Two decades (1998 to 2018) of collaborative human immunodeficiency virus clinical pharmacology capacity building in a resource constrained setting.

While important advances have been made in the prevention and treatment of Human Immunodeficiency Virus (HIV) infection, limited expertise and resource constraints to effectively manage rollout of HIV programs often contribute to poor treatment outcomes in Sub-Saharan Africa. In 1998, the University of Zimbabwe (UZ) and the University at Buffalo, State University of New York (UB), developed a collaborative clinical pharmacology capacity building program in Zimbabwe to train the next generation of HIV researchers and support rollout of the national HIV program. The collaboration was funded by research and training grants that were competitively acquired through United States of America government funding mechanisms, between 1998 and 2016. Thirty-eight research fellows were trained and a specialty clinical pharmacology laboratory was established during this period. Knowledge and skills transfer were achieved through faculty and student exchange visits. Scientific dissemination output included sixty-two scholarly publications that influenced three national policies and provided development of guidelines for strategic leadership for an HIV infection-patient adherence support group. The clinical pharmacology capacity building program trained fellows that were subsequently incorporated into the national technical working group at the Ministry of Health and Child Care, who are responsible for optimizing HIV treatment guidelines in Zimbabwe. Despite serious economic challenges, consistent collaboration between UZ and UB strengthened UZ faculty scholarly capacity, retention of HIV clinical research workforce was achieved, and the program made additional contributions toward optimization of antiretroviral therapy in Zimbabwe.

Sources of Variability and Accuracy of Performance Assessment in the Clinical Pharmacology Quality Assurance Proficiency Testing Program for Antiretrovirals.

BACKGROUND: The Clinical Pharmacology Quality Assurance (CPQA) program provides semiannual proficiency testing (PT) of antiretroviral analytes for 11 US and international clinical pharmacology laboratories (CPLs) to ensure interlaboratory comparability. In this article, we provide estimates of the main sources of variability and assess the accuracy of the algorithm for the assessment of performance. METHODS: Descriptive statistics are reported from 13 PT rounds from 2010 to 2016. Eight of the most common antiretroviral analytes were examined. Variance components analysis was used to rank the relative contributions of CPLs, antiretroviral analyte, and concentration category (low, medium, and high) to bias and variability using mixed models. Binary classification metrics of the PT assessment algorithm are calculated in comparison with a model using 95% prediction limits around estimated regression equations. RESULTS: CPLs provided 4109 reported concentrations of 65 unique samples for each of the 8 antiretroviral analytes across 13 PT rounds. Individual CPL accounted for the greatest amount of total variability (4.4%). Individual CPL and analyte combination (interaction) accounted for the greatest amount of bias (8.1%). Analyte alone accounted for 0.5% or less for total variability and bias. Overall, using a ±20% acceptance window around the final target, 97% of individual reported concentrations were scored acceptable, and 96% of antiretroviral/round scores were deemed satisfactory. Comparison with the regression model gave 100% sensitivity but only 34.47% specificity. Narrowing the acceptance window to ±15% improved specificity to 84.47% while maintaining a 99.17% sensitivity. CONCLUSIONS: The current CPQA PT scoring algorithm that use a ±20% acceptance window seems to suffer from a low specificity and may be too lenient. A stricter ±15% acceptance window would increase specificity and overall accuracy while lowering the overall pass rate by only 3%.

Paritaprevir and Ritonavir Liver Concentrations in Rats as Assessed by Different Liver Sampling Techniques.

The liver is crucial to pharmacology, yet substantial knowledge gaps exist in the understanding of its basic pharmacologic processes. An improved understanding for humans requires reliable and reproducible liver sampling methods. We compared liver concentrations of paritaprevir and ritonavir in rats by using samples collected by fine-needle aspiration (FNA), core needle biopsy (CNB), and surgical resection. Thirteen Sprague-Dawley rats were evaluated, nine of which received paritaprevir/ritonavir at 30/20 mg/kg of body weight by oral gavage daily for 4 or 5 days. Drug concentrations were measured using liquid chromatography-tandem mass spectrometry on samples collected via FNA (21G needle) with 1, 3, or 5 passes (FNA1, FNA3, and FNA5); via CNB (16G needle); and via surgical resection. Drug concentrations in plasma were also assessed. Analyses included noncompartmental pharmacokinetic analysis and use of Bland-Altman techniques. All liver tissue samples had higher paritaprevir and ritonavir concentrations than those in plasma. Resected samples, considered the benchmark measure, resulted in estimations of the highest values for the pharmacokinetic parameters of exposure (maximum concentration of drug in serum [Cmax] and area under the concentration-time curve from 0 to 24 h [AUC0-24]) for paritaprevir and ritonavir. Bland-Altman analyses showed that the best agreement occurred between tissue resection and CNB, with 15% bias, followed by FNA3 and FNA5, with 18% bias, and FNA1 and FNA3, with a 22% bias for paritaprevir. Paritaprevir and ritonavir are highly concentrated in rat liver. Further research is needed to validate FNA sampling for humans, with the possible derivation and application of correction factors for drug concentration measurements.

Interaction of disulfiram with antiretroviral medications: efavirenz increases while atazanavir decreases disulfiram effect on enzymes of alcohol metabolism.

BACKGROUND AND OBJECTIVES: Alcohol abuse complicates treatment of HIV disease and is linked to poor outcomes. Alcohol pharmacotherapies, including disulfiram (DIS), are infrequently utilized in co-occurring HIV and alcohol use disorders possibly related to concerns about drug interactions between antiretroviral (ARV) medications and DIS. METHOD: This pharmacokinetics study (n=40) examined the effect of DIS on efavirenz (EFV), ritonavir (RTV), or atazanavir (ATV) and the effect of these ARV medications on DIS metabolism and aldehyde dehydrogenase (ALDH) activity which mediates the DIS-alcohol reaction. RESULTS: EFV administration was associated with decreased S-Methyl-N-N-diethylthiocarbamate (DIS carbamate), a metabolite of DIS (p=.001) and a precursor to the metabolite responsible for ALDH inhibition, S-methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO). EFV was associated with increased DIS inhibition of ALDH activity relative to DIS alone administration possibly as a result of EFV-associated induction of CYP 3A4 which metabolizes the carbamate to DETC-MeSO (which inhibits ALDH). Conversely, ATV co-administration reduced the effect of DIS on ALDH activity possibly as a result of ATV inhibition of CYP 3A4. DIS administration had no significant effect on any ARV studied. DISCUSSION/CONCLUSIONS: ATV may render DIS ineffective in treatment of alcoholism. FUTURE DIRECTIONS: DIS is infrequently utilized in HIV-infected individuals due to concerns about adverse interactions and side effects. Findings from this study indicate that, with ongoing clinical monitoring, DIS should be reconsidered given its potential efficacy for alcohol and potentially, cocaine use disorders, that may occur in this population.

Pharmacokinetic interactions of CEP-1347 and atazanavir in HIV-infected patients.

CEP-1347 is a potent inhibitor of mixed lineage kinase (MLK), which was investigated for ameliorating HIV-associated neurocognitive disorders. CEP-1347 and atazanavir pharmacokinetics were determined when CEP-1347 50 mg twice daily was administered to HIV-infected patients (n = 20) receiving combination antiretroviral therapy including atazanavir and ritonavir (ATV/RTV, 300/100 mg) once daily continuously. Co-administration of CEP-1347 and ATV/RTV resulted with significant changes in pharmacokinetics of ATV but not RTV. Specifically, an increase in ATV accumulation ratio of 15 % (p = 0.007) and a prolongation of T(½) from 12.7 to 15.9 h (p = 0.002) were observed. The results suggested that co-administration of CEP-1347 with ATV/RTV in HIV-infected patients might result in limited impact on ATV but not on RTV pharmacokinetics.

Discordant associations between SLCO1B1 521T→C and plasma levels of ritonavir-boosted protease inhibitors in AIDS clinical trials group study A5146.

OBJECTIVE: Among HIV-positive patients prescribed ritonavir-boosted lopinavir, SLCO1B1 521T→C (rs4149056) is associated with increased plasma lopinavir exposure. Protease inhibitors (PIs) are also substrates for cytochrome P450 (CYP) 3A and ABCB1, which are induced by NR1I2. We characterized relationships between ABCB1, CYP3A4, CYP3A5, NR1I2, and SLCO1B1 polymorphisms and trough PI concentrations among AIDS Clinical Trials Group study A5146 participants. METHODS: At study entry, subjects with virologic failure on PI-containing regimens initiated new ritonavir-boosted PI regimens. We studied associations between week 2 PI plasma trough concentrations and 143 polymorphisms in these genes, including 4 targeted polymorphisms. RESULTS: Among 275 subjects with both drug concentrations and genetic data, allelic frequencies of SLCO1B1 521T→C were 15%, 1%, and 8% in whites, blacks, and Hispanics, respectively. Further analyses were limited to 268 white, black, or Hispanic subjects who initiated ritonavir-boosted lopinavir (n = 98), fosamprenavir (n = 69), or saquinavir (n = 99). Of targeted polymorphisms, SLCO1B1 521T→C tended to be associated with higher lopinavir concentrations, with a 1.38-fold increase in the mean per C allele (95% confidence interval, 0.97-1.96; n = 98; P = 0.07). With fosamprenavir, SLCO1B1 521T→C was associated with lower amprenavir concentrations, with a 35% decrease in the mean per C allele (geometric mean ratio 0.65; 95% confidence interval, 0.44-0.94; n = 69; adjusted P = 0.02). There was no significant association with saquinavir concentrations, and none of the remaining 139 exploratory polymorphisms were statistically significant after correcting for multiple comparisons. CONCLUSIONS: With ritonavir-boosted PIs, a SLCO1B1 polymorphism that predicts higher lopinavir trough concentrations seems to predict lower amprenavir trough concentrations. The mechanism underlying this discordant association is uncertain.

Clinical pharmacology quality assurance program: models for longitudinal analysis of antiretroviral proficiency testing for international laboratories.

Among National Institutes of Health HIV Research Networks conducting multicenter trials, samples from protocols that span several years are analyzed at multiple clinical pharmacology laboratories (CPLs) for multiple antiretrovirals. Drug assay data are, in turn, entered into study-specific data sets that are used for pharmacokinetic analyses, merged to conduct cross-protocol pharmacokinetic analysis, and integrated with pharmacogenomics research to investigate pharmacokinetic-pharmacogenetic associations. The CPLs participate in a semiannual proficiency testing (PT) program implemented by the Clinical Pharmacology Quality Assurance program. Using results from multiple PT rounds, longitudinal analyses of recovery are reflective of accuracy and precision within/across laboratories. The objectives of this longitudinal analysis of PT across multiple CPLs were to develop and test statistical models that longitudinally: (1) assess the precision and accuracy of concentrations reported by individual CPLs and (2) determine factors associated with round-specific and long-term assay accuracy, precision, and bias using a new regression model. A measure of absolute recovery is explored as a simultaneous measure of accuracy and precision. Overall, the analysis outcomes assured 97% accuracy (±20% of the final target concentration of all (21) drug concentration results reported for clinical trial samples by multiple CPLs). Using the Clinical Laboratory Improvement Act acceptance of meeting criteria for ≥2/3 consecutive rounds, all 10 laboratories that participated in 3 or more rounds per analyte maintained Clinical Laboratory Improvement Act proficiency. Significant associations were present between magnitude of error and CPL (Kruskal-Wallis P < 0.001) and antiretroviral (Kruskal-Wallis P < 0.001).

Therapeutic drug monitoring of protease inhibitors and efavirenz in HIV-infected individuals with active substance-related disorders.

BACKGROUND: Achieving targeted antiretroviral (ARV) plasma concentrations during long-term treatment in human immunodeficiency virus (HIV)-infected patients with substance-related disorders (SRDs) may be challenging due to a number of factors, including medication adherence, coinfection with hepatitis B or C virus, medication intolerance, and drug interactions. One approach to investigate these factors is to conduct therapeutic drug monitoring to measure ARV exposure during treatment. The objective of this study was to utilize therapeutic drug monitoring to compare efavirenz (EFV) and protease inhibitor pharmacokinetics in patients with and without SRDs. METHODS: This was a multicenter, cross-sectional open-label study in patients with HIV-1 infection receiving antiretroviral therapy (ART), with active (n=129) or without (n=146) SRD according to National Institute on Drug Abuse criteria. Two hundred seventy-five subjects who were receiving either protease inhibitor-based or EFV-based ART regimens for >6 months were enrolled at 4 HIV treatment centers with an equal distribution of SRD and non-SRD at each site. The patients were instructed during enrollment visits with regard to the importance of adherence before and after study visits. Demographics and routine clinical laboratory tests were recorded. RESULTS: Among the 275 patients, 47% had SRD with at least 1 substance. There were no significant differences between SRD and non-SRD groups for race, gender, age, or CD4 count at entry. A significantly higher proportion of patients with SRD had an entry HIV RNA plasma concentration>75 copies per milliliter compared with patients without SRD (40% vs 28%, P=0.044). Logistic regression modeling revealed an association between HIV RNA plasma concentration and African American race (P=0.017). A significantly higher proportion of SRDs also had an EFV or protease inhibitor trough concentration below the desired range (23% vs 9%, P=0.048). Significantly lower trough concentrations were noted in patients with SRDs receiving atazanavir (0.290 vs 0.976 µg/mL) or lopinavir (3.75 vs 5.30 µg/mL). CONCLUSIONS: The pharmacokinetic data indicate differences between HIV-infected patients with and without SRDs that may influence viral load suppression during long-term ART. These findings require additional investigation in a randomized design with more intensive pharmacokinetic assessment to identify individual factors that are contributing to suboptimal ARV exposure in patients with SRDs.

Cross-validation of a high-performance liquid chromatography nevirapine plasma assay in a resource-limited setting in Zimbabwe.

An international HIV pharmacology specialty laboratory (PSL) was established at the University of Zimbabwe to increase bioanalytical and investigator capacities. Quantitation of plasma nevirapine in samples from the AIDS Clinical Trials Group protocol 5279 was compared between the University of Nebraska Medical Center PSL and the University of Zimbabwe PSL. Both PSLs employed internally developed methods utilising reverse-phase high-performance liquid chromatography with ultraviolet detection. Eighty-seven percent of the cross-validation results exhibited ± 20% difference.

Quality assessment for therapeutic drug monitoring in AIDS Clinical Trials Group (ACTG 5146): a multicenter clinical trial.

In a randomized trial, AIDS Clinical Trials Group (ACTG) protocol 5146 (A5146) investigated the use of therapeutic drug monitoring (TDM) to adjust doses of HIV-1 protease inhibitors (PIs) in patients with prior virologic failure on PI-based therapy who were starting a new PI-based regimen. The overall percentage of "PI trough repeats" such as rescheduled visits or redrawn PI trough specimens increased from 2% to 5% to 10% as the process progressed from the clinical sites, the pharmacology specialty laboratory, and the study team, respectively. Cumulatively, this represents a 17% rate of failure to obtain adequate PI trough sample. While targeting a turnaround of 7 days or less from sample receipt to a drug concentration report, 12% of the received specimens required a longer period to report concentrations. The implementation of dosing changes in the TDM arm were achieved within 7 days or less for 56% of the dose change events and within 14 days or less for 77% of dose change events. This quality assurance analysis provides a valuable summary of the specific points in the TDM process that could be improved during a multicenter clinical trial including: 1) shortening the timeline of sample shipment from clinical site to the laboratory; 2) performing the collection of PI trough specimen within the targeted sampling window by careful monitoring of the last dose times and collection times by the clinicians; 3) increasing patient adherence counseling to reduce the number of samples that are redrawn due to suspecting inconsistent adherence; and 4) decreasing the time to successful TDM-based dose adjustment. The application of some of these findings may also be relevant to single-center studies or clinical TDM programs within a hospital.

Lopinavir cerebrospinal fluid steady-state trough concentrations in HIV-infected adults.

BACKGROUND: The central nervous system may act as a sanctuary site for viral replication in the setting of low antiretroviral penetration. Data on lopinavir cerebrospinal fluid (CSF) trough concentration (C(trough)) values have yet to be reported. OBJECTIVE: To describe lopinavir CSF C(trough) values and compare them with a measure of HIV susceptibility. METHODS: In a prospective, open-label design, HIV-infected adults whose regimen included lopinavir/ritonavir 400/100-mg soft-gel capsules twice daily for at least 4 weeks were enrolled. Each subject had 8 plasma lopinavir concentrations determined over a 12-hour dosing interval and 1 CSF lopinavir C(trough) value determined at the end of the study. Linear regression methods tested for associations between CSF or CSF to plasma concentration ratio and covariates including pharmacokinetic parameters and CSF protein. RESULTS: Ten patients (7 male; median [range] +/- SD age 45.3 +/- 2.8 y) completed the study. Median (intraquartile range [IQR]) lopinavir plasma 0- to 12-hour area under the curve (AUC(0-12)) and minimum concentrations were 71.3 h x microg/mL (48.4-87.6) and 3.82 microg/mL (2.76-5.34). Median (IQR) CSF C(trough), paired plasma concentration, and time since last dose were 11,200 pg/mL (6760-16,400), 5.42 microg/mL (3.88-5.85), and 9.9 hours (9.7-10.2), respectively. Median (IQR) CSF to plasma concentration ratio was 0.225% (0.194-0.324). Lopinavir CSF C(trough) was above the median 50% inhibitory concentration (IC(50)) for wild-type HIV-1 (wtHIV-1) (1900 pg/mL) in all subjects. Lopinavir plasma AUC(0-12) (r(2) = 0.65; p = 0.009) and CSF protein (r(2) = 0.26; p = 0.006) were associated with lopinavir CSF concentration, while CSF protein (r(2) = 0.66; p = 0.008) was associated with CSF to plasma concentration ratio. CONCLUSIONS: Lopinavir CSF C(trough) was above the median IC(50) for wtHIV-1 replication in all patients receiving lopinavir/ritonavir 400/100-mg soft-gel capsules twice daily.

Development and validation of an assay to measure cannabidiol and Δ9-tetrahydrocannabinol in human EDTA plasma by UHPLC-MS/MS.

The therapeutic use of cannabinoids has increased with providers often recommending cannabinoid-containing products with limited pre-clinical and clinical pharmacokinetic studies. An ultra-performance liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of cannabidiol and Δ9-tetrahydrocannabinol in human ethylenediaminetetraacetic acid (EDTA) plasma. The cannabinoids are extracted from plasma with a liquid-liquid procedure utilizing methyl tert-butyl ether. UHPLC Separation was achieved with a Waters Acquity HSS T3 column (100 × 2.1 mm, 1.8 µm) under isocratic conditions (18:82:0.02 water:methanol:formic acid v/v/v). The run time was 8.5 min. Detection of analytes was achieved using electrospray ionization and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 0.5 to 250 ng/mL for cannabidiol and Δ9-tetrahydrocannabinol. The intra- and inter-day accuracy (% bias) and precision (relative standard deviation) were <9.20% in low, medium, and high quality control samples. The validated method was applied to the analysis of donated human EDTA plasma. The assay provides an important patient monitoring capability to determine variability in clinical pharmacokinetics during use of cannabinoid-containing products.

Development and validation of a high performance liquid chromatography method to determine nevirapine in plasma in a resource-limited setting.

BACKGROUND: There are several instances where nevirapine pharmacokinetic monitoring may be useful, such as in special populations or pharmacokinetic drug interaction studies that require the ascertainment of nevirapine pharmacokinetics in the sub-Saharan region. OBJECTIVES: The main aim of this study was to produce a validated, sustainable and relevant nevirapine assay method that meets bio-analytical regulatory requirements. METHODS: The developed method utilised a Waters 2795 Alliance high performance liquid chromatography system with a 2996 photo diode array detector, an Atlantis dC18 5 micron, 3.9 mm × 150 mm analytical column and a gradient flow rate of 1 mL/min. Ultraviolet detection data were collected from 210 nm to 400 nm, extracted at 260 nm, and processed for nevirapine and internal standard peak height responses. RESULTS: The method proved to be linear (R2 0.995), precise (+1.92% - +9.69%) and accurate (-9.70% - 12.0%). Recovery for the analyte and internal standard was between 98.8% and 114%. The method showed good specificity as no interferences were caused by common African traditional medicines, anti-tuberculosis medications or other concomitant antiretrovirals nor endogenous components. CONCLUSION: The method is reproducible, relevant to our setting and uses considerably low plasma volumes with preservation of some consumables, a desirable key factor in a resource-limited setting.

Mentored postdoctoral training in Zimbabwe: A report on a successful collaborative effort.

Low-and-middle-income countries (LMICs) have high disease burdens, necessitating increased research. However, LMIC research output constitutes only 2% of global total. To increase output, researchers must be capacitated. The University of Zimbabwe (UZ) and the University at Buffalo (UB), developed and implemented the AIDS International Research Training Program (AITRP), in 2008, that focused on graduate scholars. The subsequent HIV Research Training Program (HRTP), begun in 2016, and piloted post-doctoral training to enhance research productivity at UZ. This report discusses the collaboration. As of 2016, prospective candidates applied by submitting letters of intent, research proposals, curriculum vitae and biographical sketches. The scholars research training included hypothesis and project development, completion of grant applications, research project budgets, research presentations to diverse audiences and the application of advanced statistics to research data. The first cohort of five postdoctoral scholars were trained at UZ and UB, between 2016 and 2019. Through the formalized postdoctoral training approach, scholars identified areas of focus. In 2017, one of the scholars obtained a National Institutes of Health (NIH) Emerging Global Leader Award and is now a highly-rated researcher based in South Africa. A second scholar made NIH D43 and K43 grant applications, while the remaining three are academicians and early researchers at UZ. Although research output in Africa and many LMICs is low, it can be built through cooperation similar to the UZ-UB HRTP. This manuscript reports on an effort aimed at building individual and institutional research capacity in Zimbabwe. This can serve as a model for building other similar training programs.

The design and implementation of A5146, a prospective trial assessing the utility of therapeutic drug monitoring using an inhibitory quotient in antiretroviral-experienced HIV-infected patients.

The AIDS Clinical Trials Group designed and implemented a prospective, randomized, strategy trial in antiretroviral-experienced, HIV-infected patients to evaluate the virologic impact of protease inhibitor dose escalation in response to therapeutic drug monitoring (TDM) with an inhibitory quotient, which integrates both drug exposure and viral drug resistance. In the process of developing this clinical trial, several unique challenges were identified that required innovative solutions. The major challenge was the need to integrate resistance testing, pharmacokinetic data, medication adherence, toxicity data, clinical assessments, randomization assignment, and protocol-specified clinical management in a way that could be utilized in real time by the protocol team, communicated promptly to the clinical sites, and transmitted accurately to the study database. In addition, the protocol team had to address the relative lack of commercially available TDM laboratories in the United States that were experienced in antiretroviral drug assays and a lack of familiarity with the principles of pharmacokinetic monitoring at participating clinical sites. This article outlines the rationale for the design of this strategy trial, specific barriers to implementation that were identified, and solutions that were developed with the hope that these experiences will facilitate the design and conduct of future trials of TDM.

Interaction between buprenorphine and atazanavir or atazanavir/ritonavir.

Opioid addiction and HIV disease frequently co-occur. Adverse drug interactions have been reported between methadone and some HIV medications, but less is known about interactions between buprenorphine, an opioid partial agonist used to treat opioid dependence, and HIV therapeutics. This study examined drug interactions between buprenorphine and the protease inhibitors atazanavir and atazanavir/ritonavir. Opioid-dependent, buprenorphine/naloxone-maintained, HIV-negative volunteers (n=10 per protease inhibitor) participated in two 24-h sessions to determine pharmacokinetics of (1) buprenorphine and (2) buprenorphine and atazanavir (400mg daily) or atazanavir/ritonavir (300/100mg daily) following administration for 5 days. Objective opiate withdrawal scale scores and mini-mental state examination were determined prior to and following antiretroviral administration to examine pharmacodynamic effects. Pharmacokinetics of atazanavir and atazanavir/ritonavir were compared in subjects and matched, healthy controls (n=10 per protease inhibitor) to determine effects of buprenorphine. With atazanavir and atazanavir/ritonavir, respectively concentrations of buprenorphine (p<0.001, p<0.001), norbuprenorphine (p=0.026, p=0.006), buprenorphine glucuronide (p=0.002, p<0.001), and norbuprenorphine glucuronide (NS, p=0.037) increased. Buprenorphine treatment did not significantly alter atazanavir or ritonavir concentrations. Three buprenorphine/naloxone-maintained participants reported increased sedation with atazanavir/ritonavir. Atazanavir or atazanavir/ritonavir may increase buprenorphine and buprenorphine metabolite concentrations and might require a decreased buprenorphine dose.

Simultaneous analysis of cyclophosphamide, doxorubicin and doxorubicinol by liquid chromatography coupled to tandem mass spectrometry.

A method for the simultaneous determination of cyclophosphamide (CP), doxorubicin (dox), and doxorubicinol (dol) was developed and validated to analyze 400 microL of plasma from patients receiving chemotherapeutic treatment with CP and dox. Final calibration ranges for the analytes were 0.440-60.0 microg/mL for cyclophosphamide, 7.20-984 ng/mL for dox and 3.04-104 ng/mL for dol. The samples were prepared using solid phase extraction and analyzed using a gradient separation over a Waters Symmetry C18, 2.1 by 30 mm (Milford, MA) column. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer.

Simultaneous determination of cortisol, dexamethasone, methylprednisolone, prednisone, prednisolone, mycophenolic acid and mycophenolic acid glucuronide in human plasma utilizing liquid chromatography-tandem mass spectrometry.

Chronic combination immunosuppressive regimens are commonly prescribed to renal transplant recipients. To develop an assay method for pharmacokinetic studies and therapeutic drug monitoring of multiple immunosuppressives, a liquid chromatography-tandem mass spectrometry (LC/MS/MS) approach for the simultaneous analysis of several glucocorticoids, mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) was investigated. The resultant method utilized a gradient reverse phase separation over a Symmetry C18 column using an ammonium acetate-methanol mobile phase at pH 3.5. The analytes were detected by coupling the chromatography system via electrospray to a triple quadrupole mass spectrometer. Multiple-reaction monitoring in the negative mode ion (MH-/product) was employed selecting MPA at 319.1/190.9, MPAG at 495.1/191.0, dexamethasone at 391.0/361.0, hydrocortisone at 361.1/331.1, methylprednisolone at 373.1/343.1, prednisone at 357.1/327.2, and prednisolone at 359.1/329.1. The calibration curve concentrations ranged from 3.60 ng/mL to 50 microg/mL with the lowest limit of quantitation for corticosteroids being 3.60-7.20 ng/mL and 0.656-6.75 microg/mL for MPA and MPAG, respectively. The relative standard deviation for quality control intraday variation and interday variation was between 0.76% and 9.57% for all analytes. This assay offers a versatile, unique method for multi-analyte immunosuppressive determinations during combination immunosuppression.

Determination of lopinavir cerebral spinal fluid and plasma ultrafiltrate concentrations by liquid chromatography coupled to tandem mass spectrometry.

A method for the determination of lopinavir (LPV) concentrations in cerebral spinal fluid (CSF) and plasma ultrafiltrate (UF) was developed and validated to analyze clinical specimens from patients receiving antiretroviral treatment with lopinavir/ritonavir. The CSF (400 microL sample volume) final calibration range for LPV was 0.313-25.0 ng/mL. The final calibration range for UF (50 microL sample volume) was 1.25-100 ng/mL. The samples were prepared using liquid-liquid extraction, concentrated, and analyzed using a reversed phase isocratic separation. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer. Isolation of LPV through chromatographic separation and proper selection of calibration matrix were important factors in achieving accurate results. Plasma UF was found to be an equivalent calibration matrix to CSF whereas plasma matrix produced a positive bias in samples with unknown concentrations. Artificial CSF media prepared chemically were biased and less superior than UF. Sources of plasma for the UF did not affect accuracy. Several CSF sources were tested for specificity of the method and LPV concentrations were accurately produced with atmospheric pressure chemical ionization source producing more accurate results than the electrospray source. The method successfully measured LPV concentrations in CSF that were previously undetectable by HPLC as well as UF from protein binding studies.

Buprenorphine assay and plasma concentration monitoring in HIV-infected substance users.

The availability of buprenorphine (BUP) provides an alternative approach to the treatment of opioid addiction with methadone, an agent that has many drug-drug interactions when combined with antiretroviral therapy (ART). However, due to limited long-term pharmacokinetic studies in HIV-infected patients, the clinical use of BUP, a CYP450-3A4 substrate, will require that studies be conducted to examine safety, tolerability and pharmacokinetics when these drugs are taken for chronic treatment. One clinical approach could include plasma concentration monitoring to avoid under- or overdosing BUP secondary to drug interactions with ART. The measurement of BUP and its active metabolite, norbuprenorphine (NBUP) facilitates the addition of BUP to ART in an attempt to avoid drug toxicity as described in a recent report by Bruce et al. Therefore, our objective was to validate a BUP assay and integrate its application into an ongoing antiretroviral (ARV) plasma concentration monitoring program. A chromatographic method for monitoring BUP and its active metabolite, NBUP was investigated. An assay was developed that would facilitate BUP and ARV measurement from a single 3 mL blood sample (0.75 mL plasma required) in conjunction with a previously validated multiple ARV HPLC method. The method measures BUP and NBUP over the range from 0.25 to 50 ng/mL with mass spectrometry detection. Inter- and intra-assay variation was