Submitted to: Journal of Chromatography B
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2018
Publication Date: 7/18/2018
Citation: Bukowski, M.R., Voeller, K.M., Jahns, L.A. 2018. Simple and sensitive dilute-and-shoot analysis of carotenoids in human plasma. Journal of Chromatography B. 1095:32-38. https://doi.org/10.1016/j.jchromb.2018.07.020.
DOI: https://doi.org/10.1016/j.jchromb.2018.07.020 Interpretive Summary: Carotenoids are brightly colored compounds that occur in plants. In addition to having intrinsic health benefits such as acting as precursors to vitamin A, measuring carotenoid concentrations in blood plasma is a useful way to assess vegetable and fruit intake. Previously published methods for measuring these compounds involved large volumes of plasma (0.5-1.0 mL) and multiple sample preparation steps requiring considerable time. Our work uses liquid chromatography coupled to mass spectrometry to increase the sensitivity of measurement, resulting in a technique that produces data using only 10 uL of plasma, about the same amount of plasma in a single blood drop from a finger stick. Additionally we simplified the sample preparation to a single step taking only minutes. We demonstrated that significant amounts of carotenoids are lost when samples are prepared using the previously published methods. This method will benefit nutritionists and clinical researchers by lowering significant barriers for the testing of carotenoids.
Technical Abstract: Carotenoids in human plasma are used as biomarkers of vegetable and fruit intake. Large sample volumes and intensive sample processing make measurement of these species cumbersome. We developed a dilute-and-shoot method for the quantitation of a-carotene, beta-carotene, beta-cryptoxanthin, lycopene and lutein/zeaxanthin using 10 uL of plasma. Plasma was injected into methanol containing internal standard and deproteinized by centrifugation. The carotenoids in the supernatant were separated using a YMC C-30 column and quantified by tandem mass spectrometry. The linearity for carotenoids ranged from sub-fmol to approximately 300 fmol on-column. Spike recovery experiments were used to correct for matrix effects. The method was validated using the human plasma standard NIST SRM 968e. Over 400 sample analyses were performed using the same guard and analytical columns. This method represents an improvement over current techniques because of the small sample size needed, ease of sample preparation, and improvement in the determination of carotenoid content.