CN112526015A

CN112526015A - Ultra-efficient synthetic phase chromatography for simultaneously measuring VA and VD in cod liver oil3Method (2)

Info

Publication number: CN112526015A
Application number: CN202011319887.2A
Authority: CN
Inventors: 吴鹏; 唐旭; 徐长安; 林春波; 刘源森; 林凌
Original assignee: Third Institute of Oceanography MNR
Current assignee: Third Institute of Oceanography MNR
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2021-03-19

Abstract

The invention provides a method for simultaneously measuring VA and VD in cod liver oil by ultra-efficient synthetic phase chromatography₃The method comprises the following steps: step 1) taking a cod liver oil sample, adding a first solvent to dissolve the cod liver oil sample, saponifying, extracting, combining organic layers, washing the organic layers to be neutral, drying the organic layers, concentrating, diluting the concentrate with a second solvent to a constant volume, and filtering to obtain a solution to be detected; step 2) precisely absorbing the liquid to be detected and detecting the liquid by using an ultra-high-efficiency phase-combination chromatograph, thereby realizing simultaneous quantitative and qualitative detection of VA and VD in the cod-liver oil₃. The invention adopts ultra-high performance symphysis chromatography to measure vitamin A and vitamin D in cod liver oil₃The content and the sample are dissolved by normal hexane after the pretreatment, so that the pretreatment of the sample can be greatly simplifiedProcessing steps, shortening analysis time, having the advantages of simple pretreatment, short time consumption, high sensitivity, high recovery rate and the like, and providing a new method for the quality control of the compound fish liver oil preparation.

Description

Ultra-efficient synthetic phase chromatography for simultaneously measuring VA and VD in cod liver oil3Method (2)

Technical Field

The invention relates to the technical field of drug analysis, in particular to a method for simultaneously measuring VA and VD in cod liver oil by ultra-high performance synthetic phase chromatography₃The method of (1).

Background

The fish liver oil is a fatty oil extracted from liver of nontoxic marine fish such as shark, wherein the fatty oil contains vitamin A and vitamin D₃Is the main nutrient substance in the cod liver oil. Vitamin a (vitamin a, VA), also known as retinol, is a fat-soluble vitamin essential for the growth and development of the human body and for maintaining the vital activities of the body. Vitamin A not only can maintain normal visual function to prevent nyctalopia, but also has important functions of maintaining the integrity and health of epithelial cell structure, promoting normal growth and development of organism, enhancing hematopoietic function of organism, preventing cancer, etc. Vitamin D₃Is helpful for promoting the absorption of calcium by small intestine mucosa, and can prevent rickets and calcification disorder of bone-like tissue, etc., however, the excessive intake of vitamin also causes health risk. Therefore, the determination of the vitamins in the cod liver oil has practical significance for evaluating the quality of the cod liver oil and reasonably using the cod liver oil.

There are many ways to detect fat-soluble vitamins: liquid chromatography, liquid chromatography tandem mass spectrometry, spectrophotometry, capillary electrokinetic chromatography, etc. At present, the vitamin A and the vitamin D in the liver of animals at home and abroad₃The content is measured by liquid chromatography. The existing method has more complicated steps in the standard preparation process, and the vitamin D is measured₃Extracting a saponified sample with petroleum ether, purifying by using normal phase chromatography, separating by using reverse phase chromatography, quantifying by using an external standard method, wherein each treatment step has different influences on the accuracy of a measurement result; the testing process is long in time consumption, more interference substances exist, and the vitamins are easily damaged, so that the recovery rate is reduced.

Waters ultra performance phase-compatible chromatography (UPC2) is a new type coming out in 2012Normal phase chromatography, which has the advantages of conventional high performance liquid chromatography and supercritical fluid chromatography, and is based on supercritical CO₂The main mobile phase is separated and analyzed by depending on the solvating capacity of the mobile phase, so that samples which are not suitable for gas chromatography and have high boiling points and low volatilization and are unstable under heat can be analyzed, and the analysis speed and column efficiency of liquid chromatography can be improved; the method combines a 2-micron packing technology, so that the method has higher separation capacity and higher analysis efficiency, can obtain the required system resolution and selectivity by accurately adjusting the strength, pressure and temperature of the mobile phase, and effectively regulates and controls the retention and separation of the substance to be detected. The combined phase chromatography as a new technology can completely replace normal phase liquid chromatography, and the separation cost of the combined phase chromatography is only one tenth of that of the liquid chromatography; the sensitivity of the method is higher than that of normal phase liquid chromatography, the separation effect is better, the analysis speed is high, the method can separate isomer components which are difficult to separate in the liquid chromatography, and components (such as polysaccharide) which are difficult to retain or elute in the traditional liquid chromatography can be analyzed. When volatile oil and grease samples are analyzed, the combined phase chromatography can simplify the pretreatment steps, avoid derivatization and greatly improve the detection efficiency.

In China, no literature report for simultaneously detecting the contents of vitamin A and vitamin D3 in the cod liver oil by using ultra-high performance synthetic phase chromatography is provided.

Disclosure of Invention

According to the defects of the prior art, the invention adopts ultra-high performance combined phase chromatography to measure vitamin A and vitamin D in the cod liver oil₃The content and the sample are dissolved by normal hexane after pretreatment, so that the pretreatment steps of the sample can be greatly simplified, the analysis time is shortened, the method has the advantages of simple pretreatment, short time consumption, high sensitivity, high recovery rate and the like, and a new method is provided for the quality control of the composite fish liver oil preparation.

In order to solve the technical problems, the invention adopts the following technical scheme:

on one hand, the invention provides an ultra-efficient synthetic phase chromatography for simultaneously measuring VA and VD in cod liver oil₃The method comprises the following steps:

step 1) taking a cod liver oil sample, adding a first solvent to dissolve the cod liver oil sample, saponifying, extracting, combining organic layers, washing the organic layers to be neutral, drying the organic layers, concentrating, diluting the concentrate with a second solvent to a constant volume, and filtering to obtain a solution to be detected;

step 2) precisely absorbing the liquid to be detected and detecting the liquid by using an ultra-high-efficiency phase-combination chromatograph, thereby realizing simultaneous quantitative and qualitative detection of VA and VD in the cod-liver oil₃。

Further, the detection conditions of the ultra-high performance phase-compatible chromatograph are as follows:

a chromatographic column: waters Acquity UPC2 Torus 1-AA (100 mm. times.3.0 mm, 1.7 μm), mobile phase: a is supercritical CO₂(ii) a The mobile phase B is one of methanol, ethanol, isopropanol and acetonitrile.

Further, the detection conditions of the ultra-high performance phase-compatible chromatograph are as follows: the volume ratio of the mobile phase A to the mobile phase B is 85: 15, and the equal gradient elution is adopted, wherein the collection time is 3 min; the flow rate is 1 mL/min; the sample volume is 1 mu L; the column temperature is 50 ℃; the detection wavelength of the vitamin D3 is 265nm, and the detection wavelength of the vitamin A is 325 nm; dynamic back pressure (ABPR): 10 MPa.

Further, the qualitative method is as follows: VA and VD in cod liver oil sample₃Retention time of (A) and (V) and (VD)₃The retention time of the standard products is consistent; the quantitative method is characterized in that the actual measurement concentration is obtained by comparing the peak area of the object to be measured with the concentration in a standard curve corresponding to the peak area of the standard object.

Further, the standard curve is drawn as follows:

step A) preparation of VA standard solution: weighing a vitamin A standard substance, shaking and dissolving the vitamin A standard substance through chromatographic grade n-hexane to a constant volume, then carrying out pipetting and filtering to obtain a series of vitamin A standard liquids with different concentrations;

step B) VD₃Preparation of standard solution: weighing vitamin D₃The standard substance is dissolved by shaking through chromatographic grade normal hexane to a constant volume, then liquid is transferred, and a series of vitamin D with different concentrations are obtained by filtering₃Standard solution;

step C) separately mixing VA and VD₃And injecting the standard solution into an ultra-high performance phase-matching chromatograph for detection, and drawing corresponding standard curves for peak areas with different concentrations.

Further, the concentration of the VA standard solution is 500ppm, 200ppm, 100ppm, 50ppm, 20ppm, 10ppm and 5ppm respectively.

Further, the VD₃The concentrations of the standard solutions were 500ppm, 200ppm, 100ppm, 50ppm, 20ppm, 10ppm and 5ppm, respectively.

Further, in the step 1), the first solvent is absolute ethyl alcohol, and the second solvent is n-hexane.

Further, in the step 1), potassium hydroxide solution is adopted for saponification, and the concentration of the potassium hydroxide solution is 1 g/mL; the saponification condition is 50-60 deg.C water bath for 15-45 min, preferably 30 min.

Further, in the step 1), the extraction is carried out by adopting petroleum ether, and a vibration extraction mode is adopted in the extraction process.

Further, the drying in step 1) adopts anhydrous sodium sulfate.

Further, the organic phase in step 1) was concentrated at 30-40 ℃ until 2mL of residue remained, and blown dry with nitrogen to obtain a concentrate.

Further, the filtration was performed using a 0.22 μm organic filter membrane.

1) preparing a vitamin A standard product mother solution: weighing 2mg of a vitamin A standard substance into a 2ml centrifugal tube, and dissolving chromatographic grade n-hexane to a constant volume to obtain a standard mother solution with the concentration of 1000 ppm;

2) preparing a vitamin A standard series solution: accurately transferring the obtained standard mother liquor into volumetric flasks of 0.5mL, 0.2mL, 0.1mL, 50 muL, 20 muL, 10 muL, 5 muL and 2 muL to 1mL by using a pipette, fixing the volume by using n-hexane, and filtering to obtain a series of standard solutions with different concentrations of 500ppm, 200ppm, 100ppm, 50ppm, 20ppm, 10ppm and 5 ppm;

3) preparing a vitamin D3 standard product mother solution: weighing 2mg of a vitamin A standard substance into a 2ml centrifugal tube, and dissolving chromatographic grade n-hexane to a constant volume to obtain a standard mother solution with the concentration of 1000 ppm;

4) preparing a vitamin A standard series solution: accurately transferring the obtained standard mother liquor into volumetric flasks of 0.5mL, 0.2mL, 0.1mL, 50 muL, 20 muL, 10 muL, 5 muL and 2 muL to 1mL by using a pipette, fixing the volume by using n-hexane, and filtering to obtain a series of standard solutions with different concentrations of 500ppm, 200ppm, 100ppm, 50ppm, 20ppm, 10ppm and 5 ppm;

5) dissolving: accurately weighing 1-2g of cod liver oil sample into a 50mL centrifuge tube with a plug, and adding 3-6mL of absolute ethyl alcohol;

6) saponification: adding 2-4ml of 1g/ml potassium hydroxide solution into the centrifuge tube with the plug in the step (1), shaking up and down, mixing uniformly, saponifying in a water bath kettle at 60 ℃ for 30 minutes, taking out and immediately cooling to room temperature;

7) extraction: transferring the saponified solution into a separating funnel, adding 5ml of petroleum ether into the separating funnel, performing shaking extraction, standing for layering, and transferring the lower-layer solution to another separating funnel;

8) washing: repeating the step (7) for 2-3 times, and combining ether layers. Washing the ether layer with 15ml of water, detecting the pH value of the lower aqueous phase by using a pH test paper until the ether layer is washed to be neutral, and removing the lower aqueous phase; the ether layer was dehydrated with about 3g of anhydrous sodium sulfate,

9) concentrating and fixing volume: transferring the ether layer into a rotary evaporation bottle, carrying out rotary evaporation in water bath at 40 ℃ until the ether liquid is about 2ml, taking off the rotary evaporation bottle, drying with nitrogen, metering the volume to 2ml with n-hexane, and filtering with a filter membrane to obtain the solution to be detected.

10) And (3) loading: precisely absorbing the standard series solution, and injecting into an ultra-efficient phase-combination chromatograph for detection; and precisely absorbing the filter to be detected, and injecting the filter into an ultra-efficient phase-combination chromatograph for detection.

Compared with the prior art, the invention has the following beneficial effects:

(1) VA and VD3 are measured by adopting ultra-high performance phase-coherent chromatography, the pretreatment process of the sample to be measured and the standard is simple and efficient, and the sample pretreatment time is greatly shortened; n-hexane is adopted for dissolving before measurement, so that the sample pretreatment step is greatly simplified, and the analysis time can be shortened;

(2) the supercritical carbon dioxide is used as a main mobile phase, the low-proportion organic solvent is used as an auxiliary solvent, the separation efficiency is high, the solvent is saved, the waste liquid treatment cost is reduced to a great extent, and the method is green and environment-friendly; meanwhile, the mobile phase has few residues, so that the column is not easy to be lost, and the cost is saved;

(3) the sample analysis time is short, the peak shape is good, the sample matrix is free of interference, and the method can be used as a rapid, convenient and reliable detection means for the quality control of the cod-liver oil.

Drawings

FIG. 1 standard curve and standard map; (a) a VA standard curve; (b) a VA standard map; (c) VD3 standard curve; (d) VD3 standard map.

Fig. 2 is a graph of VA and VD3 measured in the sample; (a) blank; (b) peak VA; (c) VD3 peak.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

Example 1

Weighing 2mg of vitamin A and vitamin D3 standard substances respectively in two 2ml centrifuge tubes, and dissolving with chromatographic grade n-hexane to a constant volume to obtain a standard mother solution with the concentration of 1000 ppm;

accurately transferring the obtained standard mother liquor into sample bottles of 0.5mL, 0.2mL, 0.1mL, 50 muL, 20 muL, 10 muL, 5 muL and 2 muL to 1mL respectively by using a liquid transfer gun, fixing the volume by using n-hexane, and filtering to obtain series concentration standard liquids of 500ppm, 200ppm, 100ppm, 50ppm, 20ppm, 10ppm and 5 ppm;

weighing 1.012g of cod liver oil sample, adding 3mL of absolute ethyl alcohol, then saponifying with 2mL of potassium hydroxide solution (with the concentration of 1g/mL) under the saponification condition of 60 ℃ water bath for 30 minutes, taking out, immediately cooling to room temperature, transferring the saponified solution into a separating funnel, adding 5mL of petroleum ether into the separating funnel, oscillating for extraction, standing for layering, transferring the lower layer solution into another separating funnel, adding 5mL of petroleum ether for extraction again, repeating for three times, and combining ether layers. Washing the ether layer with water, detecting the pH value of the lower aqueous phase by using a pH test paper until the ether layer is washed to be neutral, and removing the lower aqueous phase. Removing water from the washed ether layer by using 3g of anhydrous sodium sulfate, transferring the ether layer into a rotary evaporation bottle, carrying out rotary evaporation until about 2mL of ether liquid is left, taking down the rotary evaporation bottle, drying by using nitrogen, carrying out constant volume to 2mL by using n-hexane, and filtering by using a 0.22-micron organic filter membrane to obtain a liquid to be detected; and (3) loading on a machine, wherein the chromatographic conditions are as follows: a chromatographic column: waters Acquity UPC2 Torus 1-AA (100 mm. times.3.0 mm, 1.7 μm), mobile phase: a is CO2, B is methanol; CO2 isocratic with a volume fraction of 80%. Collecting time is 3 min; the flow rate is 1 mL/min; the sample volume is 1 mu L; the column temperature is 50 ℃; the detection wavelength of the vitamin D3 is 265nm, and the detection wavelength of the vitamin A is 325 nm; dynamic back pressure (ABPR): 10 MPa. The standard solution spectrum and the standard curve obtained by the conditions have good linearity. The peaks of vitamin A and vitamin D3 are not separated, so the ratio of mobile phases needs to be adjusted.

Example 2

weighing 1.124g of cod liver oil sample, adding 3mL of absolute ethyl alcohol, then saponifying with 2mL of potassium hydroxide solution (with the concentration of 1g/mL) under the condition of 60 ℃ water bath for 30 minutes, taking out, immediately cooling to room temperature, transferring the saponified solution into a separating funnel, adding 5mL of petroleum ether into the separating funnel, oscillating for extraction, standing for layering, transferring the lower layer solution into another separating funnel, adding 5mL of petroleum ether for extraction again, repeating for three times, and combining ether layers. Washing the ether layer with water, detecting the pH value of the lower aqueous phase by using a pH test paper until the ether layer is washed to be neutral, and removing the lower aqueous phase. Removing water from the washed ether layer by using 3g of anhydrous sodium sulfate, transferring the ether layer into a rotary evaporation bottle, carrying out rotary evaporation until about 2mL of ether liquid is left, taking down the rotary evaporation bottle, drying by using nitrogen, carrying out constant volume to 5mL by using n-hexane, and filtering by using a 0.22-micron organic filter membrane to obtain a liquid to be detected; and (3) loading on a machine, wherein the chromatographic conditions are as follows: a chromatographic column: waters Acquity UPC2 Torus 1-AA (100 mm. times.3.0 mm, 1.7 μm), mobile phase: a is CO2, B is methanol; CO2 isocratic with a volume fraction of 85%. Collecting time is 3 min; the flow rate is 1 mL/min; the sample volume is 1 mu L; the column temperature is 50 ℃; the detection wavelength of the vitamin D3 is 265nm, and the detection wavelength of the vitamin A is 325 nm; dynamic back pressure (ABPR): 10 MPa. The standard solution spectrum and the standard curve obtained by the conditions have good linearity. The peaks of the atlas of the sample to be tested, vitamin A and vitamin D3 are well separated, the content of vitamin A is 600.88IU/g, and the peak area is extremely small because the content of vitamin D3 in the cod liver oil is low and only 1g of the sample is taken, so that the sampling amount is increased.

Example 3

weighing 2.101g of cod liver oil sample, adding 6mL of absolute ethyl alcohol, then saponifying with 4mL of potassium hydroxide solution (with the concentration of 1g/mL) under the condition of 60 ℃ water bath for 30 minutes, taking out, immediately cooling to room temperature, transferring the saponified solution into a separating funnel, adding 10mL of petroleum ether into the separating funnel, oscillating for extraction, standing for layering, transferring the lower layer solution into another separating funnel, adding 10mL of petroleum ether for extraction again, repeating for three times, and combining ether layers. Washing the ether layer with water, detecting the pH value of the lower aqueous phase by using a pH test paper until the ether layer is washed to be neutral, and removing the lower aqueous phase. Removing water from the washed ether layer by using 3g of anhydrous sodium sulfate, transferring the ether layer into a rotary evaporation bottle, carrying out rotary evaporation until about 2mL of ether liquid is left, taking down the rotary evaporation bottle, drying by using nitrogen, carrying out constant volume to 5mL by using n-hexane, and filtering by using a 0.22-micron organic filter membrane to obtain a liquid to be detected; and (3) loading on a machine, wherein the chromatographic conditions are as follows: a chromatographic column: waters Acquity UPC2 Torus 1-AA (100 mm. times.3.0 mm, 1.7 μm), mobile phase: a is CO2, B is methanol; CO2 isocratic with a volume fraction of 85%. Collecting time is 3 min; the flow rate is 1 mL/min; the sample volume is 1 mu L; the column temperature is 50 ℃; the detection wavelength of the vitamin D3 is 265nm, and the detection wavelength of the vitamin A is 325 nm; dynamic back pressure (ABPR): 10 MPa. The standard solution obtained by the above conditions has a spectrum as shown in fig. 1, and the peaks of vitamin A and vitamin D3 are well separated, and the standard curve is also good in linearity. The atlas of the sample to be tested is shown in figure 2, the content of vitamin A is 604.9IU/g, and the content of vitamin D3 is 93.6 IU/g.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Ultra-efficient synthetic phase chromatography simultaneous determination of VA and VD in cod liver oil₃The method is characterized by comprising the following steps:

2. The ultra-high performance combined phase chromatography of claim 1 for simultaneous determination of VA and VD in cod liver oil₃The method of (3), wherein the detection conditions of the ultra-high performance phase-locked chromatograph are as follows:

a chromatographic column: waters Acquity UPC2 Torus 1-AA, specification 100mm x 3.0mm, 1.7 μm;

the mobile phase A is supercritical CO₂(ii) a The mobile phase B is one of methanol, ethanol, isopropanol and acetonitrile.

3. The ultra-high performance combined phase chromatography of claim 2 for simultaneously determining VA and VD in cod liver oil₃The method of (a), wherein the ultra-efficient color-matching is performedThe detection conditions of the spectrometer are as follows: the volume ratio of the mobile phase A to the mobile phase B is 85: 15, and the equal gradient elution is adopted, wherein the collection time is 3 min; the flow rate is 1 mL/min; the sample volume is 1 mu L; the column temperature is 50 ℃; the detection wavelength of the vitamin D3 is 265nm, and the detection wavelength of the vitamin A is 325 nm; dynamic back pressure: 10 MPa.

4. The ultra-high performance combined phase chromatography of claim 1 for simultaneous determination of VA and VD in cod liver oil₃The method of (2), wherein the qualitative method is: VA and VD in cod liver oil sample₃Retention time of (A) and (V) and (VD)₃The retention time of the standard products is consistent; the quantitative method is characterized in that the actual measurement concentration is obtained by comparing the peak area of the object to be measured with the concentration in a standard curve corresponding to the peak area of the standard object.

5. The ultra-high performance combined phase chromatography of claim 4 for simultaneously determining VA and VD in cod liver oil₃The method is characterized in that the standard curve is drawn as follows:

6. The ultra-high performance combined phase chromatography of claim 5 for simultaneously determining VA and VD in cod liver oil₃The method of (5), wherein the concentration of the VA standard solution is 500ppm, 200ppm, 100ppm, 50ppm, 20ppm, 10ppm, 5ppm, respectively;

the VD₃The standard solution concentrations were 500ppm, 200ppm, 100ppm, 50ppm, and 20ppm, respectively、10ppm、5ppm。

7. The ultra-high performance combined phase chromatography of claim 1 for simultaneous determination of VA and VD in cod liver oil₃The method is characterized in that in the step 1), the solvent I is absolute ethyl alcohol, and the solvent II is n-hexane;

in the step 1), potassium hydroxide solution with the concentration of 1g/mL is adopted for saponification; the saponification condition is 50-60 ℃ water bath for 15-45 minutes.

8. The ultra-high performance combined phase chromatography of claim 1 for simultaneous determination of VA and VD in cod liver oil₃The method is characterized in that in the step 1), petroleum ether is adopted for extraction, and a vibration extraction mode is adopted in the extraction process;

the drying in the step 1) adopts anhydrous sodium sulfate.

9. The ultra-high performance combined phase chromatography of claim 1 for simultaneous determination of VA and VD in cod liver oil₃The method is characterized in that the organic phase in the step 1) is concentrated at 30-40 ℃ until 2mL of residue is remained, and the residue is dried by nitrogen to obtain a concentrate;

the filtration is carried out by adopting a 0.22 mu m organic filter membrane.

10. Ultra-efficient synthetic phase chromatography simultaneous determination of VA and VD in cod liver oil₃The method is characterized by comprising the following steps:

3) vitamin D₃Preparing a standard mother solution: weighing vitamin A labelDissolving 2mg of a standard product in a 2ml centrifuge tube by using chromatographic grade n-hexane to a constant volume to obtain a standard mother solution with the concentration of 1000 ppm;

9) concentrating and fixing volume: transferring the ether layer into a rotary evaporation bottle, carrying out rotary evaporation in water bath at 40 ℃ until about 2ml of ether liquid remains, taking down the rotary evaporation bottle, drying by using nitrogen, metering volume by using normal hexane until 2ml of filter membrane is filtered, and obtaining a liquid to be detected;