Sample preparation techniques for the analysis of vitamin D3 in food matrices typically utilise a saponification step, either at room temperature or at elevated temperatures. A calciferol (vitamin D2 or isotope labelled vitamin D3) is generally chosen as the internal standard to compensate for changes of previtamin D3–vitamin D3 isomerisation during analysis, as well as to correct for analyte loss through complex sample preparation steps. Manufacturing practices and processing parameters contribute to previtamin D formation in food products. A significant proportion (5.6–8.3%) of the total vitamin D3 in premixes was found as previtamin D3, indicating that it is likely, depending upon storage temperature and the time since manufacture, that a vitamin D3-fortified food product will contain a similar proportion of previtamin D3 prior to analysis. Conversely, freshly prepared internal standard solutions have low previtamin D levels (< 1%). In lieu of direct measurement, this discrepancy in previtamin D content between the internal standard and analyte forms of vitamin D will lead to analytical bias. To mitigate this as a source of potential error, it is recommended that sample pretreatment steps are appropriately set and controlled. Based on this work, saponification times greater than 300, 120, or 60 min for temperatures of 60, 70, or 80 °C respectively should be employed and that saponification at room temperature be avoided.
Vitamin D Previtamin D Saponification Isomerisation
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The authors acknowledge the support of Fonterra Co-operative Group Ltd.
Compliance with Ethical Standards
Conflict of Interest
Brendon Gill declares that he has no conflict of interest. Donald Gilliland declares that he has no conflict of interest. Harvey Indyk declares that he has no conflict of interest. Jackie Wood declares that she has no conflict of interest. David Woollard declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Abernethy GA (2012) A rapid analytical method for cholecalciferol (vitamin D3) in fortified infant formula, milk and milk powder using Diels–Alder derivatisation and liquid chromatography–tandem mass spectrometric detection. Anal Bioanal Chem 403:1433–1440CrossRefGoogle Scholar
AOAC (2016a) Official methods of analysis of AOAC International 20th ed.: Method 2002.05. AOAC International, Gaithersburg, MDGoogle Scholar
AOAC (2016b) Official methods of analysis of AOAC International 20th ed.: Method 2016.05. AOAC International, Gaithersburg, MDGoogle Scholar
de Vries EJ, Zeeman J, Esser RJ, Borsje B, Mulder FJ (1979) Analysis of fat-soluble vitamins. XXIII. High performance liquid chromatographic assay for vitamin D in vitamin D3 and multivitamin preparations. J Assoc Off Anal Chem 62:1285–1291Google Scholar
DeLuca HF (2004) Overview of general physiologic features and functions of vitamin D. Am J Clin Nutr 80:1689S–1696SCrossRefGoogle Scholar
Eitenmiller RR, Ye L, Landen WO Jr (2008) Vitamin analysis for the health and food sciences, 2nd edn. CRC Press, Boca Raton, FLGoogle Scholar
Gill BD, Zhu X, Indyk HE (2015) A rapid method for the determination of vitamin D3 in milk and infant formula by liquid chromatography/tandem mass spectrometry. J AOAC Int 98:431–435CrossRefGoogle Scholar
Gill BD, Abernethy GA, Green RJ, Indyk HE (2016) Analysis of vitamin D2 and vitamin D3 in fortified milk powders and infant and nutritional formulas by liquid chromatography–tandem mass spectrometry: single-laboratory validation, First Action 2016.05. J AOAC Int 99:1321–1330CrossRefGoogle Scholar
Gomes FP, Shaw PN, Whitfield K, Koorts P, Hewavitharana AK (2013) Recent trends in the determination of vitamin D. Bioanalysis 5:3063–3078CrossRefGoogle Scholar
Gomes FP, Shaw PN, Whitfield K, Hewavitharana AK (2015) Simultaneous quantitative analysis of eight vitamin D analogues in milk using liquid chromatography–tandem mass spectrometry. Anal Chim Acta 891:211–220CrossRefGoogle Scholar
Hanewald KH, Mulder FJ, Keuning KJ (1968) Thin-layer chromatographic assay of vitamin D in high-potency preparations. Analysis of fat-soluble vitamins IX. J Pharm Sci 57:1308–1312CrossRefGoogle Scholar
Heudi O, Trisconi MJ, Blake CJ (2004) Simultaneous quantification of vitamins A, D3 and E in fortified infant formulae by liquid chromatography–mass spectrometry. J Chromatogr A 1022:115–123CrossRefGoogle Scholar
Hewavitharana AK (2013) Current status of vitamin D assays: are they reliable and sufficiently informative for clinical studies? Bioanalysis 5:1325–1327CrossRefGoogle Scholar
Higashi T, Shimada K, Toyo’oka T (2010) Advances in determination of vitamin D related compounds in biological samples using liquid chromatography–mass spectrometry: a review. J Chromatogr B 878:1654–1661CrossRefGoogle Scholar
Huang M, Winters D (2011) Application of ultra-performance liquid chromatography/tandem mass spectrometry for the measurement of vitamin D in foods and nutritional supplements. J AOAC Int 94:211–223Google Scholar
Keverling Buisman JA, Hanewald KH, Mulder FJ, Roborgh JR, Keuning KJ (1968) Evaluation of the effect of isomerization on the chemical and biological assay of vitamin D. J Pharm Sci 57:1326–1329CrossRefGoogle Scholar
Kwak BM, Jeong IS, Lee MS, Ahn JH, Park JS (2014) Rapid determination of vitamin D3 in milk-based infant formulas by liquid chromatography-tandem mass spectrometry. Food Chem 65:569–574CrossRefGoogle Scholar
Mackay C, Tillman J, Burns DT (1979) Determination of vitamin D2 in multivitamin tablets by high-performance liquid chromatography. Analyst 104:626–636CrossRefGoogle Scholar
Mulder FJ, de Vries EJ, Borsje B (1971) Chemical analysis of vitamin D in concentrates and its problems. XII. Analysis of fat-soluble vitamins. J Assoc Off Anal Chem 54:1168–1174Google Scholar
Pastore RJ, Dunnett RV, Webster GK (1997) Dimethyl sulfoxide extraction method for the liquid chromatographic analysis of microencapsulated vitamin D3. J Agric Food Chem 45:1784–1786CrossRefGoogle Scholar
Perales S, Alegría A, Barberá R, Farré R (2005) Review: determination of vitamin D in dairy products by high performance liquid chromatography. Food Sci Technol Int 11:451–462CrossRefGoogle Scholar
Schadt HS, Gössl R, Seibel N, Aebischer C-P (2012) Quantification of vitamin D3 in feed, food, and pharmaceuticals using high-performance liquid chromatography/tandem mass spectrometry. J AOAC Int 95:1487–1494CrossRefGoogle Scholar
Sliva MG, Green AE, Sanders JK, Euber JR, Saucerman JR (1992) Reversed-phase liquid chromatographic determination of vitamin D in infant formulas and enteral nutritionals. J AOAC Int 75:566–571Google Scholar
Strobel N, Buddhadasa S, Adorno P, Stockham K, Greenfield H (2013) Vitamin D and 25-hydroxyvitamin D determination in meats by LC-IT-MS. Food Chem 138:1042–1047CrossRefGoogle Scholar
Tian XQ, Hollick MF (1995) Catalyzed thermal isomerization between previtamin D3 and vitamin D3 via β-cyclodextrin complexation. J Biol Chem 270:8706–8711CrossRefGoogle Scholar