Analysis of Vitamin D in Milk and Infant Formula using UHPLC/MS/MS per AOAC Method 2011.11

By: Olga Shimelis, Reporter US Volume 33.2

Fortification of milk, dairy products, infant and adult nutritionals with vitamin D is required in many countries to provide additional dietary vitamin D. The fortification of milk in the USA, for example, is done at 400 International Units (IU) per eight ounces of milk. The recently adopted AOAC method 2011.11 describes the procedure for the analysis of vitamin D in fortified infant formula. This method was used in the current work to measure vitamin D in the fortified whole milk and in an infant formula sample. Two forms of vitamin D are recognized: vitamin D2 or ergocalciferol, and vitamin D3 or cholecalciferol. Although both forms may be present in the fortified food, vitamin D3 is more commonly used for fortification of dairy products. Vitamin D hydroxymetabolites can also be present in food products at low levels. While quantitative analysis was performed for vitamins D2 and D3, the samples were scanned for the presence of 25- hydroxy metabolites.

Method

AOAC method 2011.11 was followed for analysis of vitamin D in both milk and infant formula. Vitamin D2 and D3 standards and their deuterated analogues were used. The UHPLC column was Titan™ C18, 10 cm x 2.1 mm packed with 1.9 µm particles. The HPLC method was adopted from the standard AOAC method, including mobile phases, gradient, and flow rates. Baseline separation between vitamin D3 and D2 peaks, and between all vitamins and matrix component peaks was obtained on the Titan C18 UHPLC column. Analysis of vitamin D was done using MS/MS with APCI detection. MS transitions used in this study are shown in Table 1.

Table 1. MS Parameters Used for the Measurement of Vitamin D Forms

Compound Q1 Q3
Vitamin D3 385.5 259.2
Vitamin D3 385.5 159.2
Vitamin D2 397.5 125.2
Vitamin D2 397.5 107.2
Vitamin D3-d3 388.5 259.2
Vitamin D2-d3 400.5 125.2
25-OH-Vitamin D3 383.4 257.2
25-OH-Vitamin D2 395.2 269.2

 :

Table 2. Found Concentrations (IU/mL) for Vitamin D with % RSD (n=3)

Sample Vitamin D2 Vitamin D3
Whole milk 0.16 (41%) 4.76 (4%)
Infant forumula 0 5.29 (0.2%)

 

The sample preparation for both milk and infant formula samples was done through saponification, extraction, evaporation, and reconstitution of extracts into LC-compatible solvents. The sample preparation steps following AOAC method 2011.11 are shown in Figure 1.

Preparation Method for Milk and Nutritional Supplements

Figure 1. Preparation Method for Milk and Nutritional Supplements

Results

Chromatography of the vitamin D and hydroxyvitamin D compounds is presented in Figures 2 and 3, respectively. Analyses of vitamin D2 and D3 in extracted milk and infant formula are shown in Figures 4 and 5, respectively. No Vitamin D2 was found in infant formula while a low amount of D2 was found in milk. Concentrations of Vitamin D3 in milk and infant formula were found to be close to the expected fortification levels of at least 4.2 IU/mL (Table 2). No hydroxyvitamin D forms were found in samples of either milk or infant formula. The lowest level for calibration curve was 0.2 IU/mL which would correspond to 0.5 ng of vitamins per mL of milk.

UHPLC/MS Analysis Vitamin D Standards at 1 IU/mL on Titan™ C18

Figure 2. UHPLC/MS Analysis Vitamin D Standards at 1 IU/mL on Titan™ C18
Vitamin D3 is shown as 385/259 parent/daughter transition and vitamin D2 is shown as 397/125 transition using the same Y-scale.

Conditions
column:
Titan C18, 10 cm ~ 2.1 mm I.D., 1.9 µm (Product No. 577124-U); mobile phase: [A] 0.1% (v/v) formic acid in methanol: water (20:80); [B] 0.1% (v/v) formic acid in methanol; gradient: 60 to 90% B in 0.4 min, to 100% B in 0.3 min, held for 7.8 min, to 60% B, held for 1.5 min; flow rate: 0.25 mL/min for 5.55 min, to 0.50 mL/min for 3.84 min, to 0.25 mL/min for 0.7 min; column temp.: 25 °C; detector: MS/MS, APCI(+), MRM as indicated in Table 1; injection: 5 µL; sample: vitamin D2 and D3, 1 IU/mL

 

UHPLC/MS Analysis of 25-OH-Vitamin D Standards at 250 ng/mL on Titan C18<

Figure 3. UHPLC/MS Analysis of 25-OH-Vitamin D Standards at 250 ng/mL on Titan C18
25-OH-Vitamin D3 (red trace) is shown as 383/257 parent/daughter transition and 25-OH-vitamin D2 (blue trace) is shown as 395/269 transition using the same Y-scale.
Conditions same as in Figure 2.

 

UHPLC/MS Analysis Vitamin D in Milk on Titan C18

Figure 4. UHPLC/MS Analysis Vitamin D in Milk on Titan C18
Vitamin D3 is shown as 385/259 parent/daughter transition and vitamin D2 is shown as 397/125 transition using the same Y-scale.
Conditions as in Figure 2.

 

UHPLC/MS Analysis Vitamin D in Infant Formula on Titan C18

Figure 5. UHPLC/MS Analysis Vitamin D in Infant Formula on Titan C18
Vitamin D3 is shown as 385/259 parent/daughter transition and vitamin D2 is shown as 397/125 transition using the same Y-scale.
Conditions as in Figure 2.

 

Conclusion

Presented here is a method for the determination of nutritionally important vitamin D forms as standards, and extracted from milk and infant formula after saponification following AOAC method 2011.11.

Legal Information

Titan is a trademark of Sigma-Aldrich Co. LLC
Acrodisc is a registered trademark of Pall Corporation

Materials