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Detection of Vitamin K1 in infant formula milk powder using Venusil C18 Plus

Application Introduction

Green plants (such as cabbage, spinach, and green tea) and livers, lean meat and fat of the animal foodstuff are rich in vitamin K1.



Green plants (such as cabbage, spinach, and green tea) and livers, lean meat and fat of the animal foodstuff are rich in vitamin K1. The test of the vitamin K1in food is helpful to choosing the food which are rich in vitamin K1 and preventing the deficiency disease and other diseases caused by insufficiency intake of vitamin K1.


Material and Reagents

HPLC purity of methanol; analytically grade of ethanol, n-hexane, potassium hydroxide, potassium dihydrogen phosphate, and the ultrapure water; enzyme activity of lipase ≥ 700 U/mg.


Transfer 1-5 g homogenized sample (+/- 0.01 g, and the content of the vitamin K1 is no less than 0.05 μg) to a 50 mL centrifuge tube, add 5 mL warm water to fully dissolve. Add 5 mL phosphate buffer (pH = 8.0), mix well, add 0.2 g lipase and 0.2 g amylase (no need to add amylase in starch-free samples). Put on the cover and vortex for 2-3 min and keep it into the 37℃ ±2℃ constant temperature water bath oscillator for more than 2 hours for fully enzymolysis.

Sample Extraction

Take the sample after enzymolysis, add 10 mL ethanol and 1 g potash respectively, mix well and add 10 mL n-hexane and 10 mL water. Vortex or vibrating extraction for 10 min, centrifuge at 6000 r/min for 5 min (if emulsion happens, appropriately increase the amount of the n-hexane or water). Transfer the supernatant into the 100 mL rotary evaporator bottle. Add another 10 mL n-hexane to the subnatant, repeat the operation again and combine two supernates.

Rotary evaporate the extract to dry (dry the residue liquid under nitrogen), dissolve it to certain volume of 5 mL by methanol, mix will and filter through membrane followed by HPLC analysis.


HPLC column: Venusil C18 Plus; 5 μm, 120 Å, 4.6 × 250 mm;

Zinc reduction column: particle diameter: 50-70 μm, 4.6 × 10 mm;

Mobile phase: Aspirate 900 mL methanol, 100 mL THF and 0.3 mL acetic acid and mix well; add 1.5 g ZnCl2 and 0.5 g anhydrous sodium acetate, sonicate the mixture to completely dissolution and filter it through 0.22 mm membrane;

Detection: excitation wavelength: 243 nm; emission wavelength: 430 nm;

Flow rate: 1.0 mL/min;

Column temperature: 30 °C;

Injection: 10 mL.

Results and Discussion

Table 2 Performance of Venusil C18 Plus in analysis of 0.2 µg/ml Vitamin k1 in spiked milk powder sample


Retention Time/min



Vitamin K1




Figure 1 Chromatogram of 50 µg/mL Vitamin K1 standard solution

Figure 2 Chromatogram of blank milk powder sample

Figure 3 Chromatogram of spiked 0.20 mg/mL Vitamin K1 in milk powder sample


This application described the analysis of Vitamin K1 in infant formula milk powder. Venusil C18 Plus was adopted in this note and excellent separation was gained together with good resolution and accuracy at concentration level of 0.20 mg/mL. The results demonstrated that the analytical method meet the demands for nutrition testing of infant formula milk powder.

Ordering Information




Venusil C18 Plus

5 μm, 120 Å, 4.6   × 250 mm


Zinc Reduction

particle   diameter: 50-70 μm, 4.6 × 10 mm


1.5 mL vials

Screw neck vials,   12 × 32 mm


Caps and Septa

Screw neck cap,   center hole; red silicone/ white PTEE septa, slitted


Syringe   Filter (Nylon)

Monofilm,   13 mm, 0.22 μm


Disposable   Needle-Free injection systems

2 mL, 100/pk





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