The inaccuracy of routine serum 25-hydroxyvitamin D measurements hampers the interpretation of data in patient care and public health research. We developed and validated a candidate reference measurement procedure (RMP) for highly accurate quantitation of two clinically important 25-hydroxyvitamin D metabolites in serum, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d3-25(OH)D2 and d6-25(OH)D3] were extracted from serum via liquid-liquid extraction. The featured isotope-dilution LC-MS/MS method used reversed-phase chromatography and atmospheric pressure chemical ionization in positive ion mode. A pentafluorophenylpropyl-packed UHPLC column together with isocratic elution allowed for complete baseline resolution of 25(OH)D2 and 25(OH)D3 from their structural C-3 isomers within 12 min. We evaluated method trueness, precision, potential interferences, matrix effects, limits of quantitation, and measurement uncertainty. Calibration materials were, or were traceable to, NIST Standard Reference Materials 2972. Within-day and total imprecision (CV) averaged 1.9% and 2.0% for 25(OH)D3, respectively, and 2.4% and 3.5% for 25(OH)D2, respectively. Mean trueness was 100.4% for 25(OH)D3 and 100.3% for 25(OH)D2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D3 and 1.46/0.13 nmol/L for 25(OH)D2. When we compared our RMP results to an established RMP using 40 serum samples, we found a nonsignificant mean bias of 0.2% for total 25(OH)D. This candidate RMP for 25(OH)D metabolites meets predefined method performance specifications (≤5% total CV and ≤1.7% bias) and provides sufficient sample throughput to meet the needs of the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program.

Most transcription factors were for a long time considered as undruggable targets due to the absence of binding pockets for direct targeting. HOXA9, implicated in acute myeloid leukemia, is one of them. To date, only indirect targeting of HOXA9 expression or multi-target HOX/PBX

A series of novel benzimidazole diamidines were prepared from the corresponding dicyano analogues either by applying Pinner methodology (5a-c, 10 and 13a) or by making amidoximes intermediates that were reduced to the corresponding amidines (15a-c). The new amidines were evaluated in vitro against the protozoan parasite Trypanosoma brucei rhodesiense (T. b. r.). The thiophene analogue 5b and the N-methyl compound 15a showed superior antitrypanosomal activity compared to that of the parent I.

Methylmalonic acid (MMA), a functional indicator of vitamin B12 insufficiency, was measured in the U.S. population in the National Health and Nutrition Examination Survey (NHANES) from 1999–2004 using a GC/MS procedure that required 275 µL of sample and had a low throughput (36 samples/run). Our objective was to introduce a more efficient, yet highly accurate LC-MS/MS method for NHANES 2011–2014. We adapted the sample preparation with some modifications from a published isotope-dilution LC-MS/MS procedure. The procedure utilized liquid-liquid extraction and generation of MMA di-butyl ester. Reversed-phase chromatography with isocratic elution allowed baseline resolution of MMA from its naturally occurring structural isomer succinic acid within 4.5 min. Our new method afforded an increased throughput (≤160 samples/run) and measured serum MMA with high sensitivity (LOD = 22.1 nmol/L) in only 75 µL of sample. Mean (±SD) recovery of MMA spiked into serum (2 days, 4 levels, 2 replicates each) was 94±5.5%. Total imprecision (41 days, 2 replicates each) for three serum quality control pools was 4.9–7.9% (97.1–548 nmol/L). The LC-MS/MS method showed excellent correlation (n=326, r=0.99) and no bias (Deming regression, Bland-Altman analysis) compared to the previous GC/MS method. Both methods produced virtually identical mean (±SD) MMA concentrations [LC-MS/MS: 18.47±0.71 ng/mL (n=17), GC/MS: 18.18±0.67 ng/mL (n=11)] on a future plasma reference material compared to a GC/MS method procedure from the National Institute of Standards and Technology [18.41±0.70 ng/mL (n=15)]. No adjustment will be necessary to compare previous (1999–2004) to future (2011–2014) NHANES MMA data.