Retinol quantification

Several different quantification procedures for vitamin A have been described in the literature, some using retinol directly as a standard and some using retinyl acetate, which is converted to retinol by saponification. The latter approach is generally preferred, because crystalline all-trans-retinyl acetate is commercially available in high purity and is free from cis isomers. Commercial sources of retinol are oily preparations and are at best only about 70% pure. There are two ways of preparing a retinol standard from retinyl acetate. 

Chauveau-Duriot, B. Doreau, M. Noziere, P. Graulet, B. 2010. Simultaneous quantification of carotenoids, retinol, and tocopherols in forages, bovine plasma, and milk validation of a novel UPLC method. Anal. Bioanal. Chem. 397 777-790. 

Wang, Y. Xu, X. van Lieshout, M. West, C.E. Lugtenburg, J. Verhoeven, M.A. Creemers, A.F.L. Muhilal and van Breemen, R.B. 2000. A liquid chromatography-mass spectrometry method for the quantification of bioavailability and bioconversion of P-carotene to retinol in humans. Anal. Chem. 72 4999-5003. 

Blood collected at each time point was allowed to remain on ice for 15 min prior to centrifugation at 1800g. Plasma was transferred to 5-ml cryogenic vials and stored at -8(PC until analyzed. Plasma lipids were extracted from duplicate 2.2-g plasma aliquots for isotope ratio analysis or from 0.25-g aliquots for HPLC quantification of retinol and /3C after addition of internal standard (retinyl acetate). Plasma aliquots were de-proteinized with 1 vol of ethanol and lipids extracted with 3 vol of hexane (Optima Grade, Fisher Scientific, Rochester, NY). 

The method of Thurnham etal. (1988) was modified for the quantification of plasma j3C and retinol. Plasma extracts were dissolved in 40 /il of dimeth-ylforamide and vortexed and then 210 pil of acetonitrile/methanol/chloro-form (47/47/6, v/v/v) was added. Reconstituted samples were vortexed and sonicated for 40 sec prior to being transferred to autosampler vials and sealed under nitrogen. The HPLC system consisted of a photodiode array detector (Waters 9%, Miliipore Corp., Milford, MA) with Millennium software, a Waters 717 plus autosampler, and a Hewlett-Packard Model 1050 pump. Analytes of interest were separated using acetonitrile/methanol/ chloroform (47/47/6, v/v/v), with 0.05 M of ammonium acetate and 1% triethylamine at a flow rate of 1.2 ml/min and a 4.6 X 15-cm Spherisorb ODS-2 column (LKB Instruments Ltd., Surrey, UK) maintained at 26°C using a column heater (Timberline Instruments Ltd., Boulder, CO). This analysis does not discriminate between C-enriched and nonenriched analytes, but rather measures the total concentration of each isotopomer. The retention times of retinol, retinyl acetate (internal standard), and /3-carotene were 2.1, 2.6, and 16.9 min, respectively. Plasma concentrations of retinol and /3C were calculated using a standard curve for each analyte and an internal standard to correct for volume recovery. 

When total vitamin A is to be measured regardless of whether it is free or esterified, a saponification step can be included. However, degradation of the retinoids occurs even under mild conditions and in the presence of antioxidants. Consequently, direct extraction procedures without any digestion step, allowing the separation and separate quantification of the ester forms and of retinol itself, have gained interest in the last two decades. 

Lessin WJ, Catigani GL, Schwartz SJ (1997) Quantification of cis-trans isomers of provitamin A carotenoids in fresh and processed fruits and vegetables. J Agric Food Chem 45 3728 MacCrehan WA, Schonberger E (1987) Determination of retinol, a-tocopherol, and a-carotene in serum by liquid chromatography with absorbance and electrochemical detection. Clin Chem 33 1585... 

Fig 1. Quantification of retinal and retinol by HPLC. Retinoids were resolved by a normal-phase Dupont Zorbax-Sil Reliance cartridge column (04x4 cm) eluted at 2 mL/min with a linear gradient from 4% tetrahydrofuran to 15% tetrahydrofuran in hexane for 5 min, followed by 5 mm of 4% tetrahydrofuran in hexane 13-as-retinal (not shown) 1, 9-ci5-retinal 2, all-tran -retinal 3, 3,4,-didehydroretinal 4, 9-cw-retinol 5, all-tran -retinol, 6, 3,4,-didehydroretinol. Retinoids were detected by UV-370 nm for retinals 325 nm for retinols. 

Napoli, J. L. (1990) Quantification and characteristics of retinoid synthesis from retinol and b-carotene in tissue fractions and established cell lines. Methods in Enzymol. 189,470-482. 

Miyagi, M. Yokoyama, H. Shiraishi, H. Matsumoto, M. Ishii, H. Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performtuice hquid chromatography with a simple gradiation, J.Chromatogr.B, 2001, 757, 365-368. 

An important observation is that endogenous levels of retinoids in human embryos are quantitatively and qualitatively similar to those in Xenopus embryos, whereby the following retinoids were detected 13-cw-RA, 9-cis-RA, dill-trans-RA, all-fran -retinol, all-fran -dd-retinol and all-trans-retinal [8, 37, 39]. Until this report, dd-retinol had been detected only in chick embryos [33], but not in rodent embryos. These retinoids were detected in Xenopus embryos at levels that can be accurately quantified with a sensitive HPLC detection system. In contrast, levels of the same endogenous retinoids in rodents are low [19, 20, 21], resulting in less reliable quantification. 

Analytical Methods for the Quantification of the Retinol Binding Protein. 94... 

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