Analysis of vitamin D in cheese by APCI LC-MS/MS using the Bruker EVOQ™ LC-TQ Elite mass spectrometry system

Significance of the Topic

Vitamin D is critical for calcium homeostasis and bone health, and its deficiency or excess poses significant health risks. Cheese, a widely consumed dairy product, can be enriched with vitamin D2 and D3 to help meet dietary recommendations. Accurate determination of vitamin D content in enriched cheese is essential for regulatory compliance and consumer safety.

Objectives and Study Overview

The primary goal of this study was to develop and validate a rapid, simple, and robust analytical method for quantifying vitamin D2 and D3 in cheese. The method employs liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with an atmospheric pressure chemical ionization (APCI) interface on the Bruker EVOQ™ LC-TQ Elite system. Key performance criteria included sensitivity, linearity, precision, and recovery.

Methodology and Used Instrumentation

Sample Preparation:

• Homogenize 10 g of cheese and add 40 mL pyrogallol solution (2%) and 20 mL 50% KOH.
• Introduce nitrogen and agitate overnight for saponification.
• Extract with 30 mL hexane containing 12.5 mg/L BHT.
• Evaporate 10 mL extract, reconstitute in 1 mL acetonitrile/water (70/30), and filter through 0.45 µm PTFE.

Instrumentation:

• Bruker EVOQ™ LC-TQ Elite MS/MS with APCI source (spray current ±100 µA, probe temp 250 °C).
• Bruker Elute™ UHPLC, Intensity Solo C18 column (100 × 2.1 mm).
• Mobile phases: water +0.1% formic acid and methanol +0.1% formic acid, gradient elution over 9 min.
• MRM transitions: vitamin D3 385.5 > 259.1 (quantifier), vitamin D2 397.6 > 107.0, with confirmation ions.

Main Results and Discussion

Linearity and Calibration:
The method exhibited excellent linearity (R² > 0.999) over 0.5–1000 µg/kg, with RSD < 25%.

Sensitivity:
Limit of quantification (LOQ) was 0.5 µg/kg and limit of detection (LOD) < 0.1 µg/kg (S/N > 100).

Precision:
Repeatability studies at 0.5, 1, 10, 50, and 100 ppb yielded RSDs < 3.5%.

Accuracy:
Recovery experiments on quark and cottage cheese spiked at 250 ppb and 100 ppb showed recoveries between 90% and 95%, confirming method accuracy.

Benefits and Practical Applications

The developed protocol combines high sensitivity and throughput with minimal sample preparation, making it suitable for routine quality control in food laboratories. Its robustness allows reliable monitoring of vitamin D enrichment in cheese, ensuring compliance with EFSA dietary intake and upper limit guidelines.

Future Trends and Applications

Advances may include:

• Integration with automated sample preparation platforms for higher throughput.
• Expansion to simultaneous quantification of other fat-soluble vitamins (A, E, K).
• Application to a wider range of dairy matrices and fortified foods.
• Use of high-resolution mass spectrometry for enhanced identification of vitamin D metabolites.

Conclusion

A validated APCI-LC-MS/MS method on the Bruker EVOQ™ LC-TQ Elite enables rapid, sensitive, and accurate determination of vitamin D2 and D3 in cheese. The approach meets stringent analytical criteria and supports regulatory compliance and consumer safety in food quality control laboratories.

Reference

[1] MPI Technical Paper No. 2014/03, Determination of vitamin D in foods: Current knowledge and data gaps, New Zealand Government
[2] von Hurst PR, Stonehouse W, Coad J. Vitamin D supplementation reduces insulin resistance in South Asian women living in New Zealand. Br J Nutr. 2010;103(4):549-555.
[3] Verkaik-Kloosterman J, Seves SM, Ocké MC. Vitamin D concentrations in fortified foods and dietary supplements intended for infants. Food Chem. 2017;221:629-635.
[4] EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA). Dietary reference values for vitamin D. EFSA J. 2016;14(10):4547.
[5] Martin R, van’t Slot G. Online Analysis of 25-OH-Vitamin D2/D3 in Plasma and Serum with the EVOQ LC-Triple Quad. Bruker App Note LCMS-103; 2015.
[6] EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA). Tolerable Upper Intake Level of vitamin D. EFSA J. 2012;10(7):2813.