Highly Sensitive and Ultrafast Liquid Chromatography-tandem Mass Spectrometry Internal Standard Method for Determination of Alpha-Tocopherol in Serum and Application of Validated Method on Patient Serum Samples in Pakistan

Abstract

Objective: To optimize and validate a specific, sensitive and fast liquid Chromatography coupled to triple quadrupole Mass Spectro metric (LC-MS / MS) technique for accurate detection of serum α-tocopherol (Vitamin E) levels. Study Design: An experimental based study. Place and Duration of Study: The Clinical and Forensic Toxicology section of Chughtai Lab, Jail Road Lahore, from April to September 2022. Methodology: Methanol was used to deproteinize serum samples. The chromatographic separation was achieved using an Agilent Infini ty-Lab Poroshell 120EC-C18 column, Agilent 6470 LC-MS/MS (equipped with an Electron Spray Ionization source) in gradient elution mode using 0.1% LCMS grade formic acid in water and LCMS-grade methanol as mobile phases. Hexa-deuterated α-tocopherol was employed as internal standard to minimise matrix interferences. Results: The retention time of α-tocopherol was 3.0 ± 0.1 minutes. The linear concentrations obtained were ranged from 0.05-2 mg/dL with ≥0.985% coefficient of linearity. Detection and lower quantification limits determined were 0.025mg/dL and 0.05mg/dL, respec tively. Recovery ranged from 96.5 to 99.8% and ionization suppression was -15.2% and -15.9% at high and low concentrations of α-toco pherol in serum. Intra-day and inter-day coefficient variation values were 4.2-4.9% and 5.0-5.9%, respectively. Conclusion: An efficient and reliable tandem mass spectrometric technique for vitamin E analysis in serum was optimized, validated, and applied to 80 patient samples. This method has usefulness in clinical application for the accurate determination of vitamin E without potential matrix interferences.