Measurement of Vitamin D in Milk and Infant Formulas Using Automated Online Sample Preparation with Liquid Chromatography/ Tandem Mass Spectrometry

Significance of the topic

The accurate measurement of vitamin D in fortified foods is critical to ensure nutritional compliance and prevent deficiency or toxicity. Infants rely on properly supplemented formulas to support bone development and overall health.

Objectives and overview

This study presents an automated online sample preparation LC-MS/MS approach to quantify vitamin D2 and D3 in infant formula. The method aims to simplify sample handling, reduce solvent use, and improve throughput compared to traditional saponification and manual cleanup.

Methodology and instrumentation

• Sample preparation using the Thermo Scientific Transcend TLX-1 system with TurboFlow technology, eliminating saponification steps.
• Matrix calibration prepared in low-fat half and half spiked with deuterated internal standards.
• Extraction of powdered and liquid infant formula by ethanol/methanol, ultrasonication, centrifugation, and filtration.
• Online cleanup on TurboFlow C18-P XL column followed by separation on Hypersil GOLD analytical column.
• Detection using Thermo Scientific TSQ Access MAX triple quadrupole mass spectrometer with APCI source in positive mode and optimized gas and temperature settings.

Main results and discussion

• Representative chromatograms at 10 ng/mL displayed sharp peaks and high signal intensity for both vitamins.
• Matrix-matched calibration curves exhibited linearity over two orders of magnitude with r² above 0.99.
• Limits of quantitation were 1.0 ng/mL (0.3 μg/100 g) for D2 and 0.5 ng/mL (0.15 μg/100 g) for D3.
• Analysis of commercial infant formulas detected only vitamin D3, showing less than 15% deviation from label claims and CV below 11%.

Benefits and practical applications

• Removal of saponification and manual extraction reduces analysis time and solvent consumption.
• Automated online preparation ensures consistent recoveries and higher sample throughput.
• High specificity and sensitivity enable reliable quantification in complex dairy matrices.

Future trends and opportunities

• Extension of this workflow to other fortified food products such as dairy beverages and nutritional bars.
• Implementation of multiplexed TurboFlow systems (TLX-2 or TLX-4) for simultaneous multi-channel analysis.
• Integration with advanced software for real-time data processing and remote quality control monitoring.

Conclusion

The automated LC-MS/MS method employing TurboFlow technology offers a rapid, solvent-efficient, and reproducible solution for vitamin D measurement in infant formulas. It supports regulatory compliance and enhances nutritional quality assurance in food manufacturing.

References

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