Determination of Vitamin K1 in Vegetables

Low-fat plant samples such as fruits and vegetables are extracted with isopropanol and n-hexane to obtain Vitamin K1, which is then purified using a Vitamin K1 SPE column to remove interfering substances like chlorophyll. Vitamin K1 is separated from other impurities using a C18 liquid chromatography column. Post-column zinc reduction is applied, and detection is performed using a fluorescence detector with external standard quantification. (Spinach and apple were used in this experiment.)

Reference Standard: GB 5009.158-2016 National Food Safety Standard – Determination of Vitamin K1 in Food.

Vitamin K1 Standard Stock Solution (1 mg/mL): Weigh 50 mg (accurate to 0.1 mg) of Vitamin K1 standard into a 50 mL volumetric flask, dissolve in methanol, and make up to the mark. Transfer the solution to an amber glass container, store at -20°C, protected from light. Shelf life: 2 months.

Vitamin K1 Standard Intermediate Solution (100 μg/mL): Accurately pipette 10.00 mL of the stock solution into a 100 mL volumetric flask, dilute to the mark with methanol, and mix well. Transfer the solution to an amber glass container, store at -20°C, protected from light. Shelf life: 2 months.

Vitamin K1 Working Solution (1.00 μg/mL): Accurately pipette 1.00 mL of the intermediate solution into a 100 mL volumetric flask, dilute to the mark with methanol, and mix well.

n-Hexane-Ethyl Acetate Mixture (90:10): Measure 90 mL of n-hexane and add 10 mL of ethyl acetate, mix well.

Mobile Phase: Measure 900 mL of methanol, 100 mL of tetrahydrofuran, and 0.3 mL of glacial acetic acid. Mix, then add 1.5 g of zinc chloride and 0.5 g of anhydrous sodium acetate. Dissolve by ultrasonication and filter using a 0.22 µm organic phase filter membrane.

Weigh 5 g of homogenized sample into a 50 mL centrifuge tube, add 5 mL of isopropanol, vortex for 1 minute, and ultrasonicate for 5 minutes. Then add 10 mL of n-hexane, vortex and shake for 3 minutes, centrifuge at 6000 rpm for 5 minutes. Transfer the supernatant to a 25 mL amber volumetric flask. Add 10 mL of n-hexane to the lower layer, repeat the extraction, and combine the supernatants in the volumetric flask, make up to the mark with n-hexane. Accurately pipette 5 mL of the supernatant into a 15 mL centrifuge tube, evaporate to dryness under nitrogen, and dissolve the residue in 1 mL of n-hexane for purification.

SPE Column: Welchrom® Vitamin K1 SPE column, 2g/6mL.

Activation: Wash the column with 5 mL of n-hexane, discard the eluate.

Sample Loading: Load the sample solution, discard the eluate.

Washing: Wash with 5 mL of n-hexane, discard the eluate.

Elution: Elute with 15 mL of n-hexane-ethyl acetate mixture, collect into a 50 mL centrifuge tube, and evaporate to near dryness under nitrogen. Redissolve the residue in 5 mL of methanol and filter through a 0.22 µm membrane for HPLC analysis.

1. The sample loading speed should not be too fast, maintain a rate of 1 drop per second, and keep the packing material moist throughout the process.
2. In the final elution step, the standard specifies 6 mL which is insufficient, and the volume is increased to 15 mL in this method.
3. Spiking levels: For the apple sample, add 0.25 mL of 1 mg/L standard to 5 g of the sample for a spiking level of 0.05 μg/g, resulting in a machine reading of 10 ng/mL. For the spinach sample, add 0.05 mL of 100 mg/L standard to 5 g of the sample for a spiking level of 1 μg/g, resulting in a machine reading of 200 ng/mL (since both apple and spinach naturally contain Vitamin K1, the spiking is based on the amount of Vitamin K1 present in the matrix).
4. Minimize light exposure during the pretreatment process.

Column:Ultisil® XB-C18 4.6×250 mm, 5 μm

Zn Column: Ultisil® Zn 4.6×50 mm

Mobile Phase: 900 mL methanol, 100 mL tetrahydrofuran, 0.3 mL glacial acetic acid, 1.5 g zinc chloride, 0.5 g anhydrous sodium acetate

Flow Rate: 1.0 mL/min

Column Temperature: 30°C

Injection Volume: 10 μL

Detection Wavelength: Excitation 243 nm, Emission 430 nm

Table 1. Spiked recovery