Determination of L-hydroxyproline using hydrophilic interaction chromatography coupled to tandem mass spectrometry with lyophilized concentrated extraction in milk and dairy products

Abstract

In this study, a screening and confirmation method for the determination of l-hydroxyproline (Hyp) as a target compound in milk and dairy products using high-performance liquid chromatography with tandem mass spectrometry was developed. The samples were lyophilized after acidic hydrolysis, followed by cleanup with graphitized carbon black to remove pigments. Hyp was separated by a hydrophilic interaction chromatographic column and analyzed by high-performance liquid chromatography with tandem mass spectrometry working with multiple reaction monitoring mode using an electrospray ionization interface in a positive-ion mode. Average recoveries in spiked milk and dairy products ranged from 68.0 to 101.1% with relative standard deviations between 2.0 and 11.7% (n = 7). A reagent-matched standard calibration curve was used for quantification of Hyp, with linear correlation coefficient (R(2)) > 0.99 in the concentration range of 0.1-100 μg/mL. The LOQs were from 0.25 to 5 mg/kg, which were usually sufficient to verify the Hyp in samples. The confirmation concentration of Hyp ranged from 10 to 50 mg/kg.

Keywords: Hydrophilic interaction chromatography; Lyophilization; Milk and dairy products; l-Hydroxyproline; tandem mass spectrometry.