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Background: Therapeutic drug monitoring (TDM) using cyclin-dependent kinase inhibitors (CDK4/6is) is a novel approach for optimizing treatment outcomes. Currently, palbociclib, ribociclib, and abemaciclib are the available CDK4/6is and are primarily coadministered with letrozole. This study aimed to develop and validate an LC-MS/MS method for the simultaneous analysis of CDK4/6is, 2 active metabolites of abemaciclib (M2 and M20), and letrozole in human plasma for use in TDM studies., Methods: Sample pretreatment comprised protein precipitation with methanol and dilution of the supernatant with an aqueous mobile phase. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column (2.5 μm, 3.0 × 75 mm XP), with methanol serving as the organic mobile phase and pyrrolidine-pyrrolidinium formate (0.005:0.005 mol/L) buffer (pH 11.3) as the aqueous mobile phase. A triple quadrupole mass spectrometer was used for the detection, with the ESI source switched from negative to positive ionization mode and the acquisition performed in multiple reaction monitoring mode., Results: The complete validation procedure was successfully performed in accordance with the latest regulatory guidelines. The following analytical ranges (ng/mL) were established for the tested compounds: 6-300, palbociclib and letrozole; 120-6000, ribociclib; 40-800, abemaciclib; and 20-400, M2 and M20. All results met the acceptance criteria for linearity, accuracy, precision, selectivity, sensitivity, matrix effects, and carryover. A total of 85 patient samples were analyzed, and all measured concentrations were within the validated ranges. The percent difference for the reanalyzed samples ranged from -11.2% to 7.0%., Conclusions: A simple and robust LC-MS/MS method was successfully validated for the simultaneous quantification of CDK4/6is, M2, M20, and letrozole in human plasma. The assay was found to be suitable for measuring steady-state trough concentrations of the analytes in patient samples., Competing Interests: F. Puglisi has received honoraria from Amgen, AstraZeneca, Daiichi Sankyo, Celgene, Eisai, Eli Lilly, Exact Sciences, Gilead, Ipsen, Menarini, MSD, Novartis, Pierre Fabre, Pfizer, Roche, Seagen, Takeda, and Viatris. F. Puglisi is currently receiving research grants from AstraZeneca, EISAI, and Roche. L. Gerratana has received honoraria from AstraZeneca, Daiichi Sankyo, Eli Lilly, GlaxoSmithKline, Incyte, Novartis, Pfizer, Menarini Stemmline, and AbbVie. L. Gerratana is currently receiving research grants from Menarini Silicon Biosystems. S. Corsetti has received a travel grant from AstraZeneca. The remaining authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.)

Background: Adrenocortical carcinoma is an orphan but aggressive malignancy with limited treatment options. Cabozantinib (CAB), a tyrosine kinase inhibitor, has emerged as a new potential treatment. However, no data are available on whether and how CAB can be administered to patients undergoing hemodialysis., Methods: An liquid chromatography with tandem mass spectrometry detection method was developed and validated according to the European Medicines Agency and United States Food and Drug Administration guidelines for bioanalytical method validation. The samples were prepared using protein precipitation and online solid-phase extraction. The method was applied to clinical samples of an adrenocortical carcinoma patient receiving CAB treatment (80 mg daily). During the 10 days of observation, the patient received periodic hemodialysis on 7 days. Pharmacokinetic (PK) simulations were performed using Bayesian forecasting according to an existing population PK model for CAB., Results: Based on the PK simulation, a mean plasma trough concentration of 1375 ng/mL [90% prediction interval (PI), 601-2602 ng/mL] in the steady state at a daily dose of 80 mg was expected for CAB. However, an individual simulation involving the measured plasma levels of the patient resulted in a mean trough concentration of 348 ng/mL (90% PI, 278-430 ng/mL). The model based on individual PK parameters estimated accessible plasma levels of 521, 625, and 834 ng/mL by dose adjustment to 100, 120, and 160 mg, respectively., Conclusions: After establishing an liquid chromatography with tandem mass spectrometry detection method for therapeutic drug monitoring of CAB, our analyses involving a single patient undergoing hemodialysis indicated that higher than expected doses of CAB were required to achieve reasonable plasma concentrations. Our study demonstrates the usefulness of therapeutic drug monitoring for the evaluation of "new" drugs in patients with renal impairment., Competing Interests: O. Scherf-Clavel reported a professorship grant (Horphag Research Ltd.) and received funding from the Hector Stiftung II gGmbH. M. Kroiss received institutional grant support and travel support from Ipsen Pharma GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Under certain circumstances, clinicians treating patients with isavuconazole for invasive aspergillosis or mucormycosis may use therapeutic drug monitoring. However, the accuracy and reproducibility of the various assays used by different laboratories for the quantification of isavuconazole plasma concentrations have yet to be determined., Methods: Human plasma samples spiked with known concentrations of isavuconazole were provided to 27 European laboratories that took part in a "round-robin" test (an interlaboratory test performed independently at least 2 times; 2 rounds performed in the current study). Assay methods included liquid chromatography-tandem mass spectrometry (LC-MS/MS), LC with ultraviolet detection (LC-UV), LC with fluorescence detection (LC-FL), and bioassay. The accuracy and reproducibility compared with the known concentrations for each sample in each round were compared overall, between assays, and between laboratories., Results: Twenty-seven laboratories participated in the study (LC-MS/MS, n = 15; LC-UV; n = 9; LC-FL, n = 1; bioassay, n = 2). In round 1, for nominal concentrations of 1000, 1700, 2500, and 4000 ng/mL, the mean (SD) determined concentrations were 1007 (183), 1710 (323), 2528 (540), and 3898 (842) ng/mL, respectively. In round 2, for nominal concentrations of 1200, 1800, 2400, and 4000 ng/mL, the mean (SD) determined concentrations were 1411 (303), 2111 (409), 2789 (511), and 4723 (798) ng/mL, respectively. Over both rounds, determined concentrations were consistently within 15% of the nominal concentrations for 10 laboratories (LC-MS/MS, n = 4; LC-UV, n = 5; bioassay, n = 1) and consistently exceeded the upper 15% margin for 7 laboratories (LC-MS/MS and LC-UV, n = 3 each; LC-FL, n = 1)., Conclusions: Alignment of methodologies among laboratories may be warranted to improve the accuracy and reproducibility of therapeutic drug measurements.

Background: Apatinib is a new oral micromolecular tyrosine kinase inhibitor, which is mainly used as a third-line treatment for chemotherapy-refractory advanced metastatic gastric cancer patients. However, apatinib has shown dose titration and severe adverse reactions in clinical practice. Quantification of plasma concentrations of apatinib may be an effective method to balance the clinical efficacy and adverse reactions. The purpose of this study was to develop and validate a 2-dimensional liquid chromatography method for the measurement of apatinib in plasma., Methods: The analysis of apatinib was performed using a 2-dimensional high-performance liquid chromatography system. We precipitated the proteins with acetonitrile. The mobile phases consisted of a first-dimensional mobile phase (acetonitrile:methanol:25 mmol·L ammonium phosphate = 25:25:50, V/V/V, pH adjusted to 7.2 using phosphoric acid) and a second-dimensional mobile phase (acetonitrile:10 mmol·L ammonium phosphate = 28:72, vol/vol, pH adjusted to 3.7 using phosphoric acid). The ultraviolet detection wavelength was set at 340 nm. The temperature of the detector cell was 40°C, and the injection volume was 500 μL., Results: The range of calibration curve was 15.27-1491.48 ng/mL. The accuracy and imprecision were within ±2.23% and less than 10.22%, respectively (intraday and interday). The range of recovery was 97.45%-108.92%. The intraday and interday relative SDs (reproducibility) of high-performance liquid chromatography retention times were less than 0.18% and 0.46%, respectively. In the clinical assessment, the dose range of apatinib mesylate for patients with gastric cancer was 250-500 mg every day (2-60 days), resulting in trough plasma concentrations between 272.7 and 727.8 ng/mL., Conclusions: A simple, convenient, accurate, and robust 2-dimensional liquid chromatography method was developed and verified, which successfully determined the plasma concentrations of apatinib in patients with gastric cancer.

Background: Isavuconazole is a triazole antifungal agent for treatment of invasive aspergillosis and mucormycosis. At present, it is unclear whether a therapeutic drug monitoring (TDM) of isavuconazole would be necessary. The aim of the investigation was to validate a high-performance liquid chromatography (HPLC) assay for routine applications., Methods: An HPLC assay for routine determination of isavuconazole in plasma has been adapted and validated. The assay used the reagents and HPLC column provided by the ChromSystems HPLC Kit for TDM of itraconazole, posaconazole, and voriconazole. Isocratic flow rate was set at 1.0 mL/min. Detection was performed using a fluorescence detector with excitation wavelength set at 261 nm and emission wavelength set at 366 nm., Results: The assay was linear between 0.15 and 10 mg/L with intraday and interday imprecision and accuracy <10% (<20% at lower limit of quantification). The method was applied to routine TDM of 7 patients after hematopoietic stem cell transplantation (n = 31 samples). In these patients, trough levels ranged from 0.45 to 3.06 mg/L (median 1.44 mg/L)., Conclusions: A robust and simple HPLC assay of isavuconazole in plasma for routine TDM applications is presented.

Background: A simple, rapid, and sensitive liquid chromatography coupled with tandem mass spectrometry method has been developed and validated for the quantification of ruxolitinib, olaparib, vismodegib, and pazopanib in human plasma., Methods: After a simple protein precipitation of plasma samples, the chromatographic separation was performed using an ultraperformance liquid chromatography system coupled with mass tandem spectrometry in a positive ionization mode. The mobile phase consisted of a gradient elution of 10-mmol/L formate ammonium buffer containing 0.1% (vol/vol) formic acid (phase A) and acetonitrile with 0.1% (vol/vol) formic acid (phase B) at a flow rate at 300 µL/min., Results: Analysis time was 5.0 minutes per run, and all analytes and internal standards eluted within 1.5-1.73 minutes. The calibration curves were linear over the range from 10 to 2500 ng/mL for ruxolitinib and from 100 to 100,000 ng/mL for olaparib, vismodegib, and pazopanib with coefficients of correlation above 0.99 for all analytes. The intraday and interday coefficients of variation were below 14.26% and 14.81%, respectively, for lower concentration and below 9.94% and 6.37%, respectively, for higher concentration., Conclusions: Using liquid chromatography coupled with tandem mass spectrometry, we have developed and validated a simple and rapid assay for the simultaneous quantification of olaparib, vismodegib, pazopanib, and ruxolitinib in human plasma. This method is now part of our therapeutic drug monitoring service provision and is currently used clinically to manage patients prescribed these drugs.

Background: Direct oral anticoagulants (DOACs) are prescribed for anticoagulation in patients with atrial fibrillation and venous thromboembolic disease. Fixed doses are recommended, but measuring their serum drug concentrations as a basis for dose adjustments may be useful in some clinical settings., Methods: An ultra-high-performance liquid chromatography-tandem mass spectrometry method for the analysis of the DOACs apixaban, dabigatran, edoxaban, and rivaroxaban in human serum was developed and validated. A 100-µL serum sample was handled using a pipetting robot. Protein precipitation was performed with 375 µL of 1% formic acid in acetonitrile (vol/vol), and phospholipid removal was performed using a Waters Ostro 96-well plate. The injection volume was 1 µL, and run time was 3.0 minutes., Results: The calibration range was 5-800 nmol/L. The between-day precision relative SDs were in the range of 3.3%-10%. Recoveries ranged from 85% to 105%, and matrix effects from 88% to 102%, when corrected with internal standard. Edoxaban was, in contrast to the other DOACs, unstable when stored for more than 6 hours at 30°C. The suitability of the method was demonstrated by analyzing routine samples from 345 patients undergoing anticoagulation treatment., Conclusions: The developed method fulfilled the set validation criteria, and its suitability was demonstrated in a routine setting. The instability of edoxaban may complicate the transport of routine samples to the laboratory.

Background: A fast and easy-to-use liquid chromatography-tandem mass spectrometry method for the determination and quantification of 6 triazoles [fluconazole (FLZ), isavuconazole (ISZ), itraconazole (ITZ), hydroxy-itraconazole (OH-ITZ), posaconazole (PSZ), and voriconazole (VRZ)] in human plasma and serum was developed and validated for therapeutic drug monitoring., Methods: Sample preparation was based on protein precipitation with acetonitrile and subsequent centrifugation. Isotope-labeled analogues for each analyte were used as internal standards. Chromatographic separation was achieved using a 50 × 2.1 mm, 1.9 μm polar Hypersil Gold C18 column and mobile phase consisting of 0.1% formic acid/acetonitrile (45%/55%, vol/vol) at a flow rate of 340 μL/min. The triazoles were simultaneously detected using a triple-stage quadrupole mass spectrometer operated in selected reaction monitoring mode with positive heated electrospray ionization within a single runtime of t = 3.00 minutes., Results: Linearity of all azole concentration ranges was verified by the Mandel test and demonstrated for all azoles. All calibration curves were linear and fitted using least squares regression with a weighting factor of the reciprocal concentration. Limits of detection (μg/L/L) were FLZ, 9.3; ISZ, 0.3; ITZ, 0.6; OH-ITZ, 8.6; PSZ, 3.4; and VRZ, 2.1. The lower limits of quantitation (μg/L/liter) were FLZ, 28.3; ISZ, 1.0; ITZ, 1.7; OH-ITZ, 26.2; PSZ, 10.3; and VRZ, 6.3. Intraday and interday precisions ranged from 0.6% to 6.6% for all azoles. Intraday and interday accuracies (%bias) of all analytes were within 10.5%. In addition, we report on a 29-year-old white woman (94 kg body weight) with a history of acute myeloid leukemia who underwent stem cell transplantation. Because of diagnosis of aspergillus pneumonia, antifungal pharmacotherapy was initiated with different application modes and dosages of ISZ, and plasma concentrations were monitored over a time period of 6 months., Conclusions: A precise and highly sensitive liquid chromatography-tandem mass spectrometry method was developed that enables quantification of triazoles in plasma and serum matrix across therapeutically relevant concentration ranges. It was successfully implemented in our therapeutic drug monitoring routine service and is suitable for routine monitoring of antifungal therapy and in severely ill patients.

Background: Direct oral anticoagulants (DOACs) are prescribed for systemic anticoagulation. Fixed doses are recommended, but dose individualization may be warranted. Functional coagulation assays may be available, but their use requires knowledge of the drug taken. To provide alternative methodology for guiding dosage, we have developed and validated a liquid chromatography-mass spectrometric assay for apixaban, dabigatran, edoxaban, and rivaroxaban at the concentrations attained during therapy., Methods: Samples, calibrators, and internal quality controls (100 μL) were mixed with internal standard solution (50 μg/L both dabigatran-13C6 and rivaroxaban-13C6 in acetonitrile) and, after centrifugation (16,400g, 4 minutes), supernatant (100 μL) was injected onto a Cyclone-C18-P-XL TurboFlow column. Analytes were focused onto an Accucore PhenylHexyl (2.1 × 100 mm, 2.6 μm) analytical column and eluted using a methanol + acetonitrile (1 + 1):aqueous ammonium acetate (10 mmol/L) gradient. Data were acquired using high-resolution mass spectrometry in full-scan mode (100-2000 m/z) with data-dependent fragmentation to confirm peak identity. Calibration was linear (1-500 μg/L all analytes)., Results: Total analysis time was 6 minutes. Intra-assay imprecision (% RSD) at 1 μg/L was 2.6%, 4.2%, 17.3%, and 9.5% for apixaban, dabigatran, edoxaban, and rivaroxaban, respectively. Mean recovery was 96%-101%. No signal suppression or enhancement was observed. Apixaban, dabigatran, and rivaroxaban were stable over 3 freeze-thaw cycles, after storage at room temperature, and at 2-8°C for up to 2 weeks. Edoxaban was stable over 3 freeze-thaw cycles but showed a mean deterioration of 16% if stored at 2-8°C (2 weeks) and of 18% and 70% (1 day and 2 weeks, respectively) at room temperature., Conclusions: The method is suitable for high-throughput therapeutic drug monitoring of DOACs. The acquisition of full scan data allows for the retrospective identification of metabolites. The method can be used to identify a particular DOAC if information on the drug taken is lacking.

Background: Therapeutic drug monitoring (TDM) of antiretroviral drugs (ARVs) is used to improve the efficacy and safety of ARVs, but there is little interest for the systematic or random TDM of ARVs in the medical management of patients with acquired immune deficiency syndrome. This study aimed to evaluate a different approach and test the potential advantages of TDM as part of medical treatments when clinical problems are identified in human immunodeficiency virus-infected patients., Methods: The authors conducted a prospective, noncontrolled, cohort study on 544 human immunodeficiency virus-positive patients treated either with a protease inhibitor (PI), atazanavir/lopinavir, or with a nonnucleoside reverse transcriptase inhibitor (NNRTI), efavirenz/nevirapine. Patients who had virological failure, clinical signs of toxicity, or a risk of pharmacokinetic interactions were identified as having medication-related problems (MRPs), and they were scheduled for TDM of the PIs or NNRTIs. Cases with drug levels outside the range were subjected to intervention, and a second determination of plasma levels and viral load was scheduled to assess their response to the intervention., Results: Of the 521 treatment courses analyzed, 173 (32.4%) presented at least 1 MRP during the study. The TDM yielded abnormal results in 52.5% of the 198 identified MRP cases (95% CI: 45%-59%). The patients treated with PIs had an increased risk for having drug plasma levels that fell outside the normal range compared to those treated with NNRTIs (relative risk =1.36, 95% CI: 1.04-1.79). The TDM-guided interventions contributed to the resolution of 52.1% of the cases that involved treatment courses with MRPs and abnormal drug plasma levels., Conclusions: MRPs, including therapeutic failure, were common in the patients who were included in the study. A high proportion of the treatment courses involving such MRPs also presented abnormal plasma drug levels. The TDM-guided interventions are advantageous under these situations because they allow the continuation of treatments that would otherwise be substituted by more complex and costly alternatives.

A simple liquid chromatography-mass spectrometry method was developed and validated for quantification of sorafenib (Nexavar) in human plasma. After a solid-phase extraction procedure, the separation was performed within 2 minutes using an isocratic flow of a mobile phase consisting of formic acid/acetonitrile applied on a C18 analytical column. The analyte was detected by mass spectrometry in the single-ion monitoring mode. The method was validated according to the recommendations of the US Food and Drug Administration. The method was linear (r² > 0.99) between 10 and 10,000 ng/mL. The lower limits of detection and quantification were 5 and 10 ng/mL, respectively. Within-day and between-day imprecisions were less than 10.4%, and inaccuracy did not exceed 8.7%. The mean extraction recovery was 92.2%. The method also provided satisfactory results in terms of time stability and dilution integrity. Sorafenib plasma concentrations of the studied patient ranged between 1831 and 3459 ng/mL. This new technique is rapid, sensitive, and was applied to the determination of sorafenib plasma concentrations in a patient undergoing hemodialysis. Our results indicate that sorafenib is not cleared from plasma by hemodialysis, although analysis should be delayed after dialysis to avoid erratic fluctuations.

Background: Adequate plasma trough concentrations (Ctrough) of protease inhibitors are required to maintain antiviral activity throughout the dosing interval. Therapeutic drug monitoring is used in clinical practice to optimize dosage and avoid toxic or subtherapeutic drug exposure. The pharmacokinetic variability of Atazanavir (ATV) can be relatively large, as a result of several factors. One of the affecting factors may be hepatic impairment due to hepatitis C virus (HCV) coinfection., Methods: We collected trough plasma samples from human immunodeficiency virus (HIV)-1-infected outpatients, with and without HCV coinfection and/or cirrhosis, receiving stable highly active antiretroviral therapy containing ATV. In the total population, we mainly compared the 2 regimens: 300ATV + 100RTV OD [ritonavir (RTV), once daily (OD)] versus 400ATV OD. We used a threshold value of 0.15 μg/mL, based on the proposed therapeutic range (0.15-0.85 μg/mL). Plasma concentrations of ATV were determined by a validated assay using high-performance liquid chromatography with ultraviolet detection. A total of 214 HIV-infected outpatients were included. For each regimen, we compared 3 groups of subjects: HIV+/HCV-, HIV+/HCV+, and HIV+/HCV+ with cirrhosis., Results: In the whole study population, we observed a large variability and found suboptimal Ctrough levels (<0.15 μg/mL) in 23 subjects (2 belonging to the 300/100 OD group and 21 to the 400 OD group). For the standard dosage regimen of 300ATV + 100RTV OD, we did not find a statistical difference between HIV-infected patients without HCV coinfection versus HIV-infected patients with HCV coinfection: median 0.85 (interquartile range 0.53-1.34) and 0.95 (0.70-1.36) μg/mL, respectively. In HIV+/HCV+-infected patients with cirrhosis, we found a median Ctrough of 0.70 (0.43-1.0) μg/mL, with no statistical difference when compared with HIV+/HCV- infected patients. For the 400ATV OD (n=90) dosage regimen, the total median ATV Ctrough was 0.40 (0.23-1.0) μg/mL. In this group, we found a statistically significant difference between HIV+/HCV- and HIV+/HCV+-infected patients: median Ctrough was 0.23 (0.11-0.42) and 0.52 (0.20-1.0) μg/mL, respectively. In HIV+/HCV+ subjects with cirrhosis, the Ctrough median value was 0.42 (0.13-0.75) μg/mL, and there was a significant difference when compared with HIV patients without coinfection., Conclusions: Therapeutic drug monitoring of ATV in patients receiving unboosted regimen may be useful to identify those HIV-infected subjects, with or without HCV coinfection, who may benefit from adding low RTV doses, or the subset of patients in whom removal of RTV could be attempted without the risk of suboptimal plasma ATV exposure.

Raltegravir (RAL) is primarily metabolized by uridine diphosphate-glucorunosyl transferase 1A1 (UGT1A1). Atazanavir (ATV), a strong inhibitor of UGT1A1, has been shown to increase plasma concentrations of RAL by approximately 50% in healthy volunteers. However, the extent of this interaction has not been studied in HIV-infected patients. A pharmacokinetic study was performed in 22 HIV-infected adults treated with 400 mg RAL plus 300 mg ATV 300 twice a day. Both drugs showed high pharmacokinetic variability (RAL AUC 0-12 7649 ± 4862 ng*h/mL; ATV AUC 0-12 = 19237 ± 13136 ng*h/mL). Notably, RAL trough concentrations were significantly higher compared with those measured in HIV subjects (n = 24) on RAL plus nucleoside reverse transcriptase inhibitors (506 ± 411 versus 177 ± 262 ng/mL, P < 0.01). A significant correlation was found between RAL and ATV area under the curve (AUC) (r = 0.611, P = 0.005). Notably, patients with ATV AUC 0-12 above the mean or with concentrations exceeding the half maximal inhibitory concentration for UGT1A1 had twofold higher RAL AUCs compared with patients with lower ATV exposure. Coadministration of ATV significantly increased plasma concentrations of RAL, especially in HIV-1-infected patients exposed to high concentrations of the protease inhibitor. This pharmacokinetic drug interaction could be handled by routine measurements of ATV trough concentrations and by the assessment of plasma RAL concentrations 2 to 3 hours after the morning drug intake.

Despite being among the most potent protease inhibitors, the use of tipranavir (TPV) is hampered by a high pill burden and frequent side effects compared with other boosted protease inhibitors. A total of 10 patients receiving TPV/ritonavir (TPV/RTV) 500/200 for longer than 6 months were randomized to stay on the same dosing schedule or switch to TPV/RTV 500/100. Although all patients on TVP/RTV 500/200 remained stable for the next 12 weeks, 3 out of 5 patients who switched doses experienced benefits in terms of reducing aminotransferases and total cholesterol. Fasting triglycerides were also reduced in 2 of them. Plasma HIV-RNA remained undetectable in all patients, despite the observed decline in TPV trough concentrations.

The aim of this study was to determine the population pharmacokinetic (PK) parameters of atazanavir in adult human immunodeficiency virus-infected patients to build up a Bayesian strategy for dosage regimen individualization. This was an observational study of patients treated with the once-daily regimen atazanavir associated with 100 mg of ritonavir. Blood samples were drawn at steady state at various times ranging from 1 to 26 hours postdose. Atazanavir plasma concentrations were determined by a validated reverse-phase high-performance liquid chromatography method. PK analysis of the atazanavir population was performed using a nonlinear mixed-effects model (NONMEM version 6). One hundred eighty-seven patients were included in the study. The atazanavir doses prescribed were 300 mg (n = 169), 400 mg (n = 12), 200 mg (n = 1), and 150 mg (n = 5). The atazanavir population PK was described using a 1-compartment model with first-order absorption. Mean PK parameter estimations (95% confidence interval, coefficients of variation %) were as follows: oral clearance (CL) = 7.6 L/h (6.9-8.3; 34%), volume of distribution (V) = 80.8 L (67.4-94; 37%), and absorption constant rate (Ka) = 1.05 hours (0.01-2.09; 156%). The mean estimated half-life (T-half) was 7.5 hours (95% confidence interval: 7.2 -7.8 hours). The estimated T-half of atazanavir was in agreement with that previously reported of 8.6 and 8.8 hours. We observed a wide interpatient variability for the PK parameters, especially for Ka. This population approach allowed us to determine atazanavir PK parameters in human immunodeficiency virus-infected patients in a real-life context and to perform Bayesian analysis to predict Ctrough from samples collected at any moment during the dosing interval. This could therefore improve therapeutic drug monitoring interpretations and provide an interesting tool for correlation with virologic data.

Atazanavir (ATV) is a widely used human immunodeficiency virus (HIV)-1 protease inhibitor (PI) that, like other approved PIs, has been considered as a candidate for therapeutic drug monitoring (TDM). To provide ATV assay results that can be applied to patient management through TDM, the assay would need to perform in a manner consistent with Clinical Laboratory Improvement Amendments (CLIA) standards. To quantitate ATV concentrations in human plasma, the authors added ATV to a previously published reversed-phase high-performance liquid chromatography (HPLC) method from their laboratory. Detection was effected with use of a photodiode-array detector (PDA) collecting spectra at 248 nm. This method allows for detection of ATV to a lower limit of quantitation of 0.05 microg/mL, with an intra-assay coefficient of variation (CV%) of 8.9% or less over 5 days of testing and an interassay CV% ranging from 1.4 to 6.4%. The assay has met passing requirements for interlaboratory proficiency testing for 2 years nationally and internationally, with accuracy within +/-15% over all test samples. During 2 years, more than 100 batches of analyses have been performed and have proved the method is rugged, specific, and accurate. This assay method is currently used in the authors' clinical research program in TDM.

Because HIV medications are used in combination, it is important to develop multiplex assays to streamline the therapeutic drug monitoring process and provide rapid turnaround. This article reports full validation of an analytical method that combines atazanavir with 6 HIV-protease inhibitors (indinavir, amprenavir, saquinavir, nelfinavir, ritonavir, and lopinavir) and 2 nonnucleoside reverse transcriptase inhibitors (nevirapine and efavirenz). Using 200 microL of plasma and a simple liquid-liquid extraction method, this analytical method achieved a clean baseline and high extraction efficiencies (90.0% to 99.5%). A Zorbax C-18 (150 x 4.6 mm, 3.5 microm) analytical column was used along with a 27-minute linear gradient elution of the mobile phase to provide sharp peaks at 210 nm. This method was validated over a range of 25 to 10,000 ng/mL and is accurate (90.4% to 110.5%) and precise (precision within a day and between days ranged from 2.3% to 8.3%). Because this method is simple and inexpensive, it may have applicability in countries with low resources.

Tipranavir has recently received accelerated approval from the FDA. The initial clinical use of tipranavir will be for patients with prior virologic failure with the presence of key HIV-1 protease inhibitor mutations. In Phase III trials patients with greater virologic response also had higher trough tipranavir concentrations (BI product information 2005). In addition, hepatotoxicity was concentration-related with a higher incidence in those patients exceeding a trough plasma concentration of 48.2 microg/mL (80 microM). Therefore, tipranavir may be an HIV-1 protease inhibitor for which therapeutic drug monitoring (TDM) may be helpful in optimizing outcomes. To quantitate tipranavir concentrations in human plasma, a method using reversed phase high performance liquid chromatography (RP-HPLC) was validated. Detection was effected using a photodiode-array detector, scanning at a wavelength of 254 nm. This method allows for detection of tipranavir to a lower limit of quantitation of 0.390 microg/mL with an interday variation in control value ranging from 2.9 to 4.6%. The method is being used in a clinical therapeutic drug monitoring program that is ongoing in our laboratory.

Several liquid chromatography methods for assay of antiretroviral drugs in human plasma have been described, but none included atazanavir. The authors describe a user-friendly, validated, and rapid technique, derived from a procedure the authors have already published, allowing simultaneous quantification of amprenavir, indinavir, lopinavir, nelfinavir (and its M8 metabolite), ritonavir, saquinavir, efavirenz, and nevirapine. Assays were performed after diethyl ether liquid-liquid extraction from 250 microL plasma samples. Chromatographic separation was achieved on an X-TERRA column using a 58% water (with 3 mM pyrrolidine) and 42% acetonitrile mobile phase; 240 nm ultraviolet wavelength was used for atazanavir detection. This method has been in routine use in our laboratory for antiretroviral drug monitoring and now allows quantitative assay of a novel HIV protease inhibitor, atazanavir, with satisfactory intra- and interassay precision (CV<12%).

Several studies suggest that therapeutic drug monitoring of protease inhibitors and nonnucleoside reverse transcriptase inhibitors may contribute to the clinical outcome of HIV-infected patients. Because of the growing number of antiretroviral drugs and of drug combinations than can be administered to these patients, an accurate high-performance liquid chromatographic (HPLC) method allowing the simultaneous determination of these drugs may be useful. To date, the authors present the first simultaneous HPLC determination of the new protease inhibitor atazanavir with all the others currently in use (M8 nelfinavir metabolite included) and the 2 widely used nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine. This simple HPLC method allows the analysis all these drugs at a single ultraviolet wavelength following a 1-step liquid-liquid extraction procedure. A 500-muL plasma sample was spiked with internal standard and subjected to liquid-liquid extraction using by diethyl ether at pH 10. HPLC was performed using a Symmetry Shield RP18 and gradient elution. All the drugs of interest and internal standard were detected with ultraviolet detection at 210 nm. Calibration curves were linear in the range 50-10,000 ng/mL. The observed concentrations of the quality controls at plasma concentrations ranging from 50 to 5000 ng/mL for these drugs showed that the overall accuracy varied from 92% to 104% and 92% to 106% for intraday and day-to-day analysis, respectively. No metabolites of the assayed compounds or other drugs commonly coadministered to HIV-positive patients were found to coelute with the drugs of interest or with the internal standard. This assay was developed for the purpose of therapeutic monitoring (TDM) in HIV-infected patients.

A rapid and simple procedure for liquid chromatographic analysis of plasma 3-deazauridine (3-DU), an antineoplastic agent, was developed. The plasma was extracted with methanolic silver acetate to remove interfering ultraviolet-absorbing materials and the 3-DU partially purified on a small anion exchange column prior to chromatography on a reverse-phase (C18) column. 2'-O-Methyl-3-deazauridine was used as an internal standard. The mobile phase was 0.1 M ammonium acetate, pH 4.0, containing 5% methanol. The 3-DU was identified by its retention time and its characteristic ultraviolet absorbance ratio (280/254 nm) of 3.0. This method was used to determine the plasma concentration of 3-DU in three patients with acute leukemia. The patients received a 36-h infusion of 3-DU at a rate of 5.0 mg/kg/h. The mean steady-state plasma concentration of 3-DU at the end of the infusion was 22.1 micrograms/ml (range, 13.6-42.8 micrograms/ml) and the mean elimination half-life was 109 min (range, 92-136 min). 2,4-Dihydroxypyridine, a potential metabolite of 3-DU, was not detected in any of the plasma samples of the patients receiving a 3-DU infusion.

Ways of improving sensitivity of the radioreceptor assay to determine the plasma levels of dihydropyridine calcium antagonists were investigated. Extraction of the drug from plasma with organic solvent was found to enhance the sensitivity of the assay (method 1). Alternatively, the inhibitory effect observed when plasma is added directly to the binding assay can be counteracted by increasing the amount of membranes in the assay (method 2). Plasma levels after single oral doses of nitrendipine and nicardipine were followed with method 1. Plasma levels of isradipine were measured with methods 1 and 2 and by mass fragmentography. The data confirm that nitrendipine plasma level kinetics vary widely from patient to patient, whereas for nicardipine the drug level profile is more homogeneous. The similarity of the data obtained from the radioreceptor assay and from mass fragmentography suggests the absence of any active metabolite of isradipine.

An ultrafiltration technique, using Ultrafree Anticonvulsant Drug Filters, was compared to equilibrium dialysis for the determination of disopyramide plasma protein binding. Mean total recovery of drug was 92.4% for ultrafiltration compared with 98.4% for equilibrium dialysis. At initial plasma concentrations spanning the therapeutic range (2-8 micrograms/ml), the percentage binding of disopyramide was concentration dependent for both methods (78-38%). Initial experiments with ultrafiltration (1 ml of plasma) indicated a small (0.14%) but variable loss of total plasma protein in the ultrafiltrate (0.1-ml volume). In ultrafiltration, percentage binding of disopyramide was similar at 22 and 37 degrees C, whereas in equilibrium dialysis, binding was significantly (p less than 0.001) greater (1.4-2.7%) at 22 than at 37 degrees C. Percentage binding for plasma (1-ml volume), assessed at ultrafiltrate volumes of approximately 0.1 and 0.25 ml, was found to be significantly (p less than 0.05) different, but in practical terms the mean difference was less than 1% at any of the concentrations studied and was therefore of little consequence. Absolute values for percentage disopyramide bound by ultrafiltration and equilibrium dialysis were very similar, irrespective of the temperature. However, when these values were related in the usual way to the starting plasma drug concentration for ultrafiltration or to the final dialysed plasma concentration, ultrafiltration gave results that were consistently greater than those with equilibrium dialysis. Reasons for these differences and their implications are discussed.

Plasma disopyramide concentrations were measured in 19 patients with suspected myocardial infarction (MI) following an oral loading dose of 300 mg. Infarction was confirmed in 17 patients. In 10 patients blood samples were collected frequently over a period of 6 h, and in the remaining nine, blood samples were collected for 60 h whilst they were receiving a maintenance dose of 100 mg six hourly. There was a wide variation in the absorption of the drug, and only 8 patients achieved plasma concentrations in the range 2-4 mg/L within one hour of ingestion; 8 of the patients on maintenance therapy were within this range 24 h following the start of therapy. Although only 2 noninfarct patients were observed, they achieved the highest plasma disopyramide concentrations--above the therapeutic range--within 1 h of receiving the loading dose. Narcotic analgesia was associated with poor absorption of the drug, but this does not exclude severity of infarction or vomiting after the loading dose from contributing to the wide variation in plasma concentrations in the early stages following the loading dose. It is concluded that this regime is unsuitable for prophylactic use in acute MI.