Huili GONG, Guoxia DUAN, Lijun LIU, Shuo TANG, Cuizhi LI, Zhiyong LU

Inner Mongolia Yili Industrial Group Co. Ltd., Hohhot 010110, China

Abstract [Objectives] To make simultaneous determination of vitamin K1 (VK1) and vitamin K2 (VK2, mainly MK-4, MK-7 and MK-9) in milk and dairy products. [Methods] A high performance liquid chromatographic method was developed for the simultaneous determination of vitamin K1 (VK1) and vitamin K2 (VK2, mainly MK-4, MK-7 and MK-9) in milk and dairy products. After enzymatic digestion, the samples were extracted with hexane for VK1, MK-4, MK-7 and MK-9, subjected to gradient elution at excitation wavelength 243 nm and emission wavelength 430 nm, detected by high performance liquid chromatography with fluorescence detector and quantified by external standard method. [Results] The linearity of VK1, MK-4, MK-7 and MK-9 was good in the concentration range of 2.5-1 000 ng/mL with the correlation coefficients greater than 0.999; The relative standard deviations (RSD) of VK1, MK-4, MK-7 and MK-9 in milk powder, liquid milk and yogurt were 1.32%-5.05%, 1.10%-2.48% and 2.20%-3.47%, respectively; the recovery rates of VK1, MK-4, MK-7 and MK-9 at different levels in milk powder, liquid milk and yogurt were 81.1%-108%, 81.8%-103% and 82.1%-99.2%, respectively. [Conclusions] The method is rapid, accurate, reproducible and capable of simultaneous determination of VK1, MK-4, MK-7 and MK-9.

Key words High performance liquid chromatography (HPLC), Vitamin K1, Vitamin K2, MK-4, MK-7, MK-9

1 Introduction

Vitamin K is a class of compounds with a similar structure. According to the 2-methyl-1, 4-methanonaphthoquinone parent nucleus and different side chains at position 3[1-4], natural vitamin K can be divided into (VK1) and vitamin K2(VK2), vitamin K1is also known as chlorophyll quinone and is abundant in green leafy vegetables (e.g.cabbage, spinach, lettuce). Vitamin K2is mainly produced by microbial metabolism and is present in small amounts in meat, dairy products and fermented foods, menatetrenone 9 (MK-9)[5-8], and so on. Vitamin K1is mainly stored in the liver. Thus, it plays a pivotal role in synthesis of coagulation proteins[9-10]. In contrast, vitamin K2has important biological activities and medicinal value, and it has good efficacy in improving osteoporosis. Some studies have shown that vitamin K2has the ability to inhibit cardiovascular calcification, prevent liver cirrhosis, prevent Alzheimer’s disease, and reduce the risk of type II diabetes[11-14]. Other studies also have shown that VK2can help carboxylate primary osteocalcin secreted by osteoblasts into active osteocalcin, thus promoting the deposition of calcium ions into bone in the blood. Vitamin K2generates bone proteins, which then work with calcium to produce bone mass, accordingly increase bone density and prevent fractures[15-16]. Therefore, the content of vitamins K1and K2is of increasing interest. Currently, there are high performance liquid chromatography (HPLC) for the determination of MK-7 in multi-component formulations containing vitamin K2[17], determination of vitamin K2(Menaquinone-7) in milk powder by High Performance Liquid Chromatography[18], HPLC for the simultaneous determination of vitamin D3and vitamin K2[19], two liquid chromatographic methods for the determination of vitamin K2in health product softgels[20],etc.In contrast, there are few studies on the simultaneous determination of VK1, MK-4, MK-7 and MK-9 in milk powder, liquid milk and yogurt. Therefore, in this paper, we established a method of high performance liquid chromatography for the simultaneous determination of VK1, MK-4, MK-7 and MK-9 in milk and dairy products.

2 Materials and methods

2.1 Reagents and materialsDairy products were randomly selected from the market. Standard product: vitamin K1(C31H46O2): purity ≥ 99%, Merck Sigma Biologicals; vitamin K2standards: (C31H40O2(MK-4): purity ≥ 99.6%, Merck Sigma Biologicals; (C31H64O2(MK-7): purity ≥ 98.0%, USP Pharmaceutical Standard R&D (Shanghai) Inc. Ltd.; (C31H80O2(MK-9): purity ≥ 97.0%, Shanghai Zhihua Chemical Technology INC. Damao Chemical Reagent Factory; Zinc chloride (superior pure) Tianjin Damao Chemical Reagent Factory; potassium dihydrogen phosphate (analytical pure) Tianjin Shuisheng Fine Chemical INC.

2.2 Instruments and equipmentThe 1260 high performance liquid chromatograph (with fluorescence detector) Agilent Technologies, USA; AL204 analytical balance METTLER-TOLEDO, Switzerland; Multi Reax multi-point vortex oscillator Heidolph, Germany; pro UV ultra-pure water machine Thermo Fisher, USA; SHZ-82 water bath thermostatic oscillator Jiangsu Golden Instruments Technology INC. Ltd.; MS 3 basic vortex oscillator IKA, Germany; RVID rotary evaporator IKA, Germany; KQ-300DE ultrasonic oscillator Kunshan Ultrasonic Instruments INC.

2.3 Methods

2.3.1Sample preparation. (i) Enzymatic digestion. Accurately weighed 1-5 g (accurate to 0.01 g) of homogenized sample in a 50 mL centrifuge tube, added 5 mL of warm water (37±2) ℃ to dissolve (liquid sample directly weigh 5 g), added 5 mL of phosphate buffer (pH=8.0), mixed well, added 0.2 g of lipase, covered and vortex treated for 2-3 min. After mixing, placed the sample in a constant temperature water bath shaker at 37 ℃±2 ℃ for more than 2 h to make it fully enzymatic.

(ii) Extraction. Took out the enzymatic sample, added 10 mL of ethanol and 1 g of potassium carbonate, mixed well, added 10 mL of hexane and 10 mL of water, vortex treated or shook for 10 min, centrifuged at 6 000 r/min for 5 min, transferred the supernatant to a 100 mL vortex bottle, added 10 mL of hexane to the lower layer, repeated the operation twice, and combined the supernatant into the above vortex bottle.

(iii) Concentration. The above hexane extract was evaporated to dryness, transferred to a 5 mL volumetric flask with the methanol solution and fixed the volume, shaken well, filtered by 0.22 μm filter membrane, and the filtrate was fed into the sample.

2.3.2Chromatographic reference conditions. Chromatographic column: Agilent ZORBAX SB-C18column (150 mm×4.6 mm × 5 mm); zinc reduction column (50 mm × 4.6 mm); flow rate: 1 mL/min; detection wavelength: excitation wavelength 243 nm, emission wavelength 430 nm; injection volume: 10 μL; mobile phase: A: 1 000 mL of methanol, ice acetic acid A: 1 000 mL of methanol and 0.3 mL of glacial acetic acid, mixed well, added 1.5 g of zinc chloride and 0.5 g of anhydrous sodium acetate, and filtered with 0.22 μm organic filter membrane; B: 1 000 mL of tetrahydrofuran and 0.3 mL of glacial acetic acid, mixed well, added 1.5 g of zinc chloride and 0.5 g of anhydrous sodium acetate, and filtered with 0.22 μm organic filter membrane. The gradient elution conditions are shown in Table 1.

Table 1 Mobile phase gradient elution program

2.3.3Preparation of standard solutions. Vitamin K1standard stock solution (1 mg/mL): accurately weighed 50 mg (accurate to 0.1 mg) of vitamin K1(VK1) standard in a 50 mL volumetric flask, dissolved with methanol and fix the volume to the scale. Transferred the solution to a brown glass container and stored in a refrigerator at -20 ℃, protected from light for 2 months. It should be noted that the standard stock solution needs to be corrected for concentration before use[21].

2.3.4Preparation of vitamin K2standard stock solutions 4-methanonaphthoquinone (MK-4), 7-methanonaphthoquinone (MK-7) and 9-methanonaphthoquinone (MK-9) (100 μg/mL): accurately weigh 0.010 0 g of MK-4, MK-7 and MK-9 standards, respectively, dissolve with isopropyl alcohol solution and fix the volume in a 100 mL volumetric flask, store at -20 ℃ away from light, with a storage period of 2 months.

2.3.5MK-4, MK-7, MK-9 and VK1standard use solution (1.00 μg/mL): accurately aspirated 1.00 mL MK-4, MK-7, MK-9 standard stock solution and 1.00 mL VK1standard intermediate solution in a 100 mL volumetric flask, added methanol to the scale and shook well, and prepared as needed.

2.3.6Standard series working solution: accurately aspirated 0.025, 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, 4.00, 5.00 and 8.00 mL of MK-4, MK-7, MK-9 and VK1standard solutions, respectively in a 10 mL volumetric flask, added methanol to dissolve and fix the volume to the scale. The concentrations of MK-4, MK-7, MK-9 and VK1standard series working solutions were 2.5, 5, 10, 20, 50, 100, 200, 400, 500, 800 and 1 000 ng/mL, respectively. Prepared for later use.

2.4 Data ProcessingEXCEL was used to process and plot the data and plots involved.

3 Results and analysis

3.1 Selection of solvent for preparing reserve solutionIn the selection of the solvent for the preparation of vitamin K2standard stock solution, methanol and isopropanol were compared, and the stock solution prepared by methanol showed crystalline precipitation after freezing and storage, while isopropanol had good solubility. Therefore, isopropanol was selected as the solvent for the preparation of vitamin K2standard stock solution.

3.2Analysis of chromatographic conditions

3.2.1Selection of detection wavelengths. The excitation and emission wavelengths (λex=243 nm, λem=430 nm; λex =320 nm, λem =410 nm, and λex =326 nm, λem =432 nm) were optimized for the treated samples.

As can be seen from Fig.1, the response values of the peaks were greatest when λex=243 nm and λem=430 nm as the optimal wavelengths for the chromatographic conditions.

Fig.1 Effect of different wavelengths on the response values of chromatographic peaks

3.2.2Selection of isocratic elution program. Fig.2 shows the chromatograms of the mobile phases methanol: tetrahydrofuran=9∶1 at the flow rates of 0.5 mL/min (Fig.2A), 0.8 mL/min (Fig.2B), 1.0 mL/min (Fig.2C) and 1.5 mL/min (Fig.2D), respectively, and it can be seen from Fig.2 that the effect of the solvent on the peaks could not be changed by the adjustment of the flow rate. Moreover, there was a significant broadening of the MK-9 peak with time. The conventional isocratic elution method cannot achieve effective separation of the peaks.

Fig.3 Chromatogram with different mobile phase ratios

3.2.3Selection of gradient elution program. Since the conventional isocratic elution procedure could not achieve the effective separation of the peaks, we used a gradient elution procedure with a flow rate of 1.0 mL/min for the separation of the peaks in this study.

In Fig.3, A is the mobile phase methanol∶tetrahydrofuran=90∶10, and the mobile phase ratio was changed to 75∶25 after 7.1 min; B is the mobile phase methanol∶tetrahydrofuran=95∶5, and the mobile phase ratio was changed to 90∶10 after 6 min and 80∶20 after 9 min; C is the mobile phase methanol∶tetrahydrofuran=93∶7, and the mobile phase ratio was changed to 80∶20 after 4 min; D is the mobile phase methanol∶tetrahydrofuran=93∶7, and the mobile phase ratio was changed to 80∶20 after 7.1 min. It can be seen from Fig.3A that MK-4 was not well separated from the impurity peaks, and the baseline was not smooth when MK-7 came out; MK-7 in Fig.3B failed to come out on a smooth baseline; VK1in Fig.3C did not come out on a smooth baseline; Fig.3D extended the peak emergence time of the target peak by increasing the proportion of methanol, which made the target peak effectively separated from the impurity peaks. After analyzing for a period of time, increasing the proportion of tetrahydrofuran advanced the retention time of MK-9 peak to 18 min, and also improved the spreading phenomenon of MK-9 peak. Therefore, we used a gradient elution procedure as the optimal chromatographic conditions in this paper.

3.2.4Linearity, limit of detection and limit of quantification. As can be seen from Table 2, the correlation coefficients of the standard curves for VK1, MK-4, MK-7 and MK-9 were all greater than 0.99, and the linear ranges met the requirements.

Table 2 Standard curve and correlation coefficient

As shown in Table 3, the S/N ratios of the limit of detection of VK1, MK-4, MK-7 and MK-9 in milk powder, milk and yogurt samples were all greater than 3, which met the requirements of the limit of detection; the S/N ratios of the limit of quantification of VK1, MK-4, MK-7 and MK-9 in milk powder, milk and yogurt samples were all greater than 10, which met the requirements of the limit of quantification.

3.2.5Precision test. TheRSDvalues of milk powder, liquid milk and yogurt were calculated by the limits of quantification,constant points and 5 times the limits of quantification six times according to the experimental method.

Fig.4 Standard chromatogram of VK1, MK-4, MK-7, and MK-9

Table 3 Limit of detection and limit of quantification

As can be seen from Table 4, repeatability experiments for different samples with different contents: According to the requirements of GB/T 27417-2017, theRSD≤15% for 100 μg/kg of the measured component, and theRSD≤11% for 100 mg/kg of the measured component. As indicated in Table 4, the maximum value ofRSDof the experimental results was 5.05%<11%, and the repeatability met the requirements.

Table 4 Results of precision

(Continued)

3.2.6Accuracy test. In accordance with the experimental method, we performed six spiking experiments at the limit of quantification, constant point and 5-fold limit of quantification for milk powder, liquid milk and yogurt, and calculated the recovery results and average recovery results were for milk powder, liquid milk and yogurt.

From Table 5, it can be seen that the correctness experiments for different samples with different contents: According to the requirements of GB/T 27417-2017, the content of the measured component < 0.1 mg/kg, the recovery range was 60%-120%, and the experimental result was 81.1%-104%, the content of the measured component was in the range of 0.1-1.0 mg/kg, the recovery range was 80%-110%, and the experimental result was 81.3%-108%, so the accuracy met the requirements.

Table 5 Accuracy of results

(Continued)

3.2.7Sample results. We obtained samples of milk powder, milk and yogurt from random sampling in the market.

Table 6 Sample test results

4 Conclusions

In this study, we established a method for the simultaneous determination of VK1, MK-4, MK-7 and MK-9 in food stuffs by high performance liquid chromatography (HPLC). The samples were digested by enzymatic digestion, and VK1, MK-4, MK-7 and MK-9 were extracted with n-hexane at excitation wavelength 243 nm and emission wavelength 430 nm with gradient elution, detected by liquid chromatography fluorescence detector and quantified by external standard method. The results showed that this method has good linearity, and the reproducibility and accuracy tests met the requirements, so it is expected to provide a method for the simultaneous determination of vitamin K1, MK-4, MK-7 and MK-9 contents.