Determination of Cholecalciferol (Vitamin D3) in Selected Foods by Liquid Chromatography: NMKL Collaborative Study

Abstract

Results are presented from an NMKL (Nordic Committee on Food Analysis) collaborative study of a method for the determination of cholecalciferol (vitamin D3) in foods. The method is based on the addition of an internal standard (vitamin D2), followed by saponification and extraction with n-heptane. The fraction that contains vitamin D2/D3 is separated by preparative normal-phase liquid chromatography (LC), and the analytes are determined by reversed-phase LC with UV detection at 265 nm. The method was tested by 8 participating laboratories. In this study 6 different matrixes were analyzed for cholecalciferol content: milk, liquid infant formula (gruel), cooking oil, margarine, infant formula, and fish oil. The contents varied from 0.4 to 12 μg/100 g. Three matrixes (milk, gruel, and margarine) were fortified with vitamin D3. In the other matrixes, vitamin D3 was added at 3 different levels at the Swedish National Food Administration. The milk was analyzed as a blind duplicate, whereas the other matrixes were analyzed as split-level pairs. The recoveries from the samples with vitamin D3 added varied from 93 to 102%. The repeatability relative standard deviation (RSDr) values for accepted results varied between 2.2% (fish oil) and 7.4% (cooking oil), whereas the reproducibility relative standard deviation (RSDR) values varied between 6.8% (margarine) and 24% (cooking oil).

Vitamin D3 in High-Quality Cow Milk: An Italian Case Study

“…Known quantities of ergocalciferol (at three different concentration levels (0.01, 0.03 and 0.06 mg kg −1 )), were added to calculate the vitamin D2 recovery after sample saponification and purification. The average extraction yield was equal to 81.9%, which is within the expected recovery ranges (60- The use of an IS instead of an external calibration was already reported in an interlaboratory study by Staffas and Nyman [19]. Basically, the sample purification for vitamin D3 is a laborious protocol (sample saponification followed by solvent extraction and SPE), thus the loss of the analyte during the procedure cannot be considered constant and a correction factor cannot be determined.…”

“…As a second necessary step, vitamin D3 (the "a" peak in the top chromatogram of Figure 1) was unambiguously identified using HPLC/tandem MS (MS 2 ) under the same HPLC conditions. The mass fragmentation of the pseudomolecular peak of vitamin D3 is reported in Figure 2 The use of an IS instead of an external calibration was already reported in an interlaboratory study by Staffas and Nyman [19]. Basically, the sample purification for vitamin D3 is a laborious protocol (sample saponification followed by solvent extraction and SPE), thus the loss of the analyte during the procedure cannot be considered constant and a correction factor cannot be determined.…”

“…The IS method was chosen for a simple but crucial reason: since the IS was added directly in the milk (at a very early stage), losses along the various steps of the purification procedure were automatically compensated for [20]. In addition, the choice of this analytical approach is further supported by the following facts: (i) vitamin D2 (IS) is easily distinguished from the analyte in the HPLC trace, as reported in Figure 1; (ii) vitamin D2 exhibits similar chemical properties to vitamin D3, so it is assumed that they undergo a similar loss during sample pretreatment; (iii) the presence of vitamin D2 was excluded in all the evaluated samples; (iv) the extinction coefficient E (1%, 1 cm) at 265 nm is extremely similar for both vitamins D2 and D3 (475 and 480, respectively) [19].…”

Salting-out assisted liquid–liquid extraction coupled with high-performance liquid chromatography for the determination of vitamin D3 in milk samples

“…Saponification is commonly the first step before LLE and SPE and is used to remove neutral lipids, especially triglycerides. This method has been used for the determination of vitamin D in various types of food samples such as infant products [12,16], milk, cream and butter [17], human foods, pet foods and supplements [18], fish oil [19], beef and poultry [20], fortified orange juice [21], dietary supplements and vitamin premixes [22], bovine milk [11], vegetables [23], meat [24] and human serum [6]. Saponification using methanolic or ethanolic KOH at elevated temperatures (60-100°C) for 20-45 min was able to suppress lipids and proteins interferences [25].…”

Effect of phospholipid composition on the structure and physicochemical stability of proliposomes incorporating curcumin and cholecalciferol

“…Concentrations were obtained by absorbance readings at 425 nm in a spectrophotometer (Libra S22; Biochrom, Cambridge, UK). Regarding cholecalciferol retained in proliposomes, these values were obtained by high-performance liquid chromatography (Shmadzu; Kyoto, Japan) according to Staffas and Nyman (2003) [13].…”