Method validation and clinical application for the quantification of lopinavir, efavirenz, and ritonavir in breast milk using liquid chromatography tandem mass spectrometry.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been validated for quantification of three antiretroviral drugs (efavirenz [EFV], lopinavir [LPV], and ritonavir [RTV]) from human breast milk. The samples were extracted employing protein precipitation method using methanol as precipitating agent. The supernatant was evaporated and reconstituted before injecting into the chromatograph and separated on a biphenyl column. Calibration curves for the three tested antiretroviral drugs were linear (r ≥ 0.999) over the range examined. The inter- and intra-day coefficients of variation (CV) were ≤15% for efavirenz, lopinavir, and ritonavir. Mean recovery ranged from 96% to 105% and no major matrix effects were observed. This validated LC-MS/MS method was efficiently applied to determine EFV, LPV, and RTV concentrations in breast milk from Human Immunodeficiency Virus (HIV)-positive breastfeeding mothers. This assay requires a simple sample processing method with a short run time, making it well suited for high-throughput routine clinical or research purposes.

Evaluation of 4 quantification methods for monitoring 16 antibiotics and 1 beta-lactamase inhibitor in human serum by high-performance liquid chromatography with tandem mass spectrometry detection.

Antibiotic (ATB) prescription in an intensive care unit (ICU) requires continuous monitoring of serum dosages due to the patient's pathophysiological condition. Dosing adjustment is necessary to achieve effective targeted concentrations. Since ICUs routinely use a large number of ATBs, global monitoring needs to be developed. In the present study, we developed a global analytical method for extracting, separating and quantifying the most widely used ATBs in ICUs: amoxicillin, piperacillin, cefazolin, cefepime, cefotaxime, ceftazidime, ceftolozane, ceftriaxone, ertapenem, meropenem, ciprofloxacin, moxifloxacin, levofloxacin, daptomycin, dalbavancin, linezolid and a beta-lactamase inhibitor: tazobactam. To guarantee the robustness of the quantification, we differentiated the 16 ATBs and the beta lactamase inhibitor into 4 pools (ATB1 to ATB4), taking into account prescription frequency in the ICU, the physicochemical properties and the calibration ranges of the ATBs selected. The whole ATB was then separated with two LC columns in reversed phase: Kinetex Polar-C18 100 Å and Polar-RP-80 synergy, in less than 6.5 min. Detection was carried out by electrospray in positive ion mode, by tandem mass spectrometry (LC-MS/MS. The four quantification methods were validated according to the European guidelines on bioanalytical method validation (EMEA guide), after determining the extraction yields, matrix effects, recovery, precision, accuracy, within-run precision and between-run precision. For all analyses, bias is < 15% and is comparable to the literature and LOQs vary from 0.05 mg.L-1 for ciprofloxacin to 1.00 mg.L-1 for ceftriaxone and dalbavancin. The stability time of cefepime and piperacillin is 3 hrs and for the other ATBs 6 hrs in serum at room temperature. For long-term stability, freezing at - 80 °C guarantees 3 months of stability for ceftriaxone and dalbavancin and more than 6 months for the other ATBs.

Is the unbound concentration of atazanavir of interest in therapeutic drug monitoring?

To date, therapeutic drug monitoring (TDM) is carried out with antiretrovirals and is usually based on total concentrations (Ct ). However, for some patients, TDM does not reflect efficacy or the avoidance of toxicity as is the case for atazanavir (ATV), a HIV protease inhibitor. As the unbound concentration (Cu ) is the pharmacological active form, the aim of the study was to evaluate the value of Cu and the unbound fraction (fu , fu = Cu /Ct ) for the TDM of ATV. The variability of Cu and the corresponding fu of ATV was explored in 43 patients treated with ATV for an average of 13.5 months. Cu was determined by coupling ultrafiltration and liquid chromatography. As ATV is highly bound to alpha-1 acid glycoprotein (AAG), the correlation between fu and AAG was also evaluated. The viral load was monitored to evaluate the patients' virologic response, while total plasma bilirubin and unconjugated plasma bilirubin were used as biomarkers of ATV toxicity. Median trough Cu and Ct were 37.9 µg/L (Interquartile range (IQR) 20.6-94.9 µg/L) and 628.6 µg/L (IQR 362.7-1078.1 µg/L), respectively. fu , Cu and Ct showed high variability, but the fu variability was not correlated with the AAG level. The unbound concentration and fraction were unrelated to the virologic response (P = 0.21 and P = 0.65 for Cu and fu , respectively) nor to the unconjugated bilirubin (Pearson correlation coefficient (ρ), ρ = 0.22; P = 0.17 for Cu ). Neither total nor unbound concentrations of ATV fully explained hyperbilirubinaemia or virologic failure. From this study, we conclude that unbound ATV did not appear to be more relevant than Ct .

Quinine unbound concentration is the best marker for therapeutic drug monitoring.

Quinine monitoring should be based on unbound concentration due to variable unbound fraction in malaria patients.

[Tuberculosis: relevance of isoniazid dosage in prevention of liver side effects]. / Tuberculose: intérêt du dosage de l'isoniazide dans la prévention des effets hépatotoxiques.

OBJECTIVE: Several recent studies have established a correlation between NAT2 polymorphism and hepatotoxicity induced by isoniazid. The objective of this work was to assess the place of isoniazid dosage, marker of acetylation phenotype, in clinical practice in the department of Haute-Garonne. METHODS: Data from reportable disease of tuberculosis and the results of isoniazid dosage performed at the pharmacokinetics and clinical toxicology laboratory were used during the period 2009-2012. RESULTS: The current practice of dosage is far from being systematical: only 3.9% of patients who developed tuberculosis have benefited from isoniazid dosage. The isoniazid initial posology was adapted to the acetylation capacity for only 33.3% of patients. CONCLUSION: A decision tree was realized and used to identify populations (low metabolism) liable to benefit from isoniazid dosage.

Determination of plasma unbound fraction of voriconazole in patients treated with a prophylactic or a curative treatment.

BACKGROUND: Voriconazole (VOR) is a triazole antifungal used in the curative treatment of invasive fungal infections and the prophylactic treatment of opportunistic fungal infections in immunocompromised patients. It is a drug for which therapeutic drug monitoring (TDM) is highly recommended. METHODS: To determine the best TDM marker, the pharmacologically active form of the drug, represented by the plasma unbound concentration (Cu) and fraction (fu), has been studied using a method based on ultrafiltration and ultra performance liquid chromatography. As albumin (Alb) is a likely factor inducing fluctuations in fu, the correlation between Alb levels and fu was carried out. Similarly, correlations between trough plasma concentrations [total concentration (Ct) and Cu] and both efficacy and safety markers were determined. Efficacy evaluation was based on monitoring fungal antigens and cultures, whereas safety was monitored by measuring bilirubin levels. RESULTS: In vitro, using blank human plasma, the mean fu was determined at 32.3% ± 5.5%, whereas in patients' plasmas treated with VOR, the median (5th-95th percentiles) of the unbound VOR fraction was 22.95% (14.95%-38.42%). A high correlation was found (rho = 0.956, P < 0.001) between Ct and Cu, though there was no correlation between serum Alb levels and fu, except for some patients with severe hypoalbuminemia (<25 g/L). CONCLUSIONS: Based on the efficacy/safety correlations, a therapeutic window has been defined ranging from 4.5 to 6.5 mg/L and 1.5 and 2.0 mg/L for trough Ct and Cu, respectively. For the first time, the relevance of new pharmacokinetic parameters, such as Cu and fu, has been explored and discussed, and our results support the current TDM protocol for VOR.

Ibuprofen concentrations in human mature milk--first data about pharmacokinetics study in breast milk with AOR-10127 "Antalait" study.

BACKGROUND: Analgesics are one of the most prescribed drugs during the postpartum period to prevent and treat pain and inflammatory disease. The focus on analgesics during breastfeeding has increased because of lack of information and fatal codeine intoxication in a breastfed neonate. Ibuprofen has an advantageous benefit-risk ratio profile compared with codeine. There is a lack of information on drug transfer into human milk, thus ibuprofen intake during breastfeeding may be debated. Consequently, there is a dilemma whether to terminate breastfeeding or drug therapy. The objective of this study was to determine the relative infant dose of ibuprofen. METHODS: The first week after the delivery, each woman received ibuprofen to treat pain or inflammatory disorders (mean dose, 1012 ± 96 mg/d). Just after the third dose of ibuprofen, 1 milk sample and 2 blood samples were obtained after 1 week of breastfeeding. Ibuprofen concentrations in breast milk and blood were measured by using high-performance liquid chromatography. RESULTS: Twenty women were included after written informed consent, and 13 gave their breast milk and blood samples. The mean ibuprofen milk concentration was 360 ± 160 mcg/L. The mean fat milk concentration was 3.23 ± 1.15 g per 100 mL, and the mean milk protein concentration 0.87 ± 0.27 g per 100 mL. The ibuprofen transfer infant dose (theoretical infant dose) was 68 mcg·kg-1·d-1 (8-262 mcg·kg-1·d-1), and the relative infant dose was <0.38% (0.04%-1.53%) of the weight-adjusted maternal daily dose, which equals 0.2% of the infant dose. CONCLUSIONS: The results confirm that the transfer of ibuprofen into breast milk decreases with the protein concentration and the duration of lactation. These results suggest that the use of ibuprofen is compatible with prolonged breastfeeding after the early postpartum stage.

Influence of pre-analytical conditions on plasma ribavirin concentrations.

Ribavirin (CAS 66510-90-5) associated to peginterferon (CAS 99210-65-8) is the current standard treatment for chronic hepatitis C. Exposure to ribavirin influences the virological response and anemia. Therefore monitoring plasma concentration of ribavirin is a useful tool for individualizing ribavirin dosing regimens. Ribavirin is a substrate of several nucleoside transporters that play a role in its distribution in erythrocytes. After blood sampling, it is essential to limit this mechanism. The aim of this study was to evaluate the influence of temperature and time on ribavirin plasma concentrations. Two blood samples, collected in EDTA tubes, were taken at the same time from 23 patients. One sample was conserved on ice whereas the second one was kept at room temperature during transport to the laboratory. Upon receipt at the laboratory and at different times post-reception (from 1 to 3 h), 1.5 mL of blood from each sample was centrifuged to obtain plasma that was then stored at -20 degrees C until assay. Samples were maintained in the same conditions as during transport for the 3 h. Plasma ribavirin was analysed using an HPLC-UV system. The results showed that mean loss of ribavirin concentration, for samples kept on ice as well as at room temperature, was less than 3%, 9% and 13% after 1, 2 and 3 h, respectively. These results suggest that blood samples for ribavirin analysis can be sent at room temperature within a period of 2 h between sampling and centrifugation.