|
Abstract INTRODUCTION:
Niacin (nicotinic acid, NA), or vitamin B3, is a required nutrient that is metabolized to nicotinamide (NAM), a constituent of the essential coenzyme nicotinamide adenine dinucleotide (NAD). NA and NAM are absorbed from food; they are also available as nutritional supplements and are used therapeutically as cholesterol-lowering agents. NA can be metabolized by conjugation with glycine to form nicotinuric acid (NUA), which causes flushing. NA deficiency can result from inadequate dietary intake, carcinoid syndrome, prolonged use of the antibiotic isoniazid, or a rare congenital defect of niacin metabolism (Hartnup's disease); severe NA deficiency is termed pellagra. Niacin toxicity has been associated with ingestion of excessive amounts of NA supplements or niacin-containing energy drinks. Our aim was to develop an LC-MS/MS assay for NA and its metabolites, NAM and NUA, and to evaluate the methods’ performance.
METHODS:
Aliquots of human serum or plasma samples (50 µL), stable isotope-labeled internal standard mix (IS, nicotinic acid-13C6, nicotinamide-13C6, and nicotinuric acid-d4, 20 µL) and 250 µL of 2% formic acid were added to the wells of a 96-well plate; the plate was sealed and vortexed for 5 min. The adsorbent in wells of the cation exchange solid phase extraction (SPE) plate (Biotage) was preconditioned with methanol and 2% formic acid; samples were loaded and the adsorbent was washed with acidified water, followed by acidified methanol. The analytes were eluted using 10% ammonium hydroxide in acetonitrile; the extracts were dried and reconstituted. Instrumental analysis was performed on an Agilent series 6475 triple quadrupole mass spectrometer with electrospray ionization, using multiple reaction monitoring acquisition in positive ion mode. HPLC separation was accomplished using a Kinetex Biphenyl 2.6 µm, 3.0 x 50 mm column (Phenomenex) at 30 °C, flow rate 0.35 mL/min; injection volume 2µL. Injection to injection time was 7.5 min. Quantification was performed using a six-point calibration curve (40 – 4,000 nmol/L), with two mass transitions monitored for each analyte and IS. Evaluation of the method’s performance included assessment of precision, sensitivity, linearity, accuracy, specificity, matrix effects, dilution integrity, carryover, and correlation with a validated method of another laboratory. Blood collection containers and stability of the analytes were evaluated.
RESULTS:
We developed a method for quantifying NA, NAM, and NUA in plasma samples using LC-MS/MS and evaluated its analytical performance. The issues of poor analyte retention by SPE adsorbents and chromatographic columns were resolved through optimization of sample preparation and chromatographic separation. Given the highly polar nature of the analytes, cation exchange SPE was used for sample preparation. Due to the sensitivity of highly polar molecules' ionization to pH changes, which affects their separation using SPE, extraction recovery was optimized by maintaining tight pH control throughout all steps of sample preparation. The Kinetex biphenyl column provided the best chromatographic performance with adequate retention and separation of NA, NAM, and NUA peaks. The linear range of the assay for NA, NAM, and NUA was 40 to 4,000 nmol/L. Within-run imprecision for analysis of two plasma sample pools analyzed on one day in 10 replicates was <5%. Between-run imprecision for analysis of three plasma sample pools over nine runs was <10%. Comparison of 200 patient plasma specimens with a validated method of another laboratory showed 169 sample concentrations within the analytical measurement range for NAM (Deming regression y = 0.988*x – 14.5 nmol/L, R = 0.963). Because of the short half-lives of NA and NUA, the majority of the samples did not contain measurable amounts of NA or NUA. Out of the entire set, one sample contained NA at 157 nmol/L (130 nmol/L measured by the comparative method). NUA was present in two of the samples; the measured concentrations were 177 nmol/L and 507 nmol/L (233 nmol/L and 611 nmol/L, respectively, when measured by the comparative method). Evaluation of method accuracy for the targeted analytes using four human plasma sample pools spiked with 200 and 800 nmol/L, demonstrated within 10% agreement between the expected and the observed concentrations. We observed differences in NAM concentrations between serum and plasma samples collected from the same individuals, with lower concentrations measured in serum samples; no difference in the measured concentrations of NA and NUA was observed.
CONCLUSION:
An LC-MS/MS method was developed for quantifying vitamin B3 and its metabolites (NA, NAM and NUA) in plasma samples. Evaluation of the method performance demonstrated reliable analytical performance with adequate precision, accuracy, and robustness. NAM was present at measurable concentrations in ~70% of patient samples tested; NA and NUA were present in ≤1% of the analyzed specimens. We observed reasonably good agreement in quantitation of the three analytes with a validated method of another laboratory.
|
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
