Abstract:

Dry eye is characterized as a multifactorial disorder affecting the tear film and ocular surface， leading to symptoms of discomfort and visual disturbances， with the potential for ocular surface damage. It is among the most prevalent ocular surface disorders， with an estimated prevalence ranging from 5.0% to 50.0%， and an overall prevalence between 21.0% and 50.4% in China. Despite its high global prevalence， the fundamental mechanisms underlying the pathology of dry eye remain largely unexplored. Vitamins are crucial for maintaining ocular surface homeostasis， with vitamins A and E being physiologically present and essential for cell differentiation， development， and proper function. Deficiencies in these vitamins are frequently associated with dry eye， and systemic supplementation has been shown to benefit patients with low vitamin intake. Vitamins A and E may have potential clinical predictive value for the diagnosis and treatment of dry eye. In this study， a liquid chromatography-tandem mass spectrometry （LC-MS/MS） method was developed to simultaneously measure the concentrations of vitamins A and E in human tears. A Phenomenex Kinetex C18 column was employed for gradient elution at a flow rate of 0.4 mL/min. The mobile phase A consisted of a 0.1% formic acid aqueous solution， while mobile phase B comprised a 2 mmol/L ammonium acetate and 0.1% formic acid methanol solution. Positive atmospheric pressure chemical ionization in multiple reaction monitoring mode was utilized for MS/MS detection. The analytical method for vitamin A demonstrated a validated linear range of 5.00-300.00 ng/mL， with a limit of detection at 2.00 ng/mL. The spiked recoveries were between 98.3% and 104.7%， and intra- and inter-day precision ranged from 1.2% to 7.0%. Similarly， the method for vitamin E exhibited a validated linear range of 25.00-1 000.00 ng/mL， with a limit of detection at 6.00 ng/mL. The spiked recoveries ranged from 97.9% to 105.5%， and intra- and inter-day precision varied between 3.0% and 5.7%. Tear samples from five patients with dry eye and nine healthy volunteers were analyzed using this method. In the dry eye group， the average concentrations of vitamin A and vitamin E were 9.60 （5.13-12.54） ng/mL and 42.00 （31.75-128.00） ng/mL， respectively. In the healthy group， the average concentrations of vitamin A and vitamin E were 18.10 （12.46-21.69） ng/mL and 211.00 （181.00-459.75） ng/mL， respectively. Statistical differences in vitamin A and vitamin E levels between the dry eye group and the healthy group were identified using the Wilcoxon Mann-Whitney test （P<0.05）. This study introduces a straightforward and reliable LC-MS/MS method for the detection of tear VA and VE levels， while also investigating their association with dry eye. This offers a novel reference for understanding the potential risk factors associated with dry eye.

Key words: liquid chromatography-tandem mass spectrometry （LC-MS/MS）, tears, dry eye, vitamin A, vitamin E