Method for detecting ascorbyl palmitate content in winterized oil
By optimizing the combination of vortexing, refrigerated centrifugation, and methanol extraction with a C18 chromatographic column, the accuracy problem of ascorbate palmitate detection in winterized oil was solved, achieving efficient separation and detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CABIO BIOTECH (WUHAN) CO LTD
- Filing Date
- 2023-11-06
- Publication Date
- 2026-07-21
Smart Images
Figure CN117665153B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of oil and fat technology, and specifically provides a method for detecting the content of ascorbyl palmitate in winterized oil. Background Technology
[0002] Vitamin C palmitate, also known as ascorbate palmitate, is obtained by esterification of palmitic acid and ascorbic acid. It is an antioxidant widely used in food, medical and cosmetic fields. It has the advantages of being fat-soluble, easily absorbed by the body, having good stability, and being safe and non-toxic.
[0003] Winterization is a common method for separating oils through crystallization. Winterization removes wax from oils, resulting in winterized oil, which has a purer appearance and remains clear and transparent even at low temperatures. Adding vitamin C palmitate as an antioxidant to winterized oil can effectively inhibit oil oxidation, making the oil quality more stable and meeting people's health needs.
[0004] Common methods for detecting vitamin C palmitate in food include iodometric titration, 2,6-dichlorophenolindophenol method, thin-layer chromatography, and high-performance liquid chromatography. However, for detection scenarios with low levels of vitamin C palmitate, conventional methods are difficult to achieve high accuracy. Furthermore, winterized oils significantly remove high-melting-point fatty acids, resulting in a relatively higher content of low-melting-point fatty acids. This causes considerable interference with the detection of ascorbate palmitate, further rendering conventional methods ineffective for the quantitative detection of vitamin C palmitate in winterized oils. Summary of the Invention
[0005] The main difficulty of existing technologies for detecting ascorbyl palmitate in winterized oils lies in the fact that it is inherently difficult to achieve accurate detection of low-content ascorbyl palmitate. At the same time, winterized oils contain relatively high levels of low-melting-point fatty acids, which are difficult to effectively separate using conventional pretreatment steps. These fatty acids can severely interfere with the detection of ascorbyl palmitate during the detection process, resulting in inaccurate detection.
[0006] To address the aforementioned challenges, the present invention provides the following technical solution.
[0007] In a first aspect, the present invention provides a method for detecting the content of ascorbyl palmitate in winterized oil, comprising: adding an extractant to 150-300 mg of winterized oil sample, vortexing for 2-5 min, centrifuging at freeze to obtain the extract, and performing chromatographic analysis; the mobile phase used for chromatographic analysis is methanol-water-acetic acid with a volume ratio of (93-95):(5-7):0.1.
[0008] In the method provided by this invention, the refrigerated centrifugation conditions are: 8000~10000r / min, centrifugation at -4~-20℃ for 10~15min.
[0009] In the method provided by the present invention, the extractant is repeatedly added to the oil sample and vortexed for 2 to 5 minutes and then frozen and centrifuged to obtain the extract again. The process is repeated more than twice, preferably 2 to 3 times.
[0010] This invention reveals that different pretreatment conditions significantly affect ascorbate palmitate in winterized oil, thus influencing the determination of ascorbate palmitate content in winterized oil. In particular, when attempting to separate ascorbate palmitate, commonly used techniques such as C18 silica gel and solid-phase extraction columns cannot achieve satisfactory sample purification and separation.
[0011] The pretreatment method provided by this invention is most suitable for the separation of ascorbate palmitate from winterized oil samples, including the number of extractions, vortexing time, and refrigerated centrifugation conditions.
[0012] The pretreatment method provided by this invention is most effective when the amount of winterized oil sample used is 150-300mg. Preferably, the amount of winterized oil sample used in this invention is 150-250mg.
[0013] In the method provided by this invention, the volume-to-mass ratio of extractant to sample is 1 mL:(1-60) mg for each repetition.
[0014] In the method provided by the present invention, the extractant used is methanol, preferably methanol containing isoascorbic acid, citric acid or trichloroacetic acid.
[0015] In the method provided by this invention, the winterized oil is winterized DHA algal oil.
[0016] In the method provided by this invention, the chromatographic column used for chromatographic analysis is a C18 column, model ZORBAX SB-C18.
[0017] The method provided by this invention includes:
[0018] (1) Weigh 150-300 mg of winterized DHA algal oil, add 3-8 mL of methanol, vortex for 2-5 min, centrifuge at 9000-10000 r / min at -10--20℃ for 12-15 min to obtain extract 1;
[0019] (2) Repeatedly add 3-8 mL of methanol to the winterized DHA algal oil, vortex for 2-5 min, centrifuge at 9000-10000 r / min at -10--20℃ for 12-15 min to obtain extract 2;
[0020] (3) Repeatedly add 3-8 mL of methanol to the winterized DHA algal oil, vortex for 2-5 min, centrifuge at 9000-10000 r / min at -10--20℃ for 12-15 min to obtain extract 3;
[0021] (4) Combine extract 1, extract 2 and extract 3, mix them well, and filter them through an organic phase filter membrane to obtain a mixed extract;
[0022] (5) Use a C18 column to perform chromatographic analysis on the mixed extract obtained in step (4). The column temperature is 30-35℃, the wavelength is 250nm, the flow rate is 0.5-1.5mL / min, the injection volume is 15-25μL, the running time is 10-20min, and methanol-water-acetic acid is used as the mobile phase for elution.
[0023] The present invention also provides the application of the above method in improving the accuracy of ascorbate palmitate detection.
[0024] Based on the present invention, a method for separating ascorbyl palmitate from other fatty acids in oils is provided. In a second aspect, the present invention provides a method for extracting ascorbyl palmitate from oils, comprising: adding methanol to an oil sample and vortexing for 2 to 5 minutes, centrifuging at 8000 to 10000 r / min at -4 to -20°C for 10 to 15 minutes, and retaining the supernatant; repeating the above operation more than twice to obtain a supernatant containing ascorbyl palmitate.
[0025] The beneficial effects of this invention are as follows:
[0026] This invention achieves accurate determination of ascorbate palmitate content in oils by optimizing the pretreatment of oil samples and combining it with specific chromatographic conditions, while reducing the amount of oil sample used.
[0027] The method provided by this invention achieves an average recovery rate of over 97.22% for ascorbate palmitate in oils and fats. The limit of detection and limit of quantitation of the method provided by this invention are 0.5 mg / kg and 1.5 mg / kg, respectively. Attached Figure Description
[0028] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0029] Figure 1 This is a chromatogram of the method provided in the embodiments of the present invention.
[0030] Figure 2 This invention is the result of pretreatment using C18 silicone.
[0031] Figure 3 This invention describes the separation effect of different chromatographic columns on VC palmitate. The chromatographic column used in A is ThermoHypersil Gold (4.6*250mm, 5μm); the chromatographic column used in B is Agilent ZORBAX SB-C18 (4.6*150mm, 5μm); and the chromatographic column used in C is Agilent ZORBAX SB-C18 (4.6*250mm, 5μm). Detailed Implementation
[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0033] Citric acid (1 mg / mL) was added to the methanol solvent used in the pretreatment of this invention.
[0034] The winterized oil used in this invention is winterized DHA algal oil. Unwintered DHA algal oil contains a large amount of medium and long chain saturated fatty acids such as C16:0 / C18:0. In order to ensure that DHA algal oil does not precipitate at low temperatures, it is often subjected to a winterization process to remove these fatty acids. The content of polyunsaturated fatty acids (high melting point) in the winterized DHA algal oil sample of this invention can be as high as 70%.
[0035] Preparation of standard solutions: Accurately weigh 100 mg of VC palmitate standard into a 100 mL volumetric flask, dissolve in methanol and dilute to volume to obtain VC palmitate standard stock solution. Use the stock solution to prepare a series of standard working solutions with concentrations of 0.2 μg / mL, 0.5 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL.
[0036] The oil sample used in this embodiment of the invention contains 180 mg / kg of vitamin C palmitate.
[0037] Example 1
[0038] This embodiment provides a method for detecting the ascorbate palmitate content in winterized oil, the steps of which are as follows:
[0039] Sample pretreatment:
[0040] (1) Weigh 150mg of oil, accurate to 0.1mg, add 5mL of methanol, vortex for 5min, centrifuge at 10000r / min at -20℃ for 15min, take the supernatant into a 20mL centrifuge tube to obtain extract 1;
[0041] (2) Repeatedly add 5 mL of methanol to the lower layer of oil sample, vortex for 5 min, centrifuge at 10000 r / min at -20℃ for 15 min, take the supernatant into a 20 mL centrifuge tube to obtain extract 2;
[0042] (3) Repeatedly add 5 mL of methanol to the lower layer of oil sample, vortex for 5 min, centrifuge at 10000 r / min at -20℃ for 15 min, take the supernatant into a 20 mL centrifuge tube, and obtain extract 3.
[0043] (4) Combine extract 1, extract 2 and extract 3, mix them well, filter through a 0.45 μm organic phase membrane, and perform the determination according to the chromatographic conditions.
[0044] Chromatographic conditions: A C18 column (Agilent, ZORBAX SB-C18, 4.6*250mm, 5μm) was used at a column temperature of 35℃. Methanol-water-acetic acid (95:5:0.1) was used as the mobile phase for isocratic elution. The column was run for 15 min at a flow rate of 1 mL / min, with a wavelength of 250 nm and an injection volume of 20 μL.
[0045] Using the method of this embodiment, the content of vitamin C palmitate in winterized DHA algal oil was shown to be 180.3 mg / kg, which is consistent with the actual value. The chromatogram is shown below. Figure 1 .
[0046] Example 2
[0047] This embodiment provides a method for detecting the ascorbate palmitate content in winterized oil, the steps of which are as follows:
[0048] (1) Weigh 300mg of oil, accurate to 0.1mg, add 5mL of methanol, vortex for 2min, centrifuge at 10000r / min at -20℃ for 15min, take the supernatant into a 20mL centrifuge tube to obtain extract 1;
[0049] (2) Repeatedly add 5 mL of methanol to the lower layer of oil sample, vortex for 2 min, centrifuge at 10000 r / min at -20℃ for 15 min, take the supernatant into a 20 mL centrifuge tube to obtain extract 2;
[0050] (3) Repeatedly add 5 mL of methanol to the lower layer of oil sample, vortex for 2 min, centrifuge at 10000 r / min at -20℃ for 15 min, take the supernatant into a 20 mL centrifuge tube, and obtain extract 3;
[0051] (4) Combine extract 1, extract 2 and extract 3, mix them well, filter through a 0.45 μm organic phase membrane, and perform the determination according to the chromatographic conditions.
[0052] Chromatographic conditions: A C18 column (Agilent, ZORBAX SB-C18, 4.6*250mm, 5μm) was used at a column temperature of 35℃. Methanol-water-acetic acid (95:5:0.1) was used as the mobile phase for isocratic elution. The column was run for 15 min at a flow rate of 1 mL / min, with a wavelength of 250 nm and an injection volume of 20 μL.
[0053] Using the method of this embodiment, it was shown that the content of vitamin C palmitate in winterized DHA algal oil was 170.7 mg / kg, which is basically close to the actual value.
[0054] Example 3
[0055] This embodiment provides a method for detecting the ascorbate palmitate content in winterized oil, the steps of which are as follows:
[0056] (1) Weigh 150mg of oil, accurate to 0.1mg, add 5mL of methanol, vortex for 2min, centrifuge at 10000r / min at -20℃ for 15min, take the supernatant into a 20mL centrifuge tube to obtain extract 1;
[0057] (2) Repeatedly add 5 mL of methanol to the lower layer of oil sample, vortex for 2 min, centrifuge at 10000 r / min at -20℃ for 15 min, take the supernatant into a 20 mL centrifuge tube to obtain extract 2;
[0058] (3) Combine extract 1 and extract 2, mix them thoroughly, filter through a 0.45 μm organic phase membrane, and perform the determination according to the chromatographic conditions.
[0059] Chromatographic conditions: A C18 column (Agilent, ZORBAX SB-C18, 4.6*250mm, 5μm) was used at a column temperature of 35℃. Methanol-water-acetic acid (95:5:0.1) was used as the mobile phase for isocratic elution. The column was run for 15 min at a flow rate of 1 mL / min, with a wavelength of 250 nm and an injection volume of 20 μL.
[0060] Using the method of this embodiment, it was shown that the content of vitamin C palmitate in winterized DHA algal oil was 166.2 mg / kg, which is close to the actual value.
[0061] Example 4: Limit of detection, limit of quantitation, recovery rate, and precision
[0062] The detection limits of the methods used in Examples 1-3 were investigated. Specifically, the method provided in Example 1 was used to detect VC palmitate standard solution: the concentration corresponding to a signal-to-noise ratio of 3 (S / N=3) of the VC palmitate standard chromatographic peak was taken as the limit of detection (LOD) of the method, and the concentration corresponding to S / N=10 was taken as the limit of quantitation (LOQ) of the method. Finally, it was determined that the method of Example 1 was used to detect VC palmitate in oil samples, with an LOD of 0.5 mg / kg and an LOQ of 1.5 mg / kg.
[0063] The recovery rate and precision of the method provided in Example 1 were investigated:
[0064] The scientific validity of the method was verified by adding different amounts of standard to winterized DHA algal oil and calculating its recovery rate and precision. Specifically, vitamin C palmitate standard was added to winterized DHA algal oil in sequence at low, medium and high levels, with each sample repeated 5 times. The specific results are shown in Table 1.
[0065] Table 1. Sample spiked recovery rate
[0066]
[0067] Comparative Example 1
[0068] This comparative example provides a method for detecting the content of vitamin C palmitate in oils, the steps of which are as follows:
[0069] (1) The pretreatment steps of this comparative example are the same as those of Example 1, steps 1-4. The difference is that 2.00 g of oil sample is weighed into a 15 mL centrifuge tube, 5 mL of methanol extractant is added to dissolve it, vortex for 2 min, centrifuge at 5000 r.min for 10 min, and the supernatant is filtered through a 0.45 μm organic phase filter membrane and then determined according to the chromatographic conditions.
[0070] (2) Chromatographic conditions:
[0071] Shield RP-C18 column (250 mm x 4.6 mm, 5 μm), column temperature 30 °C, injection volume 10 μL, flow rate 1 mL·min -1The detection wavelength was 243 nm; the mobile phase was: A was phosphoric acid (1+99) solution, and B was acetonitrile-methanol (1+1) solution; gradient elution: 0-1 min, B was 40%; 1-5 min, B linearly increased from 40% to 60%; 5-12 min, B linearly increased from 60% to 95%; 12-20 min, B was 95%.
[0072] In Comparative Example 1, the optimal recovery rate of winterized oil after methanol extraction was only 70%, which was the theoretical value.
[0073] Comparative Example 2
[0074] This comparative example provides a method for detecting the content of vitamin C palmitate in oils, the steps of which are as follows:
[0075] (1) The pretreatment steps 1-4 are the same as those in Example 1.
[0076] (2) Chromatographic conditions:
[0077] Shield RP-C18 column (250 mm x 4.6 mm, 5 μm), column temperature 30 °C, injection volume 10 μL, flow rate 1 mL·min -1 The detection wavelength was 243 nm; the mobile phase was: A was phosphoric acid (1+99) solution, and B was acetonitrile-methanol (1+1) solution; gradient elution: 0-1 min, B was 40%; 1-5 min, B linearly increased from 40% to 60%; 5-12 min, B linearly increased from 60% to 95%; 12-20 min, B was 95%.
[0078] In Comparative Example 2, the optimal recovery rate of winterized oil after methanol extraction was 86%, which is the theoretical value. However, under these chromatographic conditions, the VC palmitate and the remaining low-melting-point fatty acids could not be accurately separated.
[0079] Comparative Example 3
[0080] The difference between this comparative example and Example 1 is that the pretreatment of this comparative example is as follows: the method of Example 1 is followed, but only one extract is obtained, and the determination is performed according to the chromatographic conditions.
[0081] Using the method of this embodiment, the content of vitamin C palmitate in winterized DHA algal oil was 130.3 mg / kg, which differs significantly from the actual value.
[0082] Comparative Example 4
[0083] The difference between this comparative example and Example 1 is that the pretreatment of this comparative example is as follows: the sample amount is 50mg of oil, and the other three extracts are obtained according to the method of Example 1, and the determination is carried out according to the chromatographic conditions.
[0084] Using the method of this embodiment, the content of vitamin C palmitate in winterized DHA algal oil was 132.6 mg / kg, which differs significantly from the actual value.
[0085] Comparative Example 5
[0086] The difference between this comparative example and Example 1 is that the vortex time for pretreatment in this comparative example is 10 min, and the other extractions were obtained three times according to the method of Example 1. Using the method of this comparative example, the content of VC palmitate in winterized DHA algal oil is 148.8 mg / kg, which is significantly different from the actual value.
[0087] Comparative Example 6
[0088] The difference between this comparative example and Example 1 is as follows:
[0089] In this comparative example, the pretreatment method was as follows: 150 mg of oil was weighed, 5 mL of methanol was added, and the mixture was vortexed for 5 min. Then, 100 mg, 300 mg, and 600 mg of C18 silica gel were added. The results for the VC palmitate content detection are shown below. Figure 2 .
[0090] Depend on Figure 2 The chromatogram shows that adding C18 silica gel has a purification effect, and the target peak of VC palmitate is well separated, but the extraction effect of VC palmitate is not ideal, with the detected value only 65% of the theoretical value. Figure 2 The results show that different amounts of C18 silica gel have an effect on the VC palmitate content. When the amount of C18 silica gel added is close to the amount of sample, it has little effect on the VC palmitate content. When the amount of C18 silica gel is increased (about 3 times the amount of sample), the VC palmitate content is significantly reduced, which should be because excessive silica gel has an adsorption effect on the target analyte.
[0091] Comparative Example 7
[0092] The difference between this comparative example and Example 1 is that this comparative example uses a solid-phase extraction column for oil pretreatment.
[0093] In theory, solid-phase extraction columns have a retention effect on oils. Based on the difference in retention time between the target analyte and triglycerides on the solid-phase extraction column, the target analyte can be separated, concentrated by nitrogen blowing, and quantitative analysis can be achieved.
[0094] In this comparative example, 150 mg of oil was weighed, dissolved in 5 mL of methanol, loaded onto a 10 mL centrifuge tube, and then 3 mL of methanol was added for further elution. Finally, the mixture was dried under nitrogen and reconstituted with 1 mL of methanol before being loaded onto the centrifuge.
[0095] C18 solid-phase extraction columns can purify oils, but the sample recovery rate is low, with the detected value being only half of the actual value. It is speculated that this may be due to the degradation of VC palmitate during the nitrogen blowing process caused by factors such as temperature and light. Therefore, using solid-phase extraction columns to quantify VC palmitate in winterized oils is not feasible.
[0096] Comparative Example 8
[0097] The difference between this comparative example and Example 1 is as follows:
[0098] The columns were changed to two different specifications: Agilent ZORBAX SB-C18 (4.6*150mm, 5μm) and Thermo Hypersil Gold (4.6*250mm, 5μm). The study found that all three columns achieved good separation for VC palmitate standards, but for VC palmitate in winterized oil, the Agilent ZORBAX SB-C18 (4.6*250mm, 5μm) column showed the best separation effect. (See [link to study]). Figure 3 .
[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for detecting the ascorbate palmitate content in winterized DHA algal oil, characterized in that, include: Add methanol to 150-300 mg of winterized DHA algal oil sample, vortex for 2-5 min, then freeze and centrifuge to obtain the extract. Repeat the process of adding methanol to winterized DHA algal oil sample, vortexing for 2-5 min, and freezing and centrifuging to obtain the extract again. Repeat the process at least twice and perform chromatographic analysis. The mobile phase used for chromatographic analysis is methanol-water-acetic acid with a volume ratio of (93-95):(5-7):0.1, the column temperature is 30-35℃, and the wavelength is 250 nm. The refrigerated centrifugation conditions are: 8000~10000 r / min, -4~-20℃ for 10~15 min; The chromatographic column used for the chromatographic analysis was a C18 column, model ZORBAX SB-C18, with dimensions of 4.6*250mm and 5μm.
2. The method according to claim 1, characterized in that, Repeat 2 to 3 times.
3. The method according to claim 1, characterized in that, The volume-to-mass ratio of methanol to winterized oil sample was 1 mL: (1~60) mg for each repetition.
4. The method according to any one of claims 1 to 3, characterized in that, include: (1) Weigh 150~300mg of winterized DHA algal oil, add 3~8mL of methanol, vortex for 2~5min, centrifuge at 9000~10000r / min at -10~-20℃ for 12~15min to obtain extract 1; (2) Repeatedly add 3~8 mL of methanol to the winterized DHA algal oil, vortex for 2~5 min, centrifuge at 9000~10000 r / min at -10~-20℃ for 12~15 min to obtain extract 2; (3) Repeatedly add 3-8 mL of methanol to the winterized DHA algal oil, vortex for 2-5 min, centrifuge at 9000-10000 r / min at -10--20℃ for 12-15 min to obtain extract 3; (4) Combine extract 1, extract 2 and extract 3, mix them well, and filter through an organic phase membrane to obtain a mixed extract; (5) Use a C18 column to perform chromatographic analysis on the mixed extract obtained in step (4). The column temperature is 30~35℃, the wavelength is 250nm, the flow rate is 0.5~1.5mL / min, the injection volume is 5~20μL, the running time is 10~20min, and the mobile phase is methanol-water-acetic acid with a volume ratio of (93~95):(5~7):0.
1.
5. The application of the method according to any one of claims 1 to 4 in improving the accuracy of detection of ascorbate palmitate in winterized DHA algal oil.