Extraction and Determination of Vitamin K1 in Foods by Ultrasound-Assisted Extraction, SPE, and LC-MS/MS

Vitamin K1 is one of the important hydrophobic vitamins in fat-containing foods. Traditionally, lipase is employed in the determination of vitamin K1 to remove the lipids, which makes the detection complex, time-consuming, and insensitive. In this study, the determination of vitamin K1 in fat-containing foods was developed based on ultrasound-assisted extraction (UAE), solid-phase extraction (SPE) combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS). The optimal conditions for extraction of vitamin K1 were material–liquid ratio of 1:70 (g/mL), extraction temperature of 50 °C, extraction power of 700 W, extraction time of 50 min, material-wash fluid ratio of 1:60 (g/mL), and 8 mL of hexane/anhydrous ether (97:3, v/v) as the elution solvent. Then, vitamin K1 was analyzed on a ZORBAX SB-C18 column (50 mm × 2.1 mm, 1.8 μm) by gradient elution with water (0.01% formic acid) and methanol (0.01 formic acid + 2.5 mmol/L ammonium formate) as the mobile phase. The limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.16 μg/kg, respectively. Calibration curve was linear over the range of 10–500 ng/mL (R2 > 0.9988). The recoveries at three spiked levels were between 80.9% and 119.1%. The validation and application indicated that the proposed method was simple and sensitive in determination of vitamin K1 in fat-containing foods.


Introduction
Vitamin K is an essential dietary micronutrient. Vitamin K 1 definitely accounts for more than 80% of the total vitamin K in the human diet, and most of our present knowledge on vitamin K concerns K 1 [1]. Vitamin K 1 is a fat-soluble, antihemorrhagic vitamin that is synthesized by plants, green algae, and some species of cyanobacteria. It belongs to a class of naphthoquinone derivatives with biological activity in chlorophyll [2,3]. Vitamin K 1 is stable to air and moisture, but it is sensitive to light and alkaline conditions [4]. Vitamin K 1 is essential for the formation of liver zymogen and

Effect of Ultrasonic Time
The effect of ultrasonic time on extraction efficiency was studied by varying the sonication time from 5 to 80 min. At 50 min, the peak area of vitamin K1 reached its maximum and then stabilized ( Figure 1b). This may be because vitamin K1 is stable to air and moisture, and the longer the ultrasound time, the higher the extraction efficiency. The highest efficiency and optimal extraction time occurred at 50 min.

Effect of Ultrasonic Power
Ultrasonic power is an important factor in the ultrasonic extraction process. A DTC-27J ultrasonic water bath was used for the extraction experiment. This system has a fixed frequency of 40 kHz, but the power is adjustable (maximum power is 700 W). The target compound was extracted at 100, 250, 400, 550, and 700 W. As the ultrasonic power increased, the peak area of vitamin K1 and extraction efficiency also increased ( Figure 1c). The ultrasonic power affects the cell wall of the ultrasonic fractured sample, which affects the dissolution of vitamin K1 into the extraction solvent. The ultrasonic power also affects the molecular motion efficiency of the extraction solvent, thereby affecting the contact, mutual fusion, and mixing rate of the extraction solvent with the vitamin K1 in the sample. In this study, the maximum power used for the extraction of the target compound was 700 W.

Effect of Ultrasonic Temperature
Ultrasonic time can influence compound solubility and stability. Vitamin K1 was extracted in an ultrasonic water bath at constant temperatures of 20, 35, 50, and 65 °C. As the extraction temperature

Effect of Ultrasonic Time
The effect of ultrasonic time on extraction efficiency was studied by varying the sonication time from 5 to 80 min. At 50 min, the peak area of vitamin K 1 reached its maximum and then stabilized ( Figure 1b). This may be because vitamin K 1 is stable to air and moisture, and the longer the ultrasound time, the higher the extraction efficiency. The highest efficiency and optimal extraction time occurred at 50 min.

Effect of Ultrasonic Power
Ultrasonic power is an important factor in the ultrasonic extraction process. A DTC-27J ultrasonic water bath was used for the extraction experiment. This system has a fixed frequency of 40 kHz, but the power is adjustable (maximum power is 700 W). The target compound was extracted at 100, 250, 400, 550, and 700 W. As the ultrasonic power increased, the peak area of vitamin K 1 and extraction efficiency also increased (Figure 1c). The ultrasonic power affects the cell wall of the ultrasonic fractured sample, which affects the dissolution of vitamin K 1 into the extraction solvent. The ultrasonic power also affects the molecular motion efficiency of the extraction solvent, thereby affecting the contact, mutual fusion, and mixing rate of the extraction solvent with the vitamin K 1 in the sample. In this study, the maximum power used for the extraction of the target compound was 700 W.

Effect of Ultrasonic Temperature
Ultrasonic time can influence compound solubility and stability. Vitamin K 1 was extracted in an ultrasonic water bath at constant temperatures of 20, 35, 50, and 65 • C. As the extraction temperature increased, the extraction rate of vitamin K 1 also increased ( Figure 1d). When the temperature was 50 • C, the extraction rate of vitamin K 1 was highest. A temperature increase beyond 50 • C reduced the extraction rate of vitamin K 1 . Therefore, 50 • C was chosen as the optimum ultrasonic extraction temperature.

Optimization of SPE Conditions
To improve the purification efficiency of solid-phase extraction column for vitamin K 1 , the parameters, including the material-wash fluid ratio and the elution solution ratio, were optimized. The common conditions selected were material-liquid ratio of 1/60 (g/mL), ultrasonic time of 30 min, ultrasonic power of 700 W, ultrasonic temperature of 50 • C, material-wash fluid ratio of 1/60 (g/mL), and elution solution ratio 97:3 (n-hexane : anhydrous ether, v/v).

Effect of Material-Wash Fluid Ratio
Different material-wash fluid ratios (1/15, 1/30, 1/45, 1/60, 1/75, and 1/90 g/mL) were studied. The effects of the material-wash fluid ratios on the extraction efficiency of vitamin K 1 are shown in Figure 2a. Extraction efficiency increased as volume of wash solvent was increased, but the extraction efficiency was highest when the material-wash fluid ratios were 1:60 (g/mL). However, extraction efficiency decreased after 1:60 (g/mL). A ratio of 1:60 (g/mL) generated the largest peak area and was therefore used for all subsequent work.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 13 increased, the extraction rate of vitamin K1 also increased ( Figure 1d). When the temperature was 50 °C, the extraction rate of vitamin K1 was highest. A temperature increase beyond 50 °C reduced the extraction rate of vitamin K1. Therefore, 50 °C was chosen as the optimum ultrasonic extraction temperature.

Optimization of SPE Conditions
To improve the purification efficiency of solid-phase extraction column for vitamin K1, the parameters, including the material-wash fluid ratio and the elution solution ratio, were optimized. The common conditions selected were material-liquid ratio of 1/60 (g/mL), ultrasonic time of 30 min, ultrasonic power of 700 W, ultrasonic temperature of 50 °C, material-wash fluid ratio of 1/60 (g/mL), and elution solution ratio 97:3 (n-hexane : anhydrous ether, v/v).

Effect of Material-Wash Fluid Ratio
Different material-wash fluid ratios (1/15, 1/30, 1/45, 1/60, 1/75, and 1/90 g/mL) were studied. The effects of the material-wash fluid ratios on the extraction efficiency of vitamin K1 are shown in Figure 2a. Extraction efficiency increased as volume of wash solvent was increased, but the extraction efficiency was highest when the material-wash fluid ratios were 1:60 (g/mL). However, extraction efficiency decreased after 1:60 (g/mL). A ratio of 1:60 (g/mL) generated the largest peak area and was therefore used for all subsequent work.

Effect of Elution Solution Ratio
Because of the difference in the elution ability of n-hexane and anhydrous ether, a mixed solution of n-hexane and anhydrous ether was used as the eluent. The ratio of the two solvents was optimized. An 8 mL volume of n-hexane/anhydrous ether series solution (100:0, 97:3, 94:6, 91:9, 88:12, 85:15, v/v) was eluted into a 10 mL test tube. The peak area was highest and elution efficiency was best when the elution solution ratio was 97:3 ( Figure 2b). As the proportion of anhydrous ether increased, more impurities were eluted, and the matrix effects increased, reducing the sensitivity. In this study, n-hexane/anhydrous ether (97:3, v/v) was selected as the elution solution.

Optimization of the LC-MS/MS Conditions
The choice of the mobile phase in high-performance LC-MS/MS is essential. We compared the effects of methanol-water and methanol (0.01% formic acid + 2.5 mmol/L ammonium formate)-0.01% formic acid in water solution on the peak shape and retention time of vitamin K1. The addition of an optimal amount of formic acid contributed to the symmetry of the peak shape

Effect of Elution Solution Ratio
Because of the difference in the elution ability of n-hexane and anhydrous ether, a mixed solution of n-hexane and anhydrous ether was used as the eluent. The ratio of the two solvents was optimized. An 8 mL volume of n-hexane/anhydrous ether series solution (100:0, 97:3, 94:6, 91:9, 88:12, 85:15, v/v) was eluted into a 10 mL test tube. The peak area was highest and elution efficiency was best when the elution solution ratio was 97:3 ( Figure 2b). As the proportion of anhydrous ether increased, more impurities were eluted, and the matrix effects increased, reducing the sensitivity. In this study, n-hexane/anhydrous ether (97:3, v/v) was selected as the elution solution.

Optimization of the LC-MS/MS Conditions
The choice of the mobile phase in high-performance LC-MS/MS is essential. We compared the effects of methanol-water and methanol (0.01% formic acid + 2.5 mmol/L ammonium formate)-0.01% formic acid in water solution on the peak shape and retention time of vitamin K 1 . The addition of an optimal amount of formic acid contributed to the symmetry of the peak shape and accelerated the Molecules 2020, 25, 839 5 of 12 peak time. Therefore, a methanol (0.01% formic acid + 2.5 mmol/L ammonium formate)-0.01% formic acid in water solution was selected for the mobile phase of the study.
According to tandem mass spectra of vitamin K 1 and vitamin K 1 -D 7 (see Figure 3), m/z 187 and m/z 194 were selected as quantitative ion for vitamin K 1 and vitamin K 1 -D 7 , respectively. Flow rate and injection volume can improve the separation of vitamin K 1 . The column pressure, column particle size (1.8 µm), and the optimum flow rate (200-300 µL/min) were optimized for ESI-MS/MS, and set the flow rate to 200 µL/min. The separation effects of 1 and 2 µL were investigated. The K 1 peak was good when the injection volume was 2 µL, and no leading edge and tailing peak appeared. Therefore, the flow rate used was 200 µL/min, and the injection volume was 2 µL. Chromatograms of vitamin K 1 and vitamin K 1 -D 7 are shown in Figure 4.
Molecules 2020, 25, x FOR PEER REVIEW 5 of 13 and accelerated the peak time. Therefore, a methanol (0.01% formic acid + 2.5 mmol/L ammonium formate)-0.01% formic acid in water solution was selected for the mobile phase of the study. According to tandem mass spectra of vitamin K1 and vitamin K1-D7 (see Figure 3), m/z 187 and m/z 194 were selected as quantitative ion for vitamin K1 and vitamin K1-D7, respectively. Flow rate and injection volume can improve the separation of vitamin K1. The column pressure, column particle size (1.8 μm), and the optimum flow rate (200-300 μL/min) were optimized for ESI-MS/MS, and set the flow rate to 200 μL/min. The separation effects of 1 and 2 μL were investigated. The K1 peak was good when the injection volume was 2 μL, and no leading edge and tailing peak appeared. Therefore, the flow rate used was 200 μL/min, and the injection volume was 2 μL. Chromatograms of vitamin K1 and vitamin K1-D7 are shown in Figure 4.

Linearity, Limit of Detection, and Limit of Quantification
The calibration curve of vitamin K1 was constructed by using the peak area ratio of five concentrations of vitamin K1 standard solutions (triplicates) to the internal standard of vitamin Molecules 2020, 25, x FOR PEER REVIEW 5 of 13 and accelerated the peak time. Therefore, a methanol (0.01% formic acid + 2.5 mmol/L ammonium formate)-0.01% formic acid in water solution was selected for the mobile phase of the study. According to tandem mass spectra of vitamin K1 and vitamin K1-D7 (see Figure 3), m/z 187 and m/z 194 were selected as quantitative ion for vitamin K1 and vitamin K1-D7, respectively. Flow rate and injection volume can improve the separation of vitamin K1. The column pressure, column particle size (1.8 μm), and the optimum flow rate (200-300 μL/min) were optimized for ESI-MS/MS, and set the flow rate to 200 μL/min. The separation effects of 1 and 2 μL were investigated. The K1 peak was good when the injection volume was 2 μL, and no leading edge and tailing peak appeared. Therefore, the flow rate used was 200 μL/min, and the injection volume was 2 μL. Chromatograms of vitamin K1 and vitamin K1-D7 are shown in Figure 4.     The calibration curve of vitamin K 1 was constructed by using the peak area ratio of five concentrations of vitamin K 1 standard solutions (triplicates) to the internal standard of vitamin K 1 -D 7 . The limit of detection (LOD) and limit of quantification (LOQ) were evaluated for vitamin Molecules 2020, 25, 839 6 of 12 K 1 using a calibration curve based on a signal-to-noise ratio (S/N) of 3 or 10, respectively. The LOD and LOQ were 0.05 and 0.16 µg/kg, respectively. The calibration curve indicated that the excellent linearity was obtained for vitamin K 1 within a range of 10-500 ng/mL. A typical regression equation was obtained with a correlation coefficient of 0.9988 (Table 1).

Precision and Accuracy
The precision of the method was evaluated by repeated measurements. Intra-day and inter-day precisions were both tested five times by this method under the optimized conditions, and the results were recorded as the relative standard deviation (RSD). The intra-day and inter-day precisions were 1.6-7.2% ( Table 2). The accuracy of this method was determined based on the recoveries by adding the standard solutions at three different concentrations (10, 50, and 100 µg/kg) in samples. The recoveries were calculated by comparing the total measured value of the spiked sample with the measured value of the sample, and the ratio of the difference to the spiked amount was the recovery. Recoveries ranged from 80.9% to 119.1% (Table 2). These results showed that the method had high precision, sensitivity, and accuracy for analyzing vitamin K 1 in fat-containing foods. Compared with previous studies (Table 3), the method used in this study was more sensitive and efficient for detecting vitamin K 1 in fat-containing foods.

Real Sample Analysis
This method is used to determine the content of vitamin K 1 in fat-containing foods, including rapeseed, soybean, peanut, sesame, corn flour, milk powder, corn oil, peanut oil, safflower seed oil, sesame oil, grapeseed oil, linseed oil, camellia oil, rapeseed oil, soybean oil, and olive oil (see Figure 5). Among them, the content of vitamin K 1 in soybean oil and rapeseed oil is relatively high; the content of vitamin K 1 in corn flour and peanut is relatively low. The content of vitamin K 1 in soybean oil, rapeseed oil, and olive oil summarized by Rebufa [35] was about 105-325 µg/100 g, 70-350 µg/100 g, and 12-100 µg/100 g, respectively (estimated from the picture in the paper). In this study, the average content of vitamin K 1 in soybean oil, rapeseed oil and olive oil was 118 µg/100 g, 92 µg/100 g, and 83 µg/100 g, respectively. The content of vitamin K 1 in olive oil and peanut oil determined by Woollard [36] was 93.6 µg/100 g and 1.6 µg/100 g, respectively. In this study, the average content of vitamin K 1 in olive oil and peanut oil was 92 µg/100 g and 3 µg/100 g, respectively. The comparison indicated that the similar results were obtained in this study with the previous results.

Real Sample Analysis
This method is used to determine the content of vitamin K1 in fat-containing foods, including rapeseed, soybean, peanut, sesame, corn flour, milk powder, corn oil, peanut oil, safflower seed oil, sesame oil, grapeseed oil, linseed oil, camellia oil, rapeseed oil, soybean oil, and olive oil (see Figure  5). Among them, the content of vitamin K1 in soybean oil and rapeseed oil is relatively high; the content of vitamin K1 in corn flour and peanut is relatively low. The content of vitamin K1 in soybean oil, rapeseed oil, and olive oil summarized by Rebufa [35] was about 105-325 μg/100 g, 70-350 μg/100 g, and 12-100 μg/100 g, respectively (estimated from the picture in the paper). In this study, the average content of vitamin K1 in soybean oil, rapeseed oil and olive oil was 118 μg/100 g, 92 μg/100 g, and 83 μg/100 g, respectively. The content of vitamin K1 in olive oil and peanut oil determined by Woollard [36] was 93.6 μg/100 g and 1.6 μg/100 g, respectively. In this study, the average content of vitamin K1 in olive oil and peanut oil was 92 μg/100 g and 3 μg/100 g, respectively. The comparison indicated that the similar results were obtained in this study with the previous results.

Materials and Reagents
Oilseeds such as rapeseed, soybean, peanut, and sesame were collected. After removing broken, mildew seeds and foreign matters, these samples were pulverized using a grinder and refrigerated them at 4 °C until further processing. Corn flour, infant formulas, and edible oils including soybean oils, rapeseed oils, olive oils, safflower seed oils, Camellia oils, linseed oils, grapeseed oils, sesame oils, peanut oils, and corn oils, were purchased from local supermarkets.

Materials and Reagents
Oilseeds such as rapeseed, soybean, peanut, and sesame were collected. After removing broken, mildew seeds and foreign matters, these samples were pulverized using a grinder and refrigerated them at 4 • C until further processing. Corn flour, infant formulas, and edible oils including soybean oils, rapeseed oils, olive oils, safflower seed oils, Camellia oils, linseed oils, grapeseed oils, sesame oils, peanut oils, and corn oils, were purchased from local supermarkets.

Preparation of Standard Solutions
A stock solution containing 100 µg/mL vitamin K 1 in methanol was prepared in a brown volumetric flask. A stock solution containing 100 µg/mL of vitamin K 1 -D 7 internal standard (IS) was prepared. The working standard solutions were prepared by diluting the standard stock solution with methanol. The concentration in the solution for injection was 0.10 µg/mL (IS) for all standard and samples. Calibration standards were prepared in from one stock solution of vitamin K 1 at concentrations of 0.01, 0.05, 0.10, 0.25, and 0.50 µg/mL. All prepared solutions were stored at −20 • C in darkness.

Sample Preparation
Since vitamin K 1 is sensitive to light, sample preparation was carried out under low light intensity conditions to minimize photodegradation. A 0.1 g sample (accurate to 0.001 g) was weighed and placed it in a 10 mL tube. All test portions were spiked with 0.1 mL of a 1 µg/mL IS solution. Then, 7 mL of n-hexane was added to each test tube and vortexed the solution to ensure adequate mixing. The purpose of the ultrasound-assisted extraction was to increase the fluidity of the sample and to allow the target to be rapidly and completely transferred to the extractant [20,21]. After single factor experiments, the sample in the test tube was subjected to ultrasonic-assisted extraction for 50 min in an ultrasonic generator with an ultrasonic power of 700 W and a temperature of 50 • C. The sample was then centrifuged at 4500 r/min for 6 min. The upper hexane layer was transferred to a new tube for purification.
Solid-phase extraction (SPE) was used to remove lipids and lipophilic pigments from the extracts. The SPE silica gel column was first activated with 6 mL of n-hexane, and then the extract was passed through the column at a flow rate of 1.0 mL/min. The cartridge was rinsed with 6 mL of n-hexane at a flow rate of 1.0 mL/min. Finally, 8 mL of a solution of n-hexane/diethyl ether (97:3, v/v) was added and the eluate was collected. After the eluent was dried with nitrogen, 1 mL of methanol was added, followed by vortexing. After filtration through a 0.22 µm organic filter, a 2 µL sample was injected into LC-MS instrument for analysis.
The multiple reaction monitoring (MRM) transitions were optimized by direct infusion of compounds from a standard solution containing 1 µg/mL in an automatic mode. The collision voltage and the appropriate atomization gas flow rate and drying gas flow rate were optimized. Mass spectrometric conditions were optimized as follows: DL temperature 250 • C, heat block temperature 400 • C, nebulizing gas 3 L/min (N 2 ), and drying gas 10 L/min (N 2 ). The MS/MS parameters for the analysis of compounds are shown in Table 4. Tandem mass spectra of vitamin K 1 and vitamin K 1 -D 7 are shown in Figure 3. All determinations were performed in triplicate.

Peak Identification
In the multiple reaction monitoring mode, the peak of the target in the sample was determined based on the retention time (RT) and the mass spectrum of the vitamin K 1 standard.

Statistical Analysis
All results were expressed as average values with three replicates per sample. The measurements were completed with an internal standard correction curve method. Data acquisition and processing were controlled using LabSolutions software (Kyoto, Japan). Statistical analyses were performed with Microsoft Office 2010 (Redmond, WA, USA) and GraphPad Prism software (San Diego, CA, USA).

Conclusions
An efficient, sensitive, and reliable method was developed to analyze vitamin K 1 in fat-containing foods. The method used ultrasound-assisted extraction, SPE column purification treatment, and high-performance LC-MS/MS. This method had favorable linearity, good recoveries, good intra-day and inter-day precisions, and low LOD and LOQ. Compared with the enzymatic hydrolysis method in the national standard, the method established in this paper has a simple pretreatment process, significant substrate purification effect, and accurate and reliable detection results. Based on this method, the contents of vitamin K 1 in 16 fat-containing foods such as rapeseed, soybean, and olive oil were successfully quantified. The content of vitamin K 1 in edible oil was significantly higher than that in oilseeds and other fat-containing foods. Among the tested samples, the lowest content of vitamin K 1 was corn flour, and the highest content was soybean oil. It is hoped that the above results can promote the improvement of nutrient utilization in oil-containing foods, strengthen research on high-vitamin K 1 oil-containing foods, and promote the healthy development of the food industry.