A simple and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry method to determine plasma biotin in hemodialysis patients.
A simple, rapid, and selective method for determination of plasma biotin was developed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After single-step protein precipitation with methanol, biotin and stable isotope-labeled biotin as an internal standard (IS) were chromatographed on a pentafluorophenyl stationary-phase column (2.1 × 100 mm, 2.7 μm) under isocratic conditions using 10 mm ammonium formate-acetonitrile (93:7, v/v) at a flow rate of 0.6 mL/min. The total chromatographic runtime was 5 min for each injection. Detection was performed in a positive electrospray ionization mode by monitoring selected ion transitions at m/z 245.1/227.0 and 249.1/231.0 for biotin and the IS, respectively. The calibration curve was linear in the range of 0.05-2 ng/mL using 300 μL of plasma. The intra- and inter-day precisions were all <7.1%. The accuracy varied from -0.7 to 8.2%. The developed UHPLC-MS/MS method was successfully applied to determine plasma biotin concentrations in hemodialysis patients. Copyright © 2016 John Wiley & Sons, Ltd.
Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column.
A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1h at 120 degrees C and the recovery for biocytin was 65.7+/-2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, "natto", rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.
Concentrations of biotin metabolites in human milk.
Because estimates of the biotin requirement for infants currently are based on the biotin concentration in human milk, we sought to determine whether inactive biotin metabolites are present. Samples were collected for 7 weeks post partum from 15 healthy women. Biotin and the inactive metabolites bisnorbiotin and biotin sulfoxide were measured by means of a high-performance liquid chromatography avidin-binding assay. At 8 days post partum the proportion of biotin was 44%, bisnorbiotin 48%, and biotin sulfoxide 8%. Although biotin content increased post partum (p < 0.003), the metabolites remained an important portion of the total providing evidence that accurate measurement of biotin in human milk requires an assay that is specific for biotin.