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.

OBJECTIVE

To optimize and validate a specific, sensitive and fast liquid Chromatography coupled to triple quadrupole Mass Spectrometric (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 Infinity-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, respectively. Recovery ranged from 96.5 to 99.8% and ionization suppression was -15.2% and -15.9% at high and low concentrations of α-tocopherol 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.

KEY WORDS

Vitamin E, LC-MS/MS, Tocopherol, Internal standard, Validation.