Analysis of vitamin K in food using LC-MS

Importance of the Topic

Vitamin K is a lipid-soluble vitamin essential for blood coagulation and bone metabolism. Accurate determination of its forms in foods underpins nutritional studies, regulatory compliance and quality control. Traditional HPLC techniques demand extensive sample cleanup to avoid matrix interferences, leading to longer processing times and higher labor costs. Introducing LC-MS leverages molecular specificity and streamlined preparation, meeting the growing need for efficient, high-throughput analysis in food chemistry.

Objectives and Study Overview

This work demonstrates a simplified LC-MS approach for quantifying phylloquinone (vitamin K1) and menaquinone-4 (vitamin K2) in complex food matrices. Using atmospheric pressure chemical ionization (APCI) in negative mode, the method aims to maintain high sensitivity and linear response while minimizing sample pretreatment. Margarine and fermented soy product (natto) were selected as representative matrices to evaluate method performance.

Methodology and Instrumentation

Samples (0.1 g) were extracted with 8 mL hexane and 1 mL water, vortexed and centrifuged. The upper organic phase was evaporated under reduced pressure and reconstituted in ethanol for injection. Chromatography employed a Shim-pack VP-ODS column (2.0 × 150 mm) with 25% 2-propanol in methanol at 0.2 mL/min and 40 °C. Detection was performed on an LC-MS system with APCI in negative-ion mode (probe voltage –4.0 kV, CDL 250 °C, block heater 200 °C, nebulizing gas 2.5 L/min). Calibration from 1 to 100 ppb yielded correlation coefficients of r2 = 0.99969 for phylloquinone and r2 = 0.99947 for menaquinone-4.

Main Results and Discussion

Phylloquinone and menaquinone-4 displayed clear, separate peaks in mass chromatograms and selective SIM traces. Natto samples contained 9.06 ppb phylloquinone and 9.38 ppb menaquinone-4, equating to 2.90 ng and 3.00 ng per 0.1 g. Margarine showed 58.03 ppb phylloquinone (18.56 ng/0.1 g) with no detectable menaquinone-4. Recovery rates approached 100%, confirming the efficiency of the simplified extraction procedure and the method’s robustness against matrix effects.

Benefits and Practical Applications

The LC-MS protocol markedly reduces sample preparation and analysis time compared to conventional HPLC, while maintaining high sensitivity and accuracy. Its resilience to interfering substances supports reliable nutritional labeling, routine quality assurance and regulatory testing across diverse food products with minimal manual intervention.

Future Trends and Potential Applications

Emerging advancements in tandem MS (MS/MS) and high-resolution platforms will further improve detection limits and enable simultaneous profiling of multiple vitamins. Integration with ultra-high-performance LC will shorten analysis times and increase throughput. Automated sample prep coupled with advanced data software could facilitate large-scale screening programs in food industry, clinical nutrition and epidemiological research.

Conclusion

The presented LC-APCI-MS method offers a rapid, reliable and sensitive solution for vitamin K analysis in complex food matrices. Its simplified pretreatment, high recovery and robust performance position it as a compelling alternative to traditional HPLC workflows for routine nutritional and quality control analysis.

Instrumentation

• Shim-pack VP-ODS column (2.0 × 150 mm)
• Mobile phase: 25% 2-propanol in methanol, flow rate 0.2 mL/min
• Column temperature 40 °C; Injection volume 10 µL
• APCI negative-ion mode (probe voltage −4.0 kV; CDL 250 °C; block heater 200 °C; nebulizing gas 2.5 L/min)