected reaction monitoring mass spectrometry

A specific hyphenated high performance liquid chromatography–mass spectrometric (LC–MS/MS) assay was developed for the determination of captopril in plasma. The drug was extracted from plasma using liquid–liquid extraction with a mixture of diethylether:dichloromethane. After the addition of the internal standard, samples were applied to a prepacked C₈ Waters Symmetry column. The ion trap MS/MS detector was equipped with electrospray ionization (ESI) source operating in the positive ion mode. Drug determination was accomplished monitoring captopril at molecular ion m/z 218 and MS/MS (daughter) at m/z 171.6. The method was applied to captopril determination in human plasma after the administration of captopril 50 mg tablets to healthy volunteers who have participated in a pharmacokinetic study.

The method was proved to be specific and precise by testing six different plasma batches. Linearity was established for the range of concentrations 25–3000 ng/ml with a regression factor of 0.9995. Intra-day accuracy ranged from 90.16 to 96.18%, while the intra-day precision ranged from 2.60 to 9.66% at the concentrations of 75, 1440 and 2500 ng/ml. Inter-day precision of the method ranged from 5.04 to 10.10%. This validated method of analysis was successfully applied to human plasma analyses after the administration of a single dose of 50 mg captopril tablets to healthy volunteers.     

高效液相色谱法测定大鼠血浆中吴茱萸次碱浓度

建立大鼠血浆中吴茱萸次碱的反相高效液相色谱法。以乙腈 水 (5 8∶4 2 )为流动相 ,地西泮为内标 ,在波长 3 4 5nm处检测。吴茱萸次碱浓度在 0 0 2～ 1 6μg/mL呈线性 ,相关系数为 0 999,高中低 3种浓度平均萃取回收率均大于 80 0 %     

Rapid and sensitive high-performance liquid chromatographic method for the determination of ritonavir in human plasma

Ritonavir is an HIV-1 protease inhibitor that is often used to improve the systemic availability of concurrently administered protease inhibitors by impairing their metabolism through cytochrome P450 (CYP) 3A4. Pharmacodynamic relationships between plasma ritonavir concentrations and efficacy and toxicity have also been described. To date, published high-performance liquid chromatographic (HPLC) methods for the determination of ritonavir in human plasma are often complex, requiring the use of a buffered mobile phase that contains amine-modifiers (i.e. diethylamine, triethylamine). In the method herein, ritonavir was precipitated with acetonitrile plus barium hydroxide and zinc sulphate. Chromatographic separation was accomplished using a C-18 base-deactivated (250 x 4.6 mm I.D., 5 atm particle size) analytic column with a mobile phase composed of acetonitrile:water (52:48, v/v). Quantification was performed at 239 nm. Calibration curves were linear from 0.5-25 microg/ml (R2 > 0.999); percent errors, as a measure of accuracy, were < 12.7%. Intra- and inter-assay relative standard deviations (RSD) were below 12.8%. This method provides a rapid and simple means for the accurate and precise analysis of ritonavir in human plasma. Furthermore, the assay requires neither the use of a buffered mobile phase adjusted to a specific pH, nor the addition of amine modifiers. This method has been successfully used to determine plasma ritonavir concentrations in drug interaction studies.     

Determination of loratadine in human plasma by liquid chromatography electrospray ionization ion-trap tandem mass spectrometry

A sensitive and specific liquid chromatography electrospray ionization ion-trap mass spectrometry (LC-ESI-IT-MS/MS) method has been developed and validated for the identification and quantitation of loratadine in human plasma. After the addition of the internal standard (IS), plasma samples were extracted using isooctane:isoamyl alcohol mixture. The compounds were separated on a prepacked Zorbax phenyl column using a mixture of acetonitrile, 0.20% formic acid as mobile phase. A Finnigan LCQ(DUO) ion-trap mass spectrometer connected to a Waters Alliance high performance liquid chromatography (HPLC) was used to develop and validate the method. The results were within the accepted criteria as stated in the FDA bioanalytical method validation guidance. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 0.10-10.0 ng/ml with a coefficient of determination (r(2)) of 0.9998. Accuracy for loratadine ranged from 105.00 to 109.50% at low, mid and high levels. The intra-day precision was better than 10.86%. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 0.10 ng/ml with a precision of 9.84%. The proposed method enables the unambiguous identification and quantitation of loratadine for pharmacokinetic, bioavailability or bioequivalence studies.