Simultaneous elemental detection of F and Cl offers quantitation of fluorinated and chlorinated compounds and their transformation products without compound-specific standards. Despite wide-ranging applications, this capability has been hindered by fundamental and technical shortcomings of current inductively coupled plasma (ICP)-MS methods in ion formation and isobaric interference elimination. These hurdles are alleviated here via a chemical ionization method. Fluorine and chlorine in analytes are first converted to HF and HCl by an ICP with post-plasma recombination and subsequently react with barium-containing ions supplied by a nanospray, yielding BaF + and BaCl + as elemental reporter ions. Notably, the method is readily interfaced to an Orbitrap MS which eliminates isobaric interferences at resolving powers as low as 35,000, far greater than that of current ICP−MS instruments. Moreover, the instrument is easily reverted to the ESI-MS mode for complementary molecular characterization. To demonstrate analytical capabilities, a workflow for rapid quantitation of compounds separated by reversed-phase liquid chromatography is developed using a species-independent calibration. The independent F and Cl measurements agree with each other, providing recoveries of >90% and LODs of 8−12 pmol Cl and 5− 12 pmol F on the column. The workflow along with LC-ESI-MS on the same instrument is then applied to identify and quantify invitro drug metabolites, yielding total drug-related material recoveries of >80% and quantitation of minor metabolites summing to 8% of the total drug-related compounds. These results highlight the strengths of simultaneous F and Cl speciation for rapid quantitation with applications in early drug development.
An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatographytandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D 2 (25(OH)D 2 ) and 25-hydroxyvitamin D 3 (25(OH)D 3 ). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D 2 , 25(OH)D 3 , 3-epi-25hydroxyvitamin D 3 (3-epi-25(OH)D 3 ), and 24R,25-dihydroxyvitamin D 3 (24R,25(OH) 2 D 3 ) using isotope dilution liquid chromatographytandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 1 includes results from 14 laboratories using 14 custom LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D 3 . The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D 3 and 25(OH)D 3 on assay performance, particularly with regard to mean % bias.
KeywordsTotal serum 25-hydroxyvitamin D (25(OH)D) . 25-Hydroxyvitamin D 3 (25(OH)D 3 ) . 25-Hydroxyvitamin D 2 (25(OH)D 2 ) . 3-epi-25-Hydroxyvitamin D 3 (3-epi-25(OH)D 3 ) . 24R,25-Dihydroxyvitamin D 3 (24R,25(OH) 3 D 3 ) . Liquid chromatographytandem mass spectrometry (LC-MS/MS) * Stephen A. Wise
An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of ligand binding assays (Part 2) for the determination of serum total 25-hydroxyvitamin D [25(OH)D]. Fifty single-donor samples were assigned target values for concentrations of 25-hydroxyvitamin D 2 [25(OH)D 2 ], 25-hydroxyvitamin D 3 [25(OH)D 3 ], 3-epi-25-hydroxyvitamin D 3 [3-epi-25(OH)D 3 ], and 24R,25-dihydroxyvitamin D 3 [24R,25(OH) 2 D 3 ] using isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 2 includes results from 17 laboratories using 32 ligand binding assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 50% of the ligand binding assays achieved the VDSP criterion of mean % bias ≤ |± 5%|. For the 13 unique ligand binding assays evaluated in Published in the topical collection celebrating ABCs 20th Anniversary.
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