Quantification of letermovir in human serum using high-performance liquid chromatography with diode array detection.

A simple, rapid, cost-effective and sensitive high-performance liquid chromatography method with diode array detection was developed and validated for the quantification of letermovir, a compound approved for prophylaxis of cytomegalovirus infection and disease in adult recipients of an allogeneic hematopoietic stem cell transplant. Sorafenib was used as internal standard. Samples were pre-treated by liquid-liquid extraction with tert-butyl methylether. Separation was achieved on a XTerra® RP18 column (150 × 2.1 mm, 5 µm) at 30 °C using gradient elution with a mobile phase of 20 mM ammonium bicarbonate pH 7.9 (mobile phase A) and acetonitrile:20 mM ammonium bicarbonate (9:1 v/v) (mobile phase B). Samples were eluted at a flow rate of 0.3 mL/min throughout the 20-min run. UV wavelength mode was used, letermovir and sorafenib were monitored at 260 nm. The calibration curve was linear in a concentration range of 25-5000 ng/mL with correlation coefficients ≥ 0.99. Intra-day and inter-day accuracy expressed as relative error were -11.4-20% and -7.96-10.62%, respectively. Precision expressed as coefficient of variation was 1.44-3.15% (intra-day) and 1.17-1.93% (inter-day). The method was successfully applied for analysis of 128 letermovir levels demonstrating its usefulness for letermovir monitoring in routine clinical practice.

Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma.

OBJECTIVE: Oral mitotane (o,p'-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC). AIM: Serum mitotane concentrations >14  mg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p'-dichlorodiphenylacetic acid (o,p'-DDA) and o,p'-dichlorodiphenyldichloroethane (o,p'-DDE). DESIGN: Lipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI-H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins. RESULTS: Chyle of the index patient contained 197  mg/ml mitotane, 53  mg/ml o,p'-DDA, and 51  mg/l o,p'-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction. DISCUSSION: Mitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p'-DDE was similarly distributed, but 87.9±4.2% of o,p'-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI-H295 cells and reduced ER stress marker gene expression. CONCLUSION: Mitotane absorption involves chylomicron binding. High concentrations of o,p'-DDA and o,p'-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.

Impact of benzodiazepines on posaconazole serum concentrations. A population-based pharmacokinetic study on drug interaction.

OBJECTIVES: Posaconazole is broadly used for antifungal prophylaxis and therapy. Current data suggest a concentration-dependent effect. Unlike other triazoles, cytochrome P450 is not a relevant route of biotransformation for posaconazole but glucuronidation, which might lead to a different spectrum of drug interactions. For benzodiazepines, the major metabolic pathway involves oxidation, but some, including lorazepam and temazepam, undergo conjugation to glucuronic acid. RESEARCH DESIGN AND METHODS: Since 2006 serum levels of posaconazole are determined regularly in all hospitalized patients with intake of this triazole. Here we investigate posaconazole concentration at steady state in relation to the concomitant medication of benzodiazepines. RESULTS: While similar posaconazole concentrations were determined in samples obtained from patients receiving temazepam when compared to samples without any benzodiazepine, a relevant reduction of posaconazole concentration could be observed in patients with concomitant intake of lorazepam. This difference in posaconazole concentration with or without concomittant intake of lorazepam, was consistently significant for analyses of all samples (median 336 ng/ml vs. 585 ng/ml, p 0.001), for the average concentrations (569 ng/ml vs. 276 ng/ml, p 0.039), and for patients receiving a total daily dose of 800 mg posaconazole (292 ng/ml vs. 537 ng/ml, p 0.003). There was also a similar, but not significant trend for patients with a prophylactic dosage of 200 mg posaconazole three times daily (689 ng/ml vs. 512 ng/ml, p 0.186). CONCLUSIONS: In this retrospective study, analyzing blood samples from daily clinical practice of patients in various clinical settings and with different indications for antifungal therapy, concomitant medication of lorazepam was associated with decreased posaconazole concentrations. Therefore, lorazepam but not temazepam might induce posaconazole clearance by glucuronidation.

Relevance of timing for determination of posaconazole plasma concentrations.

For posaconazole, drug monitoring is suggested, but the relevance of timing for the determination of posaconazole concentration (PC) remains unclear. We investigated the variation of PC before and 4 and 8 h after the administration of 400 mg of posaconazole. Mean concentrations were 645, 678, and 616 ng/ml. The differences between trough and maximum concentrations were below 20% in 17 and below 30% in 20 of 25 patients. Hence, untimed posaconazole plasma concentrations give reliable information for most patients.

High-performance liquid chromatographic method for the determination of sorafenib in human serum and peritoneal fluid.

PURPOSE: Sorafenib is recommended for therapy of advanced hepatocellular carcinoma and renal cell carcinoma. Preclinical data indicate a relation between dose and antitumor efficacy. In clinical trials, adverse events improve after dose reduction suggesting a dose-dependent toxicity. Given dose has a direct impact on the drug serum concentration, but the latter also can be influenced by multiple factors, including interaction and metabolisation. To enable the investigation of concentration-related effects, an easy and sensitive assay for sorafenib drug monitoring is essential. METHODS: A high-performance liquid chromatography (HPLC) analysis involving an extraction with diethyl ether followed by separation on a Pinnacle™ DB C18 column and quantitation by UV absorbance at 260 nm was established. Sorafenib concentrations in samples of serum and peritoneal fluid have been determined. RESULTS: The assay was validated for serum samples and is linear over the concentration range of 100-5,000 ng/ml with a determination coefficient of >0.999. The limit of detection is 0.25 ng/ml. The intra- and inter-day coefficients of variation were below 3.03%. Sorafenib recovery in spiked probes of peritoneal fluid was above 85%. Sorafenib concentrations in 44 serum samples and 14 probes of peritoneal fluid have been determined with a mean of 3,328 and 1,380 ng/ml, respectively (standard deviation 2,267 and 659 ng/ml). CONCLUSIONS: A sensitive and selective HPLC method for the determination of sorafenib in human serum was developed and also verified for peritoneal fluid. This method provides a useful tool for pharmacokinetic investigations as well as for therapeutic drug monitoring of sorafenib.

Simultaneous determination of voriconazole and posaconazole concentrations in human plasma by high-performance liquid chromatography.

A simple, sensitive, and selective high-performance liquid chromatographic method for the simultaneous determination of voriconazole and posaconazole concentrations in human plasma was developed and validated. Quantitative recovery following liquid-liquid extraction with diethyl ether was achieved. Linearity ranged from 0.10 to 20.0 microg/ml for voriconazole and from 0.05 to 10.0 microg/ml for posaconazole. The intra- and interday coefficients of variation were less than 8.5%, and the lower limits of quantitation were <0.05 microg/ml.

Drug monitoring of pyrimethamine during maintenance therapy of toxoplasmic encephalitis in patients with advanced HIV infection during HAART.

BACKGROUND: Prophylaxis of toxoplasmic encephalitis was performed with pyrimethamine in 6 patients with advanced HIV infection during combination therapy with protease inhibitors. MATERIAL/METHODS: Steady-state plasma pyrimethamine (PYR) levels were measured by gas chromatography. Protease inhibitor plasma concentrations were analyzed trough concentration by high pressure liquid chromatography. During a treatment period of 22I13 months a total of 93 samples from 6 patients were investigated, containing pyrimethamine and protease inhibitors. RESULTS: The mean pyrimethamine concentration was 1,108+/-459 ng/ml with a dosage of 37.5 mg/d and 1, 685+/-665 ng/ml with 50 mg/d. With the simultaneous use of indinavir (IDV), a mean pyrimethamine concentration of 2,165+/-273 ng/ml was found, significantly higher than in combination with saquinavir (SQV) and ritonavir (RTV) (1,192+/-178 ng/ml) or saquinavir and nelfinavir (NLV) (1,117+/-173 ng/ml). This effect was not considered clinically significant. Drug monitoring of protease inhibitors revealed a wide range of protease inhibitor levels. CONCLUSIONS: The statistically significant lower PYR concentrations with the SQV + RTV or SQV + NLV comedication in comparison to comedication with IDV were not considered clinically significant. Therapeutic drug monitoring of PYR and PI plasma levels can be recommended in patients during therapy with both PYR and PI, especially during therapy with a double PI regimen.

Highly sensitive gas chromatographic determination of ethanol in human urine samples.

In order to evaluate recent alcohol consumption, a very sensitive and specific gas chromatographic method for ethanol determination in human urine samples was developed. The non-invasive method was performed without any pretreatment and carried out on a Stabilwax capillary column, 30 m x 0.53 mm x 1.0 microm film thickness. Helium was used as carrier gas with a constant inlet pressure of 27.72 kPa (0.277 bar) and a flame ionization detector (FID). Quantification was performed with the use of acetonitrile as an internal standard (IS). The calibration curve was linear throughout the concentration range from 0.5 to 500 mg/l. The calculated intra- and inter-day coefficients of variation were below 8%. A clear chromatographic separation of ethanol from methanol, acetone, 1-propanol and 2-propanol was achieved.

Rapid determination of nevirapine in human plasma by gas chromatography.

A sensitive and rapid gas chromatographic method has been developed to determine the levels of the HIV-1 non-nucleoside reverse transcriptase inhibitor nevirapine in human plasma. Quantitative recovery following liquid-liquid-extraction with diethylether from 500 microl of human plasma was achieved. Subsequently, the assay was performed with a CP-Sil 5CB capillary column, 15 m x 0.32 mm x 1.0 microm film thickness with a nitrogen-phosphorous-detector (NPD), Helium 5.0 was used as carrier gas with a constant inlet pressure of 7 p.s.i. Linear standard curves were obtained for concentrations ranging from 10 to 20 000 ng/ml. The calculated intra- and inter-day coefficients of variation were below 8%.