Development and validation of an UPLC-MS/MS assay for the simultaneous quantification of seven commonly used antibiotics in human plasma and its application in therapeutic drug monitoring.

OBJECTIVE: To develop and validate an UPLC-MS/MS assay for simultaneous determination of the total concentration of ceftazidime, ciprofloxacin, flucloxacillin, piperacillin, tazobactam, sulfamethoxazole, N-acetyl sulfamethoxazole and trimethoprim, and the protein-unbound concentration of flucloxacillin, in human plasma to be used for research and clinical practice. METHODS: Sample pretreatment included protein precipitation with methanol. For the measurement of protein-unbound flucloxacillin, ultrafiltration was performed at physiological temperature. For all compounds, a stable isotopically labelled internal standard was used. Reliability of the results was assessed by participation in an international quality control programme. RESULTS: The assay was successfully validated according to the EMA guidelines over a concentration range of 0.5-100 mg/L for ceftazidime, 0.05-10 mg/L for ciprofloxacin, 0.4-125 mg/L for flucloxacillin, 0.2-60 mg/L for piperacillin, 0.15-30 mg/L for tazobactam, 1-200 mg/L for sulfamethoxazole and N-acetyl sulfamethoxazole, 0.05-10 mg/L for trimethoprim and 0.10-50 mg/L for unbound flucloxacillin. For measurement of total concentrations, the within- and between-day accuracy ranged from 90.0% to 109%, and 93.4% to 108%, respectively. Within- and between-day precision (variation coefficients, CVs) ranged from 1.70% to 11.2%, and 0.290% to 5.30%, respectively. For unbound flucloxacillin, within-day accuracy ranged from 103% to 106% and between-day accuracy from 102% to 105%. The within- and between-day CVs ranged from 1.92% to 7.11%. Results of the international quality control programme showed that the assay is reliable. CONCLUSIONS: The method provided reliable, precise and accurate measurement of seven commonly prescribed antibiotics, including the unbound concentration of flucloxacillin. This method is now routinely applied in research and clinical practice.

Highly sensitive quantification of pemetrexed in human plasma using UPLC-MS/MS to support microdosing studies.

Pemetrexed is an antifolate drug approved for the treatment of non-small-cell lung cancer and mesothelioma. Assessing pemetrexed pharmacokinetics after administration of a microdose (100 µg) may facilitate drug-drug interaction and dose individualization studies with cytotoxic drugs, without causing harm to patients. Therefore, a highly sensitive bioanalytical assay is required. A reversed-phase ultra-high performance liquid chromatography method was developed to determine pemetrexed concentrations in human ethylenediaminetetraacetic acid-plasma after microdosing. [13 C5 ]-Pemetrexed was used as the internal standard. The sample preparation involved solid-phase extraction from plasma. Detection was performed using MS/MS in a total run time of 9.5 min. The assay was validated over the concentration range of 0.0250-25.0 µg/L pemetrexed. The average accuracies for the assay in plasma were 96.5 and 96.5%, and the within-day and between-day precision in coefficients of variations was <8.8%. Extraction recovery was 59 ± 1 and 55 ± 5% for pemetrexed and its internal standard. Processed plasma samples were stable for 2 days in a cooled autosampler at 10°C. The assay was successfully applied in a pharmacokinetic curve, which was obtained as a part of an ongoing clinical microdosing study.

Placental transfer of the HIV integrase inhibitor dolutegravir in an ex vivo human cotyledon perfusion model.

OBJECTIVES: Data on fetal exposure to antiretroviral agents during pregnancy are important to estimate their potential for prevention of mother-to-child transmission (PMTCT) and possible toxicity. For the recently developed HIV integrase inhibitor dolutegravir, clinical data on fetal disposition are not yet available. Dual perfusion of a single placental lobule (cotyledon) provides a useful ex vivo model to predict the in vivo maternal-to-fetal transfer of this drug. The aim of this study was to estimate the transfer of dolutegravir across the human term placenta, using a dual-perfusion cotyledon model. METHODS: After cannulation of the cotyledons (n = 6), a fetal circulation of 6 mL/min and maternal circulation of 12 mL/min were initiated. The perfusion medium consisted of Krebs-Henseleit buffer (pH = 7.2-7.4) supplemented with 10.1 mM glucose, 30 g/L human serum albumin and 0.5 mL/L heparin 5000IE. Dolutegravir was administered to the maternal circulation (∼ 4.2 mg/L) and analysed by UPLC-MS/MS. RESULTS: After 3 h of perfusion, the mean ± SD fetal-to-maternal (FTM) concentration ratio of dolutegravir was 0.6 ± 0.2 and the mean ± SD concentrations in the maternal and fetal compartments were 2.3 ± 0.4 and 1.3 ± 0.3 mg/L, respectively. CONCLUSIONS: Dolutegravir crosses the blood-placental barrier with a mean FTM concentration ratio of 0.6. Compared with other antiretroviral agents, placental transfer of dolutegravir is moderate to high. These data suggest that dolutegravir holds clinical potential for pre-exposure prophylaxis and consequently PMTCT, but also risk of fetal toxicity.

Determination of the HCV Protease Inhibitor Telaprevir in Plasma and Dried Blood Spot by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Telaprevir is a protease inhibitor used in the treatment of hepatitis C virus infection. Analytical methods for telaprevir should separate the compound from its R-diastereomer VRT-127394, which is 30-fold less active. The objective of this work was to develop liquid chromatography-tandem mass spectrometer (LC-MS/MS) assays for telaprevir both in plasma and in dried blood spot (DBS), capable of stabilizing the equilibrium and chromatographically separating the 2 epimers. METHODS: Human plasma was acidified with formic acid and frozen within 1 hour after collection to stabilize the equilibrium between the 2 telaprevir diastereomers ex vivo in plasma. After protein precipitation, the sample was analyzed with LC-MS/MS. For the DBS assay, sampling paper was impregnated with citric acid solution to achieve stabilization of the epimers on the sampling paper. DBS samples were extracted before LC-MS/MS analysis. LC-MS/MS analysis comprised online solid-phase extraction and separation on a C18 column, with the mass spectrometer operating in TurboIonSpray-negative ionization mode and performing multiple reaction monitoring. RESULTS: The assays were linear over the concentration range of 0.1-10 mg/L in plasma and DBS. Accuracies ranged from 97% to 106% in plasma and from 93% to 99% in DBS. Within- and between-day coefficients of variation were <7.9% in plasma and <9.3% in DBS. Human whole blood samples with hematocrit values of 27%-47% gave reproducible quantitation results in the DBS assay, and spot volume did not affect results of the DBS assay either. Acidified plasma with telaprevir was stable for 5 hours at 20°C, and telaprevir on impregnated DBS paper was stable for at least 3 months at 4°C or at 20°C. CONCLUSIONS: An assay was developed and validated for the determination of telaprevir in human plasma, separating telaprevir from its R-diastereomer VRT-127394. In addition, a DBS assay was developed, which avoids immediate centrifuging, acidification, and freezing of patient samples to stabilize the equilibrium between the 2 telaprevir diastereomers.

Optimization of the rifampin dosage to improve the therapeutic efficacy in tuberculosis treatment using a murine model.

RATIONALE: The dosage of 10 mg/kg/d rifampin, as currently used in the treatment of tuberculosis (TB), is not an optimal dose. Shortening of treatment duration might be achievable using an increased rifampin dose. OBJECTIVES: Determination of optimal rifampin dosage in mice, resulting in maximum therapeutic effect and without adverse effects. Assessment of associated pharmacokinetic parameters and pharmacokinetic/pharmacodynamic indices. METHODS: A murine TB infection using a Beijing genotype Mycobacterium tuberculosis strain was established by intratracheal bacterial instillation followed by proper inhalation, while keeping mice in a vertical position. We assessed dose-dependent activity of rifampin in single-drug treatment during 3 weeks. The maximum tolerated dosage, pharmacokinetic parameters, and pharmacokinetic/pharmacodynamic index were determined. Therapeutic efficacy of a range of rifampin (R) dosages added to a regimen of isoniazid (H) and pyrazinamide (Z) was assessed. MEASUREMENTS AND MAIN RESULTS: Maximum tolerated dosage of rifampin in the murine TB was 160 mg/kg/d. Pharmacokinetic measurement in HR(10)Z and HR(160)Z therapy regimens showed for rifampin a C(max) of 16.2 and 157.3 mg/L, an AUC(0-24h) of 132 and 1,782 h·mg/L, and AUC(0-24h)/minimum inhibitory concentration ratios of 528 and 7129, respectively. A clear dose-effect correlation was observed for rifampin after 3-week single-drug treatment. Administration of HR(80)Z allowed 9-week treatment duration to be effective without relapse of infection. CONCLUSIONS: Our findings indicate that the currently used rifampin dosage in the therapy of TB is too low. In our murine TB model a rifampin dosage of 80 mg/kg/d enabled a significant reduction in therapy duration without adverse effects.

The International Interlaboratory Quality Control Program for Measurement of Antiretroviral Drugs in Plasma: a global proficiency testing program.

The International Interlaboratory Quality Control Program for Measurement of Antiretroviral Drugs in Plasma was initiated in 1999 by Radboud University Nijmegen Medical Center, The Netherlands, and continued later on in collaboration with the Dutch Association for Quality Assessment in Therapeutic Drug Monitoring and Clinical Toxicology (www.kkgt.nl). The aim of this analysis was to evaluate the first 10 years of the Program and to determine variables associated with reporting of less accurate results. Two rounds are organized annually in which blind samples are shipped to participants containing a low, medium, or high concentration of each antiretroviral drug. Any reported result that deviates more than 20% from the spiked concentration is defined as inaccurate. By the end of 2009, the number of laboratories participating in the Program had increased to 56; 44 (79%) are located in Europe. A total of 12,798 test results was available for analysis, of which 2104 (16.4%) were reported as inaccurate. Performance was best for samples containing nevirapine (mean of inadequate scores per round: 11.1%) and lopinavir (11.9%) and worst for indinavir (18.7%), atazanavir (18.9%), saquinavir (19.6%), and nelfinavir (21.3%). High and medium concentrations were less frequently reported as inaccurate than low concentrations: 13.5%, 13.0%, and 22.4%, respectively. Although the overall performance of the laboratories varied per year, a trend was visible for improvement over time with 19.9% of the results being inaccurate in 2002 (n = 20 laboratories) to 15.7% in 2009 (n = 56 laboratories). The Program provides a proficiency testing program in which laboratories are alerted to potential analytical errors while performing therapeutic drug monitoring in HIV-infected patients. Laboratories should put more effort in adequately analyzing concentrations of antiretroviral drugs with low minimum effective concentrations.

Quantification of second generation direct-acting antivirals daclatasvir, elbasvir, grazoprevir, ledipasvir, simeprevir, sofosbuvir and velpatasvir in human plasma by UPLC-MS/MS.

Direct-acting antivirals (DAA) have markedly improved the treatment of hepatitis C, with a series of DAA combinations available for treatment. A sensitive method by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous quantification of seven commonly used DAAs, daclatasvir (DAC), elbasvir (ELB), grazoprevir (GZR), ledipasvir (LDV), simeprevir (SIM), sofosbuvir (SOF), velpatasvir (VEL) and the primary analyte of interest of SOF (GS-331007) in EDTA plasma. Adequate chromatographic separation with well-defined peaks for all eight analytes was achieved with an Acquity UPLC BEH C18 column (1.7 µm 2.1 × 50 mm). Runtime for the final assay was 12 min. Protein precipitation in the sample preparation and stable isotope- labelled internal standards resulted in a robust sensitive and rapid method. Method validation was performed in accordance with the "guideline on bioanalytical method validation" of the European Medicines Agency (EMA). No interferences from endogenous substances were observed and carry-over in the blank sample was <20% of the LLOQ of each analyte and <5% of the IS after injection of the HLOQ sample. Validation was established over the range of 10-5000 µg/L for DAC and SIM, 3.0-1500 µg/L for ELB and GZR, 7.5-1500 µg/L for LDV and VEL, 5.0-2500 µg/L for SOF and 25-5000 µg/L for GS-331007. Within-day accuracy values for all analytes, ranged from 94% to 109%, and precision expressed as residual standard deviation % (RSD) was <9.3%. Between-day accuracy ranged from 99 to 107% with a RSD of <5.3%. For the lower limit of quantification, the percent deviation from the nominal concentration and the relative standard deviation was <20%. The method has been applied successfully in clinical evaluation of patients treated with DAA and to describe the pharmacokinetics of DAAs in clinical studies.

Development and validation of an UPLC-MS/MS bioanalytical method for simultaneous quantification of the antiretroviral drugs dolutegravir, elvitegravir, raltegravir, nevirapine and etravirine in human plasma.

Dolutegravir, elvitegravir, raltegravir, nevirapine and etravirine are antiretroviral drugs used as part of combined antiretroviral treatment for HIV-infection. For quantification of these drugs in human K2EDTA plasma samples an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) bioanalytical method was developed and validated. Stable isotope labeled internal standards were used for each analyte. Simple protein precipitation with methanol was implemented to prepare plasma samples of at least 50 µL. The method was validated for dolutegravir, elvitegravir, raltegravir, nevirapine and etravirine over the ranges 9.7-9700, 52-10,470, 9.7-9730, 73-14,680 and 15-3010 ng/mL, respectively. Within-run and between-run accuracy and precision were within ±15% of the nominal concentration for quality controls at high, medium and low concentrations, and within ±20% at the lower limit of quantification for all analytes. Recovery was ≥76% and reproducible. Long-term stability of patient plasma samples was demonstrated for at least 12 months at -40 °C (4 months for etravirine). Currently, this robust method with a run time of 10 min is used in clinical research and for therapeutic drug monitoring of these frequently used antiretroviral drugs.

Determination of protein-unbound, active rifampicin in serum by ultrafiltration and Ultra Performance Liquid Chromatography with UV detection. A method suitable for standard and high doses of rifampicin.

Rifampicin is the most important antibiotic in use for the treatment of tuberculosis (TB). Preclinical and clinical data suggest that higher doses of rifampicin, resulting in disproportionally higher systemic exposures to the drug, are more effective. Serum concentrations of rifampicin are the intermediary link between the dose administered and eventual response and only protein-unbound (free) rifampicin is pharmacologically active. The objective of this work was to develop an ultra performance liquid chromatography assay for protein-unbound rifampicin in serum with ultrafiltration, carried out at a sample temperature of 37°C, suitable for measurement of concentrations achieved after currently used and higher doses of rifampicin. Human serum was equilibrated at 37°C and ultrafiltrated at the same temperature in a Centrifree YM-30 ultrafiltration device, followed by dilution of the ultrafiltrate with methanol and ascorbic acid. Unbound rifampicin was analyzed using ultra performance liquid chromatography with a BEH C18 column, isocratic elution and ultra-violet (UV) detection. The run time was 5min. The assay was linear over the concentration range of 0.065-26mg/L rifampicin in ultrafiltrate. Accuracies for measurement of rifampicin in ultrafiltrate were 97% and 102% at the higher and lower limits of quantitation. Accuracy of the ultrafiltration process cannot be established, as it is not possible to spike blank serum with known amounts of protein-unbound rifampicin. Within- and between-day precision of the method including ultrafiltration as well as after ultrafiltration were within prespecified limits (CV<10%). Dilution of the ultrafiltrate with methanol and ascorbic acid kept rifampicin in solution and prevented it from degradation. Rifampicin loss during the ultrafiltration process and variation in analytical results when using two different batches of ultrafiltration devices were both limited. Processed ultrafiltrate samples were stable for 3days in the autosampler. The developed method can be applied in pharmacokinetic research, studying exposure-response relationships for rifampicin when administered at higher than currently used doses.

Lopinavir/ritonavir reduces lamotrigine plasma concentrations in healthy subjects.

BACKGROUND: Limited data are available about the effect of lopinavir and low-dose ritonavir on glucuronidation. Lamotrigine undergoes glucuronidation. We studied the effect of lopinavir/ritonavir on the pharmacokinetics of lamotrigine and vice versa. METHODS: Twenty-four healthy subjects received 50 mg lamotrigine once daily on days 1 and 2 and 100 mg twice daily on day 3 through day 23. Lopinavir (400 mg twice daily)/ritonavir (100 mg twice daily) was added on day 11. Depending on the decrease in lamotrigine trough level between days 10 and 20, either the study was stopped (<20% decrease) or a dose increase was applied from day 23 to day 31, as follows: increase to 150 mg lamotrigine twice daily if there was a 20% to 33% decrease, increase to 200 mg twice daily if there was a 34% to 66% decrease, and increase to 300 mg twice daily if there was a greater than 66% decrease. On days 10, 20, and 31, 12-hour pharmacokinetic curves were drawn. RESULTS: The mean decrease in lamotrigine trough level between days 10 and 20 was 55.4% (n = 18). A dose increment to 200 mg lamotrigine twice daily was used in all subjects. The area under the plasma concentration-time curve (AUC) values of lamotrigine on day 20 (with lopinavir/ritonavir) and day 10 (without lopinavir/ritonavir) were bioinequivalent, with a point estimate of 0.50 (90% confidence interval, 0.47-0.54). After dose adjustment of lamotrigine to 200 mg twice daily, the AUC on day 31 (n = 15) was bioequivalent to that on day 10, with a point estimate of 0.91 (90% confidence interval, 0.82-1.02). The median AUC ratios of lamotrigine 2N-glucuronide to lamotrigine on day 10 and day 20 were 0.57 (interquartile range, 0.39-0.75) and 1.12 (interquartile range, 0.87-1.31). Pharmacokinetic parameters for lopinavir/ritonavir were similar to historical controls. CONCLUSION: Lopinavir/ritonavir decreases the AUC of lamotrigine, probably by induction of glucuronidation. A dose increment to 200% of the initial lamotrigine dose is needed to achieve concentrations similar to those with lamotrigine alone. Lamotrigine does not appear to affect the pharmacokinetics of lopinavir/ritonavir.

Pharmacokinetics and safety/tolerability of higher oral and intravenous doses of rifampicin in adult tuberculous meningitis patients.

High-dose intravenous (i.v.) rifampicin improved the outcome of tuberculous meningitis (TBM) in a previous study. Unfortunately, i.v. rifampicin is not available in many high-endemic settings. This study examined exposures to and safety of higher oral rifampicin doses compared with i.v. rifampicin. Thirty adult Indonesian TBM patients were randomised to rifampicin 750 mg (ca. 17 mg/kg) orally, 900 mg (ca. 20 mg/kg) orally or 600 mg (ca. 13 mg/kg, as used previously) i.v. over 1.5 h for 14 days, combined with other TB drugs. The pharmacokinetics of rifampicin was assessed in the critical phase of TBM treatment (≤3 days after treatment initiation) and at ≥9 days. In the first days of treatment, the geometric mean (range) plasma AUC0-24 values following rifampicin 750 mg orally, 900 mg orally and 600 mg i.v. were 131.4 (38.1-275.1), 164.8 (66.9-291.2) and 145.7 (77.7-430.2) mg⋅h/L, respectively; Cmax values were 14.3 (6.1-22.2), 16.2 (5.7-28.3) and 24.7 (13.9-37.8) mg/L. CSF concentrations correlated with plasma exposures. After ≥9 days, AUC0-24 values had decreased to 100.1, 101.2 and 94.9 mg⋅h/L. Transient grade 3 ALT increases (8/30 patients) and one grade 4 ALT increase occurred, not related to rifampicin exposure. Higher oral rifampicin doses resulted in approximately similar plasma AUC0-24 but lower plasma Cmax values compared with 600 mg i.v. over 1.5 h. Exposures to rifampicin varied substantially and decreased due to autoinduction. Liver function disturbances occurred in this severely ill population. Future studies should examine even higher rifampicin doses in TBM treatment.