A Rapid High-Performance LC-MS/MS Method for Therapeutic Drug Monitoring of Voriconazole, Posaconazole, Fluconazole, and Itraconazole in Human Serum.

BACKGROUND: To achieve therapeutic efficacy and prevent toxicity simultaneously, therapeutic drug monitoring has been increasingly adopted for antifungal agents with narrow therapeutic indexes. We herein report the development and validation of an accurate, simple, fast, and cost-effective clinical test with high-performance LC-MS/MS to simultaneously quantify voriconazole, posaconazole, fluconazole, and itraconazole in human serum. METHODS: Mixed with extraction solution and internal standard, 100 µL serum samples were centrifuged for protein precipitation. Diluted supernatant was injected onto a Phenomenex® Luna C8 (2) 50 × 2 mm (3 µm) column and was analyzed with a Prominence Shimadzu high-performance liquid chromatography (HPLC) system coupled with a SCIEX QTRAP 4000 mass spectrometer in a positive ionization mode with multiple reaction monitoring. The total analytical run time was 3 min. RESULTS: The assay is linear for voricoanzole (0.01-10 µg/mL), posaconazole (0.02-40 µg/mL), fluconazole (0.2-200 µg/mL), and itraconazole (0.02-20 µg/mL). The intraday CVs ranged from 1.9% to 3.8% (n = 20); the interday CVs ranged from 2.7% to 5.4% (n = 20). Method comparison study (n = 39 or 40) demonstrated good correlation with reference laboratories (R >0.99) with average biases ranging from -7.2% to 17.5%. The recoveries for each analyte were above 90%, and matrix effects ranged from 95% to 112%. CONCLUSIONS: The method is acceptable for routine therapeutic drug monitoring of these antifungal agents in clinical laboratories for better therapeutic outcome.

A sensitive LC-MS/MS method for the quantification of urinary 8-iso-prostaglandin F2α (8-iso-PGF2α) including pediatric reference interval.

BACKGROUND: Oxidative stress has been implicated in numerous diseases, including arthritis, atherosclerosis, Alzheimer's disease, cancer, diabetes, hypertension, and inflammation. 8-iso-prostaglandin F2α, a member of the F2 isoprostane family, has been well-accepted as a valuable biomarker for the assessment of oxidative stress. METHODS: We report the development and validation of an ultra-sensitive LC-MS/MS assay for urinary 8-iso-PGF2α measurements in pediatric population. RESULTS: The assay was linear from 0.024 to 20nmol/l (R(2)=0.99). Recoveries were above 85% and matrix effects were below 5%. The variability was determined at nmol/l concentration: the intra-day variability (%CV) ranged from 3.9% to 4.5% (n=20); and the inter-day variability ranged from 4.3% to 5.7% (n=20). The accuracy of our laboratory developed test was evaluated with a clinical reference laboratory (n=39), and a correlation coefficient of 0.9257 was observed. Reference interval were established to be <0.5ng/mg creatinine in a group of pediatric population (2months-18years, n=123). CONCLUSIONS: The precision of the assay will allow for accurate assessment of oxidative stress, and is acceptable for patient testing, particularly in pediatric population.

Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS).

Cobalamin (Vitamin B12) plays an essential role both in the conversion of methylmalonyl-CoA to succinyl-CoA and in the synthesis of methionine (Met) from homocysteine (Hcy). Elevations of total homocysteine (tHcy), Met, methylmalonic acid (MMA), and 2-methylcitric acid (2MCA) are indicative of disorders in these related pathways, and can clinically present as methylmalonic acidemia, cobalamin defects or deficiency, propionic acidemia, homocystinuria, and hypermethioninemia. We have developed a fast, sensitive, and simple method for the simultaneous detection of plasma tHcy, MMA, Met, and 2MCA using liquid chromatography mass spectrometry (LC/MS/MS). All analytes were directly determined without the need of derivatization. Both positive and negative modes were used to achieve the best sensitivity and specificity. The two stereo isomers of 2MCA (2S, 3S) and (2R, 3S) were successfully separated and were designated as 2MCA1 and 2MCA2. The assays were linear up to a concentration of 800 µMol/l for tHcy, 2,000 µMol/l for Met, 80 µMol/l for MMA, 40 µMol/l for 2MCA1, and 40 µMol/l for 2MCA2 (80 µMol/l for total 2MCA), respectively. The recovery was between 84.42 % and 120.05 %. The intra-assay coefficient of variations (CVs) ranged from 2.1 % to 6.9 % (n = 20), and the inter-assay CVs ranged from 2.7 % to 11.6 % (n = 20). Reference intervals were established and verified (n = 125). A total of 15 patients with variable disorders in related pathway were successfully confirmed. The assay can be performed either in diagnostic laboratories or as second-tier, follow-up test in newborn screening laboratories.A fast, sensitive, and simple LC/MS/MS method was developed successfully for the simultaneous detection of plasma total homocysteine, methylmalonic acid, methionine, and 2-methylcitric acid for diagnosis of disorders in related pathways.