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.
Clinical risk assessment of biotin interference with a high-sensitivity cardiac troponin T assay
Abstract Objectives Biotin >20.0 ng/mL (81.8 nmol/L) can reduce Elecsys® Troponin T Gen 5 (TnT Gen 5; Roche Diagnostics) assay recovery, potentially leading to false-negative results in patients with suspected acute myocardial infarction (AMI). We aimed to determine the prevalence of elevated biotin and AMI misclassification risk from biotin interference with the TnT Gen 5 assay. Methods Biotin was measured using an Elecsys assay in two cohorts: (i) 797 0-h and 646 3-h samples from 850 US emergency department patients with suspected acute coronary syndrome (ACS); (ii) 2023 random samples from a US laboratory network, in which biotin distributions were extrapolated for higher values using pharmacokinetic modeling. Biotin >20.0 ng/mL (81.8 nmol/L) prevalence and biotin 99th percentile values were calculated. AMI misclassification risk due to biotin interference with the TnT Gen 5 assay was modeled using different assay cutoffs and test timepoints. Results ACS cohort: 1/797 (0.13%) 0-h and 1/646 (0.15%) 3-h samples had biotin >20.0 ng/mL (81.8 nmol/L); 99th percentile biotin was 2.62 ng/mL (10.7 nmol/L; 0-h) and 2.38 ng/mL (9.74 nmol/L; 3-h). Using conservative assumptions, the likelihood of false-negative AMI prediction due to biotin interference was 0.026% (0-h result; 19 ng/L TnT Gen 5 assay cutoff). US laboratory cohort: 15/2023 (0.74%) samples had biotin >20.0 ng/mL (81.8 nmol/L); 99th percentile biotin was 16.6 ng/mL (68.0 nmol/L). Misclassification risk due to biotin interference (19 ng/L TnT Gen 5 assay cutoff) was 0.025% (0-h), 0.0064% (1-h), 0.00048% (3-h), and <0.00001% (6-h). Conclusions Biotin interference has minimal impact on the TnT Gen 5 assay’s clinical utility, and the likelihood of false-negative AMI prediction is extremely low.
Characterization of a sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate in human placental choriocarcinoma cells.
The characteristics of the uptake of the vitamin pantothenate into JAr human placental choriocarcinoma cells were investigated and these cells were found to accumulate the vitamin against a concentration gradient by a Na(+)-dependent process. Substitution of Na+ with over other monovalent cations abolished the uptake completely. The transport process showed no preference for any particular anion. Kinetic analysis indicated the presence of a single saturable transport system with a Michaelis-Menten constant of 2.1 +/- 0.2 microM and a maximal velocity of 341 +/- 12 pmol/mg of protein per 10 min. The dependence of the uptake rate of pantothenate on Na+ concentration exhibited sigmoidal kinetics, indicating interaction of more than one Na+ ion with the transporter. The Hill coefficient for this process was calculated to be 1.6. The Na+/pantothenate coupling ratio being greater than unity suggests that the transport process is electrogenic, resulting in net transfer of positive charge across the membrane. This was confirmed in plasma membrane vesicles prepared from JAr cells where the uptake of pantothenate was found to be significantly stimulated by valinomycin-induced inside-negative K(+)-diffusion potential. Substrate specificity studies showed that, in addition to pantothenate, the transporter interacts with two other vitamins, namely biotin and lipoate. The characteristics of pantothenate uptake in the placental cell line BeWo was also investigated. These cells were also found to express a pantothenate transport system similar to that expressed in the JAr cells.