KR20220017280A - Analytical method to detect Vitamin K and Vitamin D in food using QuEChERS - Google Patents

Abstract

The present invention relates to a method for simultaneously detecting vitamin K and vitamin D in a sample, which includes: (a) a step of obtaining an extract by adding an extraction solvent to a sample and performing agitation; (b) a step of purifying the extract by dispersive solid phase extraction (d-SPE); and (c) a step of analyzing vitamin K and vitamin D in the extract purified in the above step (b) by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). In the present invention, optimization is performed in view of the type of the extraction solvent in the process of sample pretreatment, pretreatment and purification reagent contents, and so on as factors affecting the efficiency of extraction. As a result, the method of the present invention ensures high vitamin K and vitamin D recovery rates and is eco-friendly with the extraction time and solvent volume thereof reduced by approximately 90% or more from previously suggested methods.

Description

Method for simultaneous detection of vitamin K and vitamin D in food {Analytical method to detect Vitamin K and Vitamin D in food using QuEChERS}

The present invention relates to a method for simultaneously detecting vitamin K and vitamin D in a sample, and more particularly, the method comprising: (a) adding an extraction solvent to a sample and stirring to obtain an extract; (b) performing dispersive solid phase extraction (d-SPE) on the extract to purify the extract; and (c) analyzing vitamin K and vitamin D in the extract purified by step (b) by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS), vitamin K and It relates to a method for simultaneous detection of vitamin D.

Vitamin K (Vitamin K) is a type of fat-soluble vitamin and is divided into vitamin K1 (phylloquinone, phylloquinone) and vitamin K2 (menaquinone, menaquinone) according to the chain shape. Vitamin K1 is mainly synthesized in plants and is found in many green leafy vegetables because it is directly involved in photosynthesis. Vitamin K1 is the precursor to most vitamin K found in nature, and gut bacteria can convert vitamin K1 to vitamin K2. Vitamin K2 has recently been reported to have the effect of preventing cardiovascular diseases and increasing calcium absorption, and studies for application to age-friendly foods for the elderly are active.

On the other hand, the analysis method of vitamin K1, D2, and D3 is prescribed in the general test method of the Food and Food Additives Code, but the analysis method of vitamin K2 is not prescribed, and the analysis method of vitamin K and vitamin D, one of the fat-soluble vitamins, is also defined differently. The volume of the solvent used for extraction and analysis is also large as 5 to 100 mL, and the extraction time is also relatively long (30 minutes to 2 hours). In addition, in recent years, the demand for food with fortified nutrients has increased due to the imbalance in nutritional intake of modern people and the aging of the population. It is being gradually strengthened.

Therefore, the inventors of the present invention consider the situation that the importance of the efficacy and research of vitamin K is increasing and the systematic management of foods fortified with nutrients at the national level is being strengthened. As a result of earnest efforts to develop a method capable of simultaneously detecting/analyzing vitamins K1, D2 and D3, which are fat-soluble vitamins, along with vitamin K2, the present invention was completed. The detection method of the present invention is a factor affecting extraction efficiency, and as a result of optimization in consideration of the type of extraction solvent and the content of the purification reagent in the pre-treatment process of the sample, the recovery rate of vitamin K and vitamin D is high, but it has been previously proposed The extraction time and solvent volume are reduced by more than 90% compared to the previous method, which is environmentally friendly.

SUMMARY OF THE INVENTION The present invention aims to solve the above problems and other problems related thereto.

An exemplary object of the present invention comprises the steps of (a) adding an extraction solvent to a sample and stirring to obtain an extract; (b) performing dispersive solid phase extraction (d-SPE) on the extract to purify the extract; and (c) analyzing vitamin K and vitamin D in the extract purified by step (b) by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS), vitamin K and To provide a method for simultaneous detection of vitamin D.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem for solving the above-mentioned problems, and another problem not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

One aspect of the present invention for achieving the above object provides a method for simultaneously detecting vitamin K and vitamin D in a sample. More specifically, one aspect of the present invention for achieving the above object, (a) adding an extraction solvent to the sample and stirring to obtain an extract; (b) performing dispersive solid phase extraction (d-SPE) on the extract to purify the extract; and (c) analyzing vitamin K and vitamin D in the extract purified by step (b) by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS), vitamin K and A method for simultaneous detection of vitamin D is provided.

In the detection method of the present invention, in performing the sample pretreatment (QuEChERS pretreatment) step and liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry, LC-APCI-MS) step, extraction As factors affecting the efficiency, it was optimized in consideration of the type of extraction solvent and the content of the purification reagent in the pretreatment process of the sample. About 90% or more of the solvent volume is reduced, which is environmentally friendly.

Hereinafter, the method according to the present invention will be described step by step.

The detection method of the present invention includes (a) adding an extraction solvent to a sample and stirring to obtain an extract. In addition, step (a) may include adding magnesium sulfate (MgSO4) and NaCl and centrifuging.

As used herein, the term "sample" suffices as long as it contains or is thought to contain vitamin K and/or vitamin D, that is, vitamin K1, K2, D2 and/or D3, and more specifically, the sample includes meat, sausage, Bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes are included. The sample may be pre-treated to be applied to dispersive solid phase extraction (d-SPE) or pre-treatment with QuEChERS, which may be liquefied, but is not limited to aqueous solution. In addition, various additives (eg, antifoaming agents) may be added to the sample by those skilled in the art to comply with the dispersed solid phase extraction method (d-SPE) or QuEChERS pretreatment.

In the present invention, the term "extraction solvent" is added to the sample prior to the extraction step using the salting out effect through magnesium sulfate (MgSO ₄ ) in the pretreatment process of the sample, and may mean acetonitrile. In an embodiment of the present invention, four extraction solvents acetonitrile (CH ₃ CN), methanol (CH ₃ OH), ethanol, and acetonitrile / methanol mixed solvent (1:2, v / v) are used to compare the extraction efficiency As a result, when extracted with a mixed solvent of ethanol and acetonitrile/methanol, the peak separation was not well and the extraction efficiency could not be accurately confirmed, whereas when acetonitrile was used, the recovery value was relatively higher than that of methanol ( Fig. 1).

In the present invention, (a) adding an extraction solvent to the sample and stirring to obtain an extract, that is, the "extraction step" is to perform organic solvent layer separation using the salting-out effect in the pretreatment of the sample (QuEChERS pretreatment). In the present invention, the content of the pretreatment reagent is set as a variable that can affect the extraction efficiency. In an embodiment of the present invention, as a result of optimizing the contents of magnesium sulfate (MgSO ₄ ) and NaCl as the pretreatment reagent added using the reaction surface analysis method, the MgSO ₄ content is 0.5% to 60% of the concentration of the sample, preferably is 30% to 35% of the sample, more preferably 33.3%, and the content of NaCl is 4% to 15% of the sample concentration, preferably 5% to 10% compared to the sample, more preferably 8.3%. It was confirmed that it exhibited the highest vitamin K content (FIG. 3).

In the present invention, the term "vitamin K" is a type of fat-soluble vitamin, essential for the synthesis of blood clotting factor protein, and may be vitamin K1 (phylloquinone, phylloquinone) or vitamin K2 (menaquinone, menaquinone) depending on the form of the chain. have. In addition, in the present invention, "vitamin D" is one of the fat-soluble vitamins, plays an important role in maintaining calcium homeostasis in the body, and depending on the structure, vitamin D2 (ergocalciferol, ergocalciferol) mainly found in fungal plants and mainly found in animals It may be vitamin D3 (cholecalciferol, cholecalciferol). The vitamin K or vitamin D may be naturally present or artificially added as a component in the sample or food of the present invention, but is not particularly limited thereto.

The detection method of the present invention includes (b) performing a dispersive solid phase extraction (d-SPE) on the extract to purify the extract. In addition, the step (b) may include the addition of magnesium sulfate (MgSO ₄ ) and primary secondary amine (PSA) and centrifugation.

In the present invention, "dispersive solid phase extraction (d-SPE)" is a pretreatment method for food and agricultural products, and unless otherwise specified in the present invention, those skilled in the art will It can be carried out according to the method described above, and is not particularly limited. Specifically, the dispersed solid phase extraction method includes an extraction step using a salting out effect through magnesium sulfate (MgSO ₄ ) and a dispersive solid phase extraction (d-SPE) step of purifying by directly adding an adsorbent, and in the present specification In this case, it may mean QuEChERS pretreatment or QuEChERS extraction process of the sample.

In the present invention, (b) the step of purifying by performing the dispersion solid phase extraction method, that is, the "purification step" is used to perform the d-SPE step of removing organic acids and moisture by directly adding an adsorbent to the sample in the QuEChERS pretreatment. , In the present invention, the content of the purification reagent is set as a variable that can affect the extraction efficiency, and the purification reagent may include magnesium sulfate (MgSO ₄ ) and PSA (primary secondary amine). In an embodiment of the present invention, as a result of optimizing the contents of magnesium sulfate (MgSO ₄ ) and PSA as a purification reagent added using a reaction surface analysis method, the MgSO ₄ content is 5% to 25% of the sample concentration, preferably 10 % to 20%, more preferably 15%, and when the content of PSA is 0.5% to 5% of the sample, preferably 1% to 4%, more preferably 2.5%, the highest vitamin K content It was confirmed that it shows (FIG. 3).

In addition, in applying the dispersed solid phase extraction method or the QuEChERS extraction process according to the present invention, a person skilled in the art may selectively add various compounds known in the art as needed.

The detection method of the present invention (c) liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry, LC- APCI-MS).

In the present invention, "liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry, LC-APCI-MS" is preferably used for the detection and quantification of vitamin K and vitamin D. can Atmospheric pressure chemical ionization (APCI) is an ionization method used to detect molecules of low polarity, which are not suitable to obtain sufficient amounts of ions in electrospray ionization (ESI), in which single charged ions are mainly produced. Specifically, in an embodiment of the present invention, LC-APCI-MS/MS analysis was performed after 0.22㎛ PVDF syringe filter on the supernatant obtained after pre-treatment with QuEChERS for the sample.

Another aspect of the present invention for achieving the above object provides a kit capable of simultaneously detecting vitamin K and vitamin D from a sample according to the method of the present invention. Specifically, the kit according to the present invention may include a tool to which the dispersed solid phase extraction method or the QuEChERS extraction process can be applied, and instructions describing a method for detecting and discriminating a sample, vitamin K and vitamin D, respectively. In addition, the kit according to the present invention contains acetonitrile as an extraction solvent, magnesium sulfate (MgSO ₄ ) and NaCl as a pretreatment reagent, magnesium sulfate and PSA as a purification reagent, and the optimal content of the pretreatment reagent and the purification reagent is as described above. It's like a bar.

Simultaneous detection method of the present invention is a factor affecting extraction efficiency, and as a result of optimization in consideration of the type of extraction solvent, the content of pretreatment reagent and purification reagent, etc. in the pretreatment process of the sample, the recovery rate of vitamin K and vitamin D is high Also, it is characterized in that it is eco-friendly by reducing the extraction time and solvent volume by about 90% or more compared to the previously proposed method.

In order to more fully understand the drawings cited herein, a brief description of each drawing is provided.
1 shows the results of comparative analysis of the recovery rate of vitamin K when each extraction solvent is used to optimize the pretreatment process of the sample.
Figure 2 shows the optimization of the extraction process using the reaction surface analysis (RSM), respectively, A and B are magnesium sulfate (MgSO ₄ ) and NaCl content in the extraction step, C and D are magnesium sulfate (MgSO ₄ ) in the purification step, respectively. ) and PSA content.
3 shows calibration curves of vitamin K1 and vitamin K2, vitamin D2, and vitamin D3 as a result of verification of the detection method according to the present invention.
4 shows the MRM chromatograms of vitamins K1 and K2, vitamin D2 and vitamin D3 respectively detected according to the method of the present invention.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[Test method]

1. Pretreatment of Vitamin K Fortified Formulations (QuEChERS)

After diluting the vitamin K-enhanced formulation to a concentration of 500 ng/mL, put 2 mL into a 50 mL centrifuge tube, add 10 mL of acetonitrile, and shake sufficiently to mix. After adding the Extraction Kit (MgSO ₄ 4 g, NaCl 1 g) to a 50 mL centrifuge tube, centrifuge at 3,000 rpm for 5 minutes, and 6mL of the supernatant was transferred to dispersive SPE (900 mg of MgSO ₄ , 150 mg of primary secondary amine). After putting in the centrifugal centrifugation again at 3,000 rpm for 5 minutes. Then, the supernatant was again analyzed by LC-APCI-MS/MS after 0.22㎛ PVDF syringe filter.

2. Validation for Vitamin K Assay

Relative calibration curves were obtained using vitamin K1 and K2 stock solutions. The recovery rate experiment was measured by adding 5, 10, and 500 μg/L of vitamins K1 and K2 (standard substances) to the vitamin K fortified formulation. For LC-APCI-MS/MS instrument analysis for vitamin K, the linearity of 1-1000 μg/L standard solution was confirmed. And to verify the precision, the test solution of the vitamin K standard material obtained after extraction was repeatedly measured 6 times, inter-day (1 section per day, 3 days) and intra-day (1 day, 3 sections).

3. Liquid Chromatography-Atmospheric Pressure Chemiionization Mass Spectrometry (LC-APCI-MS) Instrument Conditions

After pre-treatment of the vitamin K-enhanced formulation, it was analyzed using Waters' ACQUITY UPLC system and Xevo TQ MS mass spectrometer for instrumental analysis, and separated through UPLC BEH C ₁₈ column (1.7 μm, 2.1 × 100 mm, Waters). . LC APCI-MS/MS analysis conditions are shown in Tables 1 and 2 below.

Instrument Waters Xevo TQ MS Column UPLC BEH C ₁₈
(1.7 μm, 2.1 × 100 mm, Waters) mobile phase A: 90% ACN
B: MeOH flow rate 0.5 mL/min gradient
(A:B) 0 min
0.5 min
2.5 min
4.5 min
5 min
6 min 100 : 0
100 : 0
0 : 100
0 : 100
100 : 0
100 : 0 Oven temp. 40℃ Injection vol. 15 μl

Compound vitamin K1 vitamin K2 vitamin D2 vitamin D3 Ionization mode APCI Q1 (m/z) 451.5 445.4 397.5 385.4 Q3 (m/z) 128
187.1 81
187.1 107
397.4 92.8
107.03
67.4 RT (min) 3.93 3.06 3.07 3.10 Cone (V) 20 Probe Temp. (℃) 450 Gas flow (L/hr) 250 Collision (eV) 20 20 17 20 Dwell time (s) 0.1

4. Optimization of extraction conditions using response surface methodology (RSM)

The experimental design for the extraction conditions was carried out with the Box-Behnken design (BBD). The content of MgSO ₄ (X ₁ ) and NaCl (X ₂ ) of the extraction kit, MgSO ₄ (X ₃ ) and primary secondary amine (PSA, X ₄ ) of the Dispersive SPE kit were set as independent variables and each was coded (Table). 4), extraction was performed under 27 extraction conditions (Table 4). In addition, as a dependent variable affected by these independent variables, the extraction yield was measured and the value was used for regression analysis.

Variables Factors Level X _It's Low (-1) Middle (0) High (+1) extraction kit MgSO ₄ (mg) X ₁ 1000 4000 7000 NaCl (mg) x ₂ 500 1000 1500 Dispersive SPE kit MgSO ₄ (mg) X ₃ 500 900 1300 PSA (mg) X ₄ 50 150 250

Exp. No Extraction condition extraction kit Dispersive SPE kit MgSO ₄ (X _One ) NaCl (X ₂ ) MgSO ₄ (X ₃ ) PSA (X ₄ ) One 7000 1000 900 250 2 1000 1500 900 150 3 7000 1000 1300 150 4 7000 1000 900 50 5 1000 1000 1300 150 6 4000 1500 500 150 7 1000 1000 500 150 8 1000 1000 900 250 9 4000 1000 500 250 10 7000 1500 900 150 11 4000 1000 1300 250 12 1000 1000 900 50 13 4000 500 900 50 14 4000 1000 900 150 15 4000 1000 900 150 16 4000 500 1300 150 17 1000 500 900 150 18 4000 1500 1300 150 19 7000 1000 500 150 20 7000 500 900 150 21 4000 1000 500 50 22 4000 500 500 150 23 4000 1000 1300 50 24 4000 1000 900 150 25 4000 1500 900 250 26 4000 500 900 250 27 4000 1500 900 50

[Experiment result]

1. Optimization of QuEChERS extraction process

(1) Selection of extraction solvent

For the selection of the extraction solvent, two substances acetonitrile (CH ₃ CN) and methanol (CH ₃ OH) were used, and extraction efficiencies were compared. As a result, when acetonitrile was used, the recovery value was relatively higher than when methanol was used ( FIG. 1 ).

(2) Optimization of extraction conditions using response surface analysis (RSM)

In order to optimize the QuEChERS conditions, the process was optimized using the response surface analysis method. In accordance with the Box-Behnken design, independent variables were set as MgSO ₄ and NaCl contents of the extraction kit, MgSO ₄ and primary secondary amine (PSA) contents of dispersive SPE kit, respectively, and the dependent variable was set as vitamin K content.

As a result, the highest vitamin K content was shown when the content of MgSO ₄ and NaCl of the extraction kit was 4000 mg and 1000 mg, respectively, and the highest vitamin K content was when the content of MgSO ₄ and PSA of the dispersive SPE kit was 900 mg and 150 mg, respectively. The K content was shown (FIG. 2). The coefficient of determination (R ² ) of this model was 0.9436 and the p value of the coefficient of determination was less than 0.0001, proving the reliability of the regression model.

2. Nutritional analysis method validation of vitamin K-fortified formulations (method validation)

(One) Linearity, Range, Limit of Detection (LOD), Limit of Quantitation (LOQ) evaluation

To verify the established analytical method, the vitamin K standard solution mixture is added to the blank sample (formulation matrix) to which the vitamin K-fortified formulation is not added, extracted in the same way as the sample, the external calibration curve is measured, and the linearity and detection limit (limit of detection, LOD) and limit of quantification (LOQ) were confirmed. The vitamin K standard solution mixture was serially diluted to obtain a total of 6 concentrations including the initial standard solution mixture, and a calibration curve of each indicator component was obtained as an area value versus concentration. The evaluation of the linearity range, LOD, and LOQ of each material is listed in the table, and R ² was all 1.0000, indicating excellent linearity.

Analyte Regression equation Correlation coefficient (R ² ) Linear range
(μg/L) LOD LOQ Vitamin K1 y = 1341.55x + 48.74 1.0000 0.5 - 100 0.04 0.11 Vitamin K2 y = 464.32x + 92.34 1.0000 0.5 - 250 0.03 0.10 Vitamin D2 y = 62.80x - 10.46 1.0000 1.0 - 250 0.25 0.77 Vitamin D3 y = 320.80x + 84.33 1.0000 0.5 - 250 0.06 0.17

(2) Evaluation of Precision, Stability, and Accuracy (Recovery)

Precision was evaluated by intra-day and inter-day precision by repeating measurements three times at three concentrations as shown in Table 6 below. In addition, parallel precision (repeatability) was evaluated with 6 repeated measurements of the vitamin K-fortified formulation. Stability was evaluated by measuring the same sample stored at room temperature within 30 hours at intervals of 6 times. Accuracy was evaluated by adding a known amount of the diluted standard solution mixture to the vitamin K-fortified formulation to be analyzed, and using this as a sample, 3 concentrations, and repeating this 3 times, applying the test method of this verification performance, all samples were RSD < 5% was evaluated.

Analyte Precision RSD (%) Accuracy Concentration
(μg/L) Intra-
day
(n=3) Inter-
day
(n=3) repeatability
RSD (%)
(n=5) Spiked
(μg/L) Detected
(μg/L) Recovery
(%) RSD
(%) Emulsion
(Soybean oil) Vit. K1 sample 5.36
(10.71) 5.70
(11.81) 106.37 ± 0.53
(110.24 ± 2.54) 0.5
(2.3) One 1.24 2.20 1.50 4.88 4.89 100.26 ± 0.30 0.3 10 0.99 1.11 9.75 9.78 100.27 ± 2.02 2.0 50 0.64 1.15 14.63 14.62 99.99 ± 1.44 1.4 Vit. K2 sample 5.23
(10.46) 5.31
(11.44) 101.53 ± 5.75
(109.35 ± 6.33) 5.8 One 1.10 2.23 2.51 4.17 4.42 106.20 ± 6.33 6.0 10 0.20 1.02 8.33 8.40 100.81 ± 1.68 1.7 50 0.17 0.76 12.50 12.49 99.92 ± 0.81 0.8 Emulsion
(Soybean oil) Vit. D2 sample 5.23
(10.46) 5.87
(10.92) 112.26 ± 2.09
(104.35 ± 5.41) 10.8
(5.2) One 2.34 3.72 5.48 4.17 4.37 104.89 ± 3.01 2.9 10 4.50 6.43 8.33 9.00 107.97 ± 4.35 4.0 50 2.27 3.62 12.50 13.02 104.15 ± 1.28 1.2 Vit. D3 sample 5.23
(10.46) 5.42
(10.92) 103.58 ± 0.71
(104.38 ± 3.40) 3.3 One 2.38 4.14 2.62 4.25 4.19 98.47 ± 0.41 0.4 10 1.24 1.90 8.50 8.96 105.36 ± 2.56 2.4 50 0.45 1.45 12.75 13.33 104.57 ± 2.89 2.8

3. Comparison of recovery rate of vitamin K1 when using the vitamin K1 (2nd method) analysis method of the Health Functional Foods Association and the detection method of the present invention

(One) Vitamin K1 (Second Method) Analysis Method of Health Functional Foods Association: Quantitative method by high-performance liquid chromatography

1) Reagent

Solvents: methanol, dichloromethane, isooctane, acetone and isopropanol (for high-speed liquid chromatography); conditioning solution (dissolve isopropanol in isooctane to 0.01%); elution solvent (mobile phase, dissolving dichloromethane and isopropanol in isooctane to 15% and 0.02%, respectively); ammonia water (36%); Anhydrous sodium sulfate

2) Preparation of standard solution

Since the vitamin K1 standard contains trans and cis isomers, separate each isomer, calculate the peak area ratio of the cis and trans forms to the whole (contains about ≤20% of the inactive cis form), and store in a dark place. For the standard stock solution, the vitamin K1 standard was dissolved in isooctane to make 5 μg/mL, and the standard solution was used by diluting the standard stock solution with isooctane to prepare a calibration curve.

3) Preparation of test solution

Saponification and extraction: A certain amount of sample (containing 3.5 μg of vitamin K1) was accurately taken, placed in a 500 mL separatory funnel, 4 mL of aqueous ammonia was added, and then mixed by shaking for 60 seconds. To this, 60 mL of methanol was added, mixed for 30 seconds, a mixture of 100 mL of dichloromethane and 50 mL of isooctane was added, and the mixture was shaken well for extraction. After separating the layers, the lower layer was transferred to a 1,000 mL empty flask, and a mixture of 100 mL of dichloromethane and 50 mL of isooctane was added for extraction. The empty flask was washed with 20 mL of acetone, and the operation of concentrating the solution was repeated 3 times, then filled with nitrogen and sealed while blocking light.

Purification of test solution: 20 mL of conditioning solution was injected into the purification column, eluted slowly, and the solution was discarded. The residue was dissolved with 10 mL of conditioning solution and passed through a column to adsorb vitamin K1. Wash the empty flask twice with the same solution, 5mL each, put the washed solution into the column, elute with the conditioning solution, and discard the solution. Then, the elution solvent was passed through the column in different amounts depending on the fat content of the sample (if the fat content of the sample was 0.72 to 0.80 g, 100 mL of solvent was used, and if it was 0.72 g or less, the amount of the eluent was determined through a preliminary experiment). The eluate was received in a 250 mL empty flask, and concentrated under reduced pressure at 75°C or lower, and the concentrated solution was divided several times with 1 to 2 mL of isooctane, washed several times, and adjusted to 5 mL. However, in the purification column, 5.0 g of 60 to 200 mesh silica (activated overnight in an oven at 100° C.) is charged in a purification column (1.0 cm id × 30 cm, glass column), and 2.0 g of anhydrous sodium sulfate thereon (in an oven at 100° C.) activation overnight) was added.

4) Test operation

Measurement conditions of high-speed liquid chromatography:

Item Condition device Shiseido HPLC system column Capcell-pak C18 UG120V (4.6 x 250 mm, 5 μm) Post column: peakman SP Zinc column (3.0 x 35 mm) detector FLD detector (excitation wavelength: 243 nm, measurement wavelength: 430 nm) mobile phase In methanol:dichloromethane = 9:1, 1.37 g of zinc chloride, 0.41 g of anhydrous sodium acetate, and 0.3 g of acetic acid were dissolved in 5 mL of methanol and added. flow rate 0.8 mL/min injection volume 20 μL column temperature 35 ℃ analysis time 20 mins

Quantitative test: Calculate the content of vitamin K1 in the sample using the calibration curve obtained for the peak area or height of the standard solution obtained by injecting 20 μL of each test solution and standard solution. The content of vitamin K1 is calculated as the sum of the concentrations of each isomer, and only trans-vitamin K1 is calculated for formulas containing corn oil.

formula:

(2) Evaluation of recovery rate of vitamin K-fortified formulations

A known amount of the diluted standard solution mixture was added to the vitamin K-enhanced formulation to be analyzed, and this was evaluated by applying the test method of the present invention at 3 concentrations and 3 repetitions as a sample, and all samples were evaluated to be less than RSD < 5%.

As a result, the recovery rate of the vitamin K1-enhanced formulation using the existing 3-5 vitamin K1 (method 2) analysis method of the existing health functional food industry was only about 46.5% (emulsion) and 49.0% (SLN), within the international standard (CODEX) range. It was found that it did not reach the acceptable value (80 to 120%). On the other hand, the detection method of the present invention showed about 99.99 to 100.27% in the emulsion depending on the concentration (5, 10, 50 μg/L) in the case of vitamin K1, and in the case of vitamin K2, the concentration (5, 10, 50 μg) /L) in the emulsion according to the appearance of about 99.92 to 106.20%, it was confirmed that all were accepted in the range (80 to 120%) specified by CODEX (Table 6).

Sample Analyte Health functional food industry standard analysis method Recovery (%) RSD (%) Emulsion Vit K1 46.47 ± 0.31 0.66 Vit K2 no method SLN Vit K1 49.00 ± 1.39 4.78 Vit K2 no method

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (7)

(a) adding an extraction solvent to the sample and stirring to obtain an extract;
(b) purifying the extract by performing dispersive solid phase extraction (d-SPE);
(c) Analysis of vitamin K and vitamin D in the extract purified by step (b) by liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry, LC-APCI-MS A method for simultaneous detection of vitamin K and vitamin D in a sample, comprising the step of: The method of claim 1, wherein the extraction solvent is acetonitrile. The method of claim 1 , wherein step (a) includes adding magnesium sulfate (MgSO ₄ ) and NaCl and centrifuging. The method according to claim 3, wherein the magnesium sulfate is contained in a concentration of 30% to 35% of the sample, and NaCl is contained in a concentration of 5% to 10% compared to the sample. The method of claim 1 , wherein step (b) includes adding magnesium sulfate (MgSO ₄ ) and primary secondary amine (PSA) and centrifuging. The method of claim 5 , wherein the magnesium sulfate is contained in a concentration of 10% to 20% relative to the sample, and PSA is contained in a concentration of 1% to 4% relative to the sample. The method according to claim 1, wherein the vitamin K is vitamin K1 and/or vitamin K2, and the vitamin D is vitamin D2 and/or vitamin D3.

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