Abstract:

Vitamins K₁ and K₂ are both essential fat-soluble vitamins for the human body. K₁ was once used in cosmetics for its efficacy in improving dark circles by promoting periorbital circulation. However， due to its potential to cause severe allergic reactions， it is now banned in cosmetics in China and the European Union. K₂， particularly the MK-7 form， has a similar structure to K₁ but features a different side chain. It has gained attention for its skin benefits， including soothing， antioxidant， and anti-aging properties. Although there are no reports of its use in cosmetics in China yet， it has been registered three times as a new cosmetic ingredient， indicating significant market potential. Most existing methods for detecting vitamins K₁ and K₂ focus on pharmaceuticals and dietary supplements， with few studies addressing their analysis in cosmetics， especially vitamin K₂. Given the structural similarity between the two compounds， a reliable method for their simultaneous determination in cosmetic products is needed. However， cosmetic matrices are complex and often interfere with analysis. Conventional sample preparation techniques， such as liquid-liquid extraction and solid-phase extraction， are time-consuming and labor-intensive. The QuEChERS method offers a faster， simpler， and more cost-effective alternative. In this study， we selected six common types of cosmetics—water-based liquids， emulsions， creams， gels， powders， and oils， to develop and validate a simultaneous quantification method for vitamins K₁ and K₂. A QuEChERS-based sample preparation method coupled with high performance liquid chromatography （HPLC） was developed for the simultaneous quantification of vitamins K₁ and K₂ in cosmetics. To enhance specificity and confirmation capability， a complementary method using high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） was also established. Samples were first pre-dispersed with saturated sodium chloride solution， extracted by n-hexane under ultrasonication， and purified using QuEChERS pretreatment technique （containing 150 mg MgSO₄， 50 mg PSA， and 25 mg C₁₈）. For HPLC analysis， separation was performed on a CAPCELL PAK C₁₈ AQ column （250 mm×4.6 mm， 5 µm） using methanol-isopropanol （80∶20， volume ratito） as the mobile phase， with detection at 270 nm. For HPLC-MS/MS confirmation， an ACQUITY UPLC BEH C₁₈ column （50 mm×2.1 mm， 1.7 µm） was employed， with methanol containing 0.05% （volume fraction） formic acid and 5 mmol/L ammonium formate as the mobile phase. Electrospray ionization in positive mode （ESI⁺） and multiple reaction monitoring （MRM） were used for detection. Both HPLC and HPLC-MS/MS methods demonstrated excellent performance for the determination of vitamins K₁ and K₂. In the HPLC method， both analytes showed good linearity over the range of 0.1–50 μg/mL （r>0.999）， with limits of detection （LOD） and quantification （LOQ） of 0.3 μg/g and 1.0 μg/g， respectively. The spiked recoveries ranged from 93.2%-104.5% with RSDs below 5%. For the HPLC-MS/MS method， linearity was observed in the range of 0.005–0.5 μg/mL （r>0.999）， with LOD and LOQ values of 0.02 μg/g and 0.05 μg/g， respectively. Recoveries in this case fell within 89.4%–108.2%， accompanied by RSDs of less than 10%. The method was successfully applied to analyze 30 cosmetic samples spanning six different matrix types. Neither vitamin K₁ nor K₂ was detected in any sample. The proposed methodology is rapid， simple， sensitive， and accurate， making it suitable for routine determination of vitamins K₁ and K₂ in diverse cosmetic products. It offers reliable technical support for quality control and regulatory compliance and demonstrates the utility of QuEChERS sample preparation for the analysis of other cosmetic ingredients.

Key words: QuEChERS, high performance liquid chromatography （HPLC）, high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）, cosmetic, vitamin K₁, vitamin K₂