Quantitative analyses of β‐carotene and retinol in serum and feces in support of clinical bioavailability studies

Abstract

Among more than 50 provitamin carotenoids, beta-carotene is the most metabolically active source of retinol. Despite diets rich in fruits and vegetables containing beta-carotene, vitamin A deficiency is the leading cause of blindness and childhood mortality in developing countries. In addition, the uncertainty of beta-carotene bioconversion into vitamin A suggests that new data are needed to update the nutritional guidelines in developed countries. Previously, we reported the development of a carotene/retinol plateau isotopic enrichment method (CarRet PIE) for the determination of beta-carotene bioavailability and bioconversion into retinol, which utilizes positive ion atmospheric pressure chemical ionization (APCI) liquid chromatography/mass spectrometry (LC/MS). While seeking to validate the CarRet PIE using a mass balance approach requiring fecal measurements of beta-carotene and retinol, interference was encountered that required substantial modifications of the LC/MS assay. Here we report a new LC/MS assay that is based on the detection of molecular anions of beta-carotene using negative ion APCI with a reversed-phase C30 column for HPLC separation. Sample preparation required saponification to eliminate interfering triglycerides. The limit of detection (LOD) of beta-carotene was 0.25 pmol calculated on the basis of an injection of 20 microL of 0.0125 microM beta-carotene, and the limit of quantitation (LOQ) was 1.0 pmol based on the injection of 20 microL of 0.050 microM beta-carotene. The linear range was 1.1 to 2179 pmol on-column. The wide linear range and low LOD and LOQ of this assay facilitated the sensitive and selective quantitative analysis of beta-carotene in both serum and fecal samples in support of an on-going clinical investigation of beta-carotene bioavailability and bioconversion into vitamin A.

An LC/MS/MS method for stable isotope dilution studies of β-carotene bioavailability, bioconversion, and vitamin A status in humans

“…In contrast, the application of liquid chromatography mass spectrometry (LC/MS) to the analysis of retinoid and carotenoid tracers offers the advantages of high sensitivity and selectivity without the need for hydrolysis and derivatization ( 17,(27)(28)(29)(30). However, isolation of carotenoids and retinoids from the plasma matrix is frequently carried out individually leading to separate injections, use of different LC systems, MS ionization methods (APCI/ESI) and modes (positive/negative) (11)(12)(13)(14)(15)(16)(17)(18). The current method of plasma retinyl esters is progressively and irreversibly transferred to the denser LDL fraction resulting in 32% of the plasma retinyl esters localized to the LDL fraction 12 h after fat load ( 34 ).…”

“…Fasted subjects were cannulated via the antecubital vein and blood was drawn into 10 ml EDTA Vacutainer tubes (Becton Dickinson). Subjects then received the dual isotopic oral dose of 2 mg [ of carotenoids/retinoids from the plasma matrix for MS analysis often involves extensive and time-consuming extraction/purifi cation procedures that have included: saponifi cation, solid-phase extraction, preparative HPLC, and, in the case of GC-MS analysis, further conversion to tert -butyl-dimethylsilyl derivatives (11)(12)(13)(14)(15)(16)(17)(18). The aim was to develop an analytical method that involved a simplifi ed extraction procedure, sensitive MS/MS for detection of physiological doses of stable isotopes, and short LC runtimes so as to be suitable for high-throughput of samples from human intervention studies.…”

Food Matrix Effects on Bioaccessibility of β-Carotene Can be Measured in an in Vitro Gastrointestinal Model

“…19,20 β-Carotene and [ 13 C 10 ]β-carotene were monitored in negative ion mode at mass-to-charge ratios ( m / z ) 536 and 546, respectively. 20 Isotopic enrichment of β-carotene in the filtrates was the proportion of labeled β-carotene to total β-carotene. These analyses were performed at the University of Illinois.…”

Effects of Season, Size and Parasitism by the Acanthocephalan, Profilicollis altmani, on the Carotenoid Concentration and Composition of the Pacific Mole Crab, Emerita analoga

“…Therefore, these studies are easily reproducible and can be built upon readily. HPLC methods have also been utilized to successfully quantify the carotenoids of less commonly studied organisms, such as red coral, human serum, red yeasts, and quail retina (Zhu et al 2006, Cvejic et al 2007, Toomey and McGraw 2007, Weber et al 2007). …”