Chemistry Tales

Background: Choline and L‑carnitine are classified as pseudo-vitamins because of their conditionally essential status. As they are involved in multiple physiological metabolic pathways in the human body, they are routinely fortified in infant and adult nutritional formulas. Objective: The performance of an LC‑MS/MS method for the analysis of choline and carnitine, compared with enzymatic methods in routine use for the analysis of total carnitine and total choline, is described. Method: Powder samples were reconstituted, with release of carnitine and choline facilitated by both acid and alkaline hydrolysis and the extract analyzed by LC‑MS/MS. Quantitation was by internal standard technique using deuteriumlabeled carnitine and deuterium-labeled choline. Results: Method range, specificity, sensitivity, precision, recovery, accuracy, and ruggedness were assessed for milk powders, infant formulas, and soy- and milk-based nutritional products. Spike recoveries of 94.0–108.4% were obtained for both total carnitine and choline, and no statistical bias (Δ = 0.05) between measured results and certified values (choline: P = 0.36; free carnitine: P = 0.67) was found for NIST 1849a certified reference material (NIST 1849a). Precision, as repeatability relative standard deviation (RSD), was 2.0% RSDr for total carnitine and 1.7% RSDr for total choline. Equivalent results for total choline and total carnitine were obtained by LC‑MS/MS and enzymatic methods (n = 30). Conclusions: The described LC‑MS/MS method is fit for purpose for routine product compliance release testing environments. This validation study has confirmed that alternative enzymatic assays can be used with confidence in laboratories in which LC‑MS/MS platforms are unavailable. Highlights: An LC‑MS/MS method was evaluated and found to be fit-for-purpose for routine product compliance release testing of infant formula. The LC‑MS/MS method was compared with enzymatic methods for the analysis of total carnitine and total choline. Alternative enzymatic assays can be used with confidence in laboratories in which LC‑MS/MS platforms are unavailable.

Reflectance colorimetry was investigated to measure the colour of whole and skim milk powders, infant formulas, an adult nutritional product, unsalted and salted butters. The colorimeter gave precise results and was cost effective, illustrating the benefits of the adoption of a colorimeter rather than the current practice of using reference charts and powders. New Zealand dairy products are yellower compared with other dairy products because of their relatively high naturally occurring β‑carotene content.

Background: Aflatoxins are secondary metabolites produced by a number of species of Aspergillus fungi. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and is found in the milk of cows fed with feed spoilt by Aspergillus species. AFM1 is carcinogenic, especially in the liver and kidneys, and mutagenic, and is also an immunosuppressant in humans. Objective: A high-throughput method for the quantitative analysis of AFM1 that is applicable to liquid milk, cheese, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), and whey powder (WP) was developed and validated. Method: AFM1 in cheese, milk, and protein products is extracted using 1% acetic acid in acetonitrile with citrate salts. The AFM1 in the resulting extract is concentrated using RIDACREST/IMMUNOPREP ONLINE cartridges followed by quantification by HPLC–fluorescence. Results: The method was shown to be accurate for WP, WPC, WPI, MPC, liquid milk, and cheese, with acceptable recovery (81–112%) from spiked samples. Acceptable precision for WP, WPC, WPI, MPC, liquid milk, and cheese was confirmed, with repeatabilities of 4–12% RSD and intermediate precisions of 5–13% RSD. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. An international proficiency scheme (FAPAS) cheese sample showed that this method gave results that were comparable with those from other methods. Conclusions: A method for high-throughput, routine testing of AFM1 is described. The method was subjected to single laboratory validation and was found to be accurate, precise, and fit-for-purpose. Highlights: An automated online immunoaffinity cleanup HPLC-fluorescence method for milk proteins, cheese, and milk was developed and single-laboratory validated. It allows for high-throughput analysis of AFM1 and can be used for the analysis of AFM1 in whey protein products.

Vitamin K is a dietary component that, as a cofactor, has critically important physiological roles in the body. As it is present in milk at very low levels, it is fortified at higher levels in paediatric milk-based formulae. Currently, the predominant analytical strategy for both food database surveys and routine compliance testing is high-performance liquid chromatography with post-column zinc reduction and fluorescence detection. This study presents a single-laboratory validation of a pre-column reduction modification that provides enhanced sensitivity (> 2×) and increased analytical throughput. Accuracy was confirmed by analysis of a certified reference infant formula and milk proficiency samples, and by comparison against the reference method for a wide range of dairy products containing vitamin K1 at both natural and supplemented levels. The method offers several performance advantages and is suitable for routine product compliance release testing.

Background: Direct measurement of the bioavailable α‑tocopherol content presents a significant analytical challenge and requires chiral separation of the α‑tocopherol stereoisomers. Objective: The objective of the study was to validate an analytical method for the analysis of α‑tocopherol stereoisomers in infant formulas and dairy products. Method: Samples were saponified at elevated temperature and lipophilic components were extracted into an organic solvent, with subsequent chromatographic separation of the α‑tocopherol stereoisomers achieved by HPLC with a chiral column and fluorescence detection. Results: The method was shown to be accurate, with spike recoveries of 91.9–108.8% for RRR‑α‑tocopherol and 90.1–104.7% for α‑tocopherol, with no statistical bias against NIST 1849a certified reference material (P‑value = 0.54) and an HPLC-UV analytical method (P‑value = 0.48). Acceptable precision was confirmed, with repeatabilities estimated at 3.5% RSDr (HorRat = 0.6) for RRR‑α‑tocopherol and 4.6% RSDr (HorRat = 0.4) for α‑tocopherol. Conclusions: A straightforward chiral chromatographic method for the analysis of stereoisomeric forms of α‑tocopherol is described. In a single analytical run, the method can quantify: (i) the total α‑tocopherol content; (ii) the nutritionally important RRR‑α‑tocopherol and/or 2R,4′‑ambo,8′‑ambo‑α‑tocopherol contents; (iii) the amount of all‑rac‑α‑tocopherol, all‑rac‑α‑tocopheryl acetate, or all‑rac‑α‑tocopheryl succinate fortified into the product. Highlights: An accurate and precise chiral chromatographic method for the analysis of isomeric forms of α‑tocopherol is described. The method is able to distinguish between natural and synthetic tocopherol sources. The method is accurate and precise and is suitable either for routine product compliance testing during product manufacture or as a possible reference method.