20. Vitamin Analysis

20.4.1.1  Overview

Because of their relative simplicity, accuracy, and precision, the chemical methods, in particular the chromatographic methods using HPLC/UHPLC, are preferred (see Chap. 13). Numerous vitamins are now commonly measured by HPLC (e.g., A, D, E, K, C, various B vitamins), many as official methods and some unofficial. Liquid chromatography in combination with mass spectrometry (MS) (see Chap. 11) has added a new dimension to vitamin analysis. In general, LC-MS or electrospray ionization (ESI) LC-MS/MS methods are available for each fat- and water-soluble vitamin. Detection by MS leads to increased sensitivity as well as unequivocal identification and characterization of the vitamin. The LC-MS assays have become a mainstay of accurate, cost-effective vitamin analyses. For example, LC-MS is commonly employed for verification of vitamin D content of products with difficult matrices (i.e., comparing results to those with standard LC analysis, e.g., AOAC Method 2012.11, Simultaneous Determination of Vitamins D₂ and D₃ in Infant Formula and Adult/Pediatric Nutritional Formula) and LC-MS/MS for folate (AOAC Method 2013.13, Folate in Infant Formula and Adult/Pediatric Nutritional Formula by a UHPLC-MS/MS assay vs. the microbiological method).

Standard HPLC is commonly employed as an official method of analysis for vitamins A (e.g., AOAC Method 992.04, 50.1.02), E (e.g., AOAC Method 992.03, 50.1.04), and D (e.g., AOAC Method 2002.05, 45.1.22A) and as a quality control method for vitamin C. While HPLC/UHPLC involves a high capital outlay, it is applicable to most vitamins and lends itself in some instances to simultaneous analysis of several vitamins and/or vitamers (i.e., isomers of vitamins). Implementation of multi-analyte procedures for the analysis of water-soluble vitamins can result in assay efficiency with savings in time and materials. To be useful, a simultaneous assay must not lead to loss of sensitivity, accuracy, and precision when compared to single-analyte methods. In general terms, multi-analyte methods for water-soluble vitamin assay of high-concentration products including pharmaceuticals, supplements, and vitamin premixes are quite easily developed. Though the applicability of HPLC has been demonstrated to a wide variety of biological matrices with no or only minor modifications in some cases, one must always bear in mind that all chromatographic techniques, including HPLC, are separation and not identification methods. Therefore, during adaptation of an existing HPLC method to a new matrix, establishing evidence of peak identity and purity is an essential step of the method adaptation or development.

20.4.1.2  Vitamin A

Vitamin A is sensitive to ultraviolet (UV) light, air (and any prooxidants, for that matter), high temperatures, and moisture. Therefore, steps must be taken to avoid any adverse changes in this vitamin due to such effects. Steps include: (1) using low actinic glassware, nitrogen, and/or vacuum, (2) avoiding excessively high temperatures, (3) working in subdued artificial light, and (4) adding pyrogallol as an antioxidant prior to saponification.

HPLC methods are considered the only acceptable methods to provide accurate food measurements of vitamin A activity. For example, in the HPLC method of vitamin A (i.e., retinol isomers) in milk and milk-based infant formula (AOAC Method 992.04, 50.1.02) [2], the test sample is saponified with ethanolic KOH, vitamin A (retinol) is extracted into organic solvent, and then concentrated. Vitamin A isomers – all-trans-retinol and 13-cis-retinol – levels are determined by HPLC on a silica column (i.e., normal phase). Vitamin A also can be analyzed using reversed-phase HPLC columns.

20.4.1.3  Vitamin D

Vitamin D is typically analyzed by HPLC with a UV-Vis detector (some version of AOAC Method 2002.05) but by HPLC-MS for verification of analyte presence, as needed. Protection against oxidation is done as described for vitamin A above. For the HPLC-UV-Vis analysis, an internal standard (vitamin D₂) is added to the sample that is subjected to basic hydrolysis then saponified in ethanolic KOH. This sample is extracted with heptane, and the heptane organic phase is evaporated to dryness. The reconstituted sample is subjected to a semi-preparative normal-phase HPLC column, from which the fractions are collected, concentrated, and diluted in acetonitrile-methanol. These samples are subjected to a reversed-phase HPLC column with UV detection to quantitate the D₃. A separate sample is tested in parallel to confirm the absence of endogenous D₂.

20.4.1.4  Vitamin E (Tocopherols and Tocotrienols)

Vitamin E is present in foods as eight different compounds: all are 6-hydroxychromans. The vitamin E family is comprised of α-, β-, γ-, and δ-tocopherol, characterized by a saturated side chain of three isoprenoid units and the corresponding unsaturated tocotrienols (α-, β-, γ-, and δ-). Like vitamins A and D, vitamin E must be protected from oxidation during sample preparation and is commonly analyzed by HPLC. Typically a normal or reversed-phase HPLC column is connected to a fluorescence detector: E _x λ = 290 nm and E _m λ = 330 nm (E _x, excitation; E _m, emission) (for fluorescence spectroscopy, see Chap. 7, Sect. 7.​3) [14]. An example chromatogram is depicted in Fig. 20.1. Vitamin E is quantitated by external standards from peak area by linear regression.

20.4.2.1  Vitamin C

The vitamin (l-ascorbic acid and l-dehydroascorbic acid) is very susceptible to oxidative deterioration, which is enhanced by high pH and the presence of ferric and cupric ions. For these reasons, the entire analytical procedure needs to be performed at low pH and, if necessary, in the presence of a chelating agent. Mild oxidation of ascorbic acid results in the formation of dehydroascorbic acid, which is also biologically active and is reconvertible to ascorbic acid by treatment with reducing agents such as β-mercaptoethanol and dithiothreitol. Two AOAC official methods for vitamin C are described below, but vitamin C also can be analyzed in infant formula and adult/pediatric nutritional formula by HPLC with UV detection (AOAC Method 2012.21) and UHPLC with UV detection (AOAC Method 2012.22).

20.4.2.1.1 2,6-Dichloroindophenol (DCIP)Titrimetric Method

This method is specified as an AOAC official method (AOAC Method 967.21, 45.1.14) [2] for vitamin preparation and juices (i.e., fruits), but it is sometimes used as a secondary method for other foods, because it is a more rapid method than the microfluorometric method (described in Sec. 20.4.2.1.2) applicable to other foods. In the DCIP method, l-ascorbic acid is oxidized to l-dehydroascorbic acid by the oxidation-reduction indicator dye, DCIP. At the endpoint, excess unreduced dye appears rose pink in acid solution (see Figs. 20.2 and 20.3). With colored samples such as red beets or heavily browned products, the rose-pink endpoint is impossible to detect by the human eye. In such cases it, therefore, needs to be determined by observing the change of transmittance using a spectrophotometer with the wavelength set at 545 nm.

20.4.2.2  Thiamine (Vitamin B₁) Thiochrome Fluorometric Method

While thiamine can be quantitated by HPLC, it is still commonly analyzed by the longtime official thiochrome fluorometric procedure (AOAC Method 942.23) [2]. Following sample extraction with dilute acid, enzymatic hydrolysis of thiamine’s phosphate esters, and chromatographic cleanup (i.e., purification), thiamine is oxidized to thiochrome, which is fluorescent. This method is based on the fluorescence measurement of thiochrome in the test solution compared to that from an oxidized thiamine standard solution.

20.4.2.3  Riboflavin (Vitamin B₂) Fluorometric Method

Like other B vitamins, riboflavin can be analyzed by HPLC, but its natural fluorescence allows for measurement based on this characteristic. Following sample extraction, cleanup, and compensation for the presence of interfering substances, riboflavin is determined fluorometrically, compared to a riboflavin standard (AOAC Method 970.65, 45.1.08) [2].