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Updated: Jul 12, 2026
Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)
Published on: June 30, 2020
Clinical validation of an automated extraction protocol and liquid chromatography single quadrupole mass spectrometry
Rowan Hellier1, Lesley Heirene1, Stuart J Moat1,2
1Department of Medical Biochemistry, Immunology and Toxicology, University Hospital of Wales, Cardiff, Wales, UK.
Objectives:
Quantitation of plasma vitamins A and E is essential for assessing nutritional status. Traditional methods typically involve liquid-liquid extraction followed by high-performance liquid chromatography with UV detection (HPLC-UV). In this study, we evaluated an automated sample extraction-method paired with liquid chromatography-single quadrupole mass spectrometry (LC-MS) as an alternative analytical approach.
Methods:
Plasma samples were extracted using Oasis PRiME HLB µElution Plates automated on a Tecan Liquid-Handling Platform and analysed using LC-MS. Injection-to-injection time was 5-min. Method performance was assessed by comparison with the Chromsystems™ Vitamin A and E HPLC-UV Kit, using patient specimens (n=70), ClinChek® Controls, and external quality assessment (EQA) materials.
Results:
The developed method demonstrated acceptable inter-assay imprecision (CV≤5 %). The bias compared to the HPLC-UV method for vitamin A was 5.67 % and -0.15 % for vitamin E. Good agreement was observed for both vitamins (concordance correlation coefficients (CCC) >0.950). The assay was linear up to 12 μmol/L for vitamin A and 125 μmol/L for vitamin E. Using EQA materials, the mean bias for both analytes was <4 %. The lower limit of quantification (LLOQ) was 0.16 μmol/L for vitamin A and 0.47 μmol/L for vitamin E. Extracts stored at 4 °C and -20 °C were stable for 5 and 14 days, respectively. The analytical column demonstrated good retention time stability (<3.0 % change for both analytes) and was suitable for >1,500 injections.
Conclusions:
This method demonstrated robust analytical performance and good agreement with HPLC-UV. Our approach confers practical advantages over a manual based extraction and HPLC-UV analysis including analytical selectivity and workflow time efficiency.
