Method for simultaneously detecting multiple water-soluble vitamins in ART solution

Abstract

The invention provides a method for simultaneously detecting various water-soluble vitamins in a human assisted reproduction culture solution, which can simultaneously detect at least four vitamins, including vitamin B1, vitamin B2, vitamin B5 and vitamin B6. According to the method, the content of the water-soluble vitamins in the human assisted reproduction culture solution can be effectively monitored, and the safety of the embryo culture process is ensured.

Description

Technical Field

[0001] The invention belongs to the field of detection and analysis, and specifically relates to a method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium. Background Art

[0002] Human Assisted Reproductive Culture Medium (ART Solution) is used to provide a material environment for the in vitro growth of human embryos. It contains a variety of nutrients required for embryonic development. Vitamins are biologically active substances that play a regulatory role in the process of cell metabolism. Among them, B vitamins are directly involved in the metabolism of carbohydrates and amino acids, which have an important impact on the development of embryos. ART solution is managed as a Class III medical device in my country. Clinical safety is an important indicator. The detection of the content of various nutrients is an important means of quality control and safety evaluation of ART solution.

[0003] At present, most of the detection of vitamins is for food, medicine, etc. The detection methods for B vitamins include microbiological method, spectrophotometry, liquid chromatography-mass spectrometry, capillary electrophoresis, high performance liquid chromatography, etc. Among them, the microbiological method uses the quantitative correspondence between bacterial growth and vitamins for detection. It has high sensitivity and accuracy, but it has the defects of long detection cycle, complex operation, and many accidental factors. Spectrophotometry is easily interfered by coexisting substances in the detection matrix. When there are many types of substances in the matrix, the detection accuracy is low. Liquid chromatography-mass spectrometry has many advantages such as high specificity, high sensitivity, high selectivity, simple pretreatment process, good separation effect, and fast analysis speed, but its equipment cost is high, operation is difficult, and maintenance cost is high, which makes it unsuitable for routine use in factories. Capillary electrophoresis has the advantages of short analysis cycle, less sample consumption, simple sample pretreatment, and less pollution, but it is greatly affected by the sample matrix, has many analysis conditions, low universality, and is less used. High performance liquid chromatography is currently the most common method for determining B vitamins, and it has the advantages of fast speed, high sensitivity and high accuracy.

[0004] The above-mentioned B vitamin detection method is used in the field of food and medicine. Compared with the ART solution, the sample matrix is ​​quite different. When using liquid chromatography to detect the content of B vitamins therein, finding suitable sample pretreatment conditions, chromatographic conditions such as chromatographic columns, mobile phases, elution methods, etc. to effectively separate B vitamins has always been a difficulty in the industry. Summary of the invention

[0005] The purpose of the present invention is to provide a method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium, aiming to improve the separation effect of water-soluble vitamins in human assisted reproductive culture medium by providing suitable pretreatment conditions, mobile phase and chromatographic column, thereby improving the accuracy and sensitivity of the detection results.

[0006] To achieve the above object, the present invention provides a method for simultaneously detecting multiple water-soluble vitamins in a human assisted reproductive culture medium, wherein the water-soluble vitamins include at least vitamin B1, vitamin B2, vitamin B5 and vitamin B6, and the vitamin B6 includes pyridoxal hydrochloride, characterized in that the method comprises the following steps: pre-treating the human assisted reproductive culture medium to obtain a sample working solution; and performing liquid chromatography analysis on the sample working solution; the liquid chromatography analysis conditions are:

[0007] Chromatographic column: C18 chromatographic column, column temperature 25℃~45℃;

[0008] Mobile phase: sodium acetate buffer-methanol, flow rate 0.8mL / min~1.5mL / min;

[0009] Elution conditions: gradient elution;

[0010] Detector: UV detector or diode array detector;

[0011] Wherein, the gradient elution includes a first elution stage, a second elution stage, a third elution stage and a fourth elution stage which are performed continuously; in the first elution stage, the volume ratio of sodium acetate buffer to methanol is constant, and the volume percentage of sodium acetate buffer is greater than the volume percentage of methanol; in the second elution stage, the volume percentage of sodium acetate buffer decreases according to a first change rate; in the third elution stage, the volume percentage of sodium acetate buffer decreases to zero according to a second change rate, and the second change rate is greater than the first change rate; in the fourth elution stage, the volume percentage of sodium acetate buffer is constant at zero.

[0012] Optionally, the first change rate is 5.8-7.25, and the second change rate is 8-20.

[0013] Optionally, the duration of the first elution stage is 4 min to 5 min, and the volume ratio of sodium acetate buffer to methanol is 98:2 to 95:5; the duration of the second elution stage is 8 min to 10 min, and at the end of the second elution stage, the volume ratio of sodium acetate buffer to methanol is 40:60; the duration of the third elution stage is 2 min to 5 min; the duration of the fourth elution stage is 2 min to 5 min.

[0014] Optionally, the sodium acetate buffer solution is prepared by weighing sodium acetate trihydrate according to a ratio, and dissolving the sodium acetate trihydrate in water to obtain a sodium acetate aqueous solution; or,

[0015] The preparation method of sodium acetate buffer is as follows: sodium acetate trihydrate is weighed according to the ratio, sodium acetate trihydrate is dissolved in water to obtain a sodium acetate aqueous solution, and glacial acetic acid is added to the sodium acetate aqueous solution to adjust the pH;

[0016] The concentration of the sodium acetate buffer is 0.05 mol / L to 0.25 mol / L, and the pH value at 25° C. is 6-7.5.

[0017] Optionally, the pretreatment step includes: placing a first predetermined amount of human assisted reproductive culture medium in an ultrafiltration tube, centrifuging and taking a target filtrate, filtering the target filtrate through a water filter membrane to obtain the sample working solution.

[0018] Optionally, the specification of the ultrafiltration tube is 10K, the centrifugal speed is 6000r / min to 10000r / min, and the centrifugal time is 5min to 10min; the pore size of the water filter membrane is 0.22μm or 0.45μm.

[0019] Optionally, before placing the human assisted reproductive culture fluid in the ultrafiltration tube, the following steps are also included: adding a second predetermined amount of human assisted reproductive culture fluid into the ultrafiltration tube, centrifuging at 6000r / min to 10000r / min for 5min to 10min, and discarding the filtrate.

[0020] Optionally, the detection wavelength of vitamin B5 is 210nm~215nm, the detection wavelength of vitamin B1 and vitamin B2 is 260nm~270nm, and the detection wavelength of vitamin B6 is 210nm~215nm or 260nm~270nm.

[0021] Optionally, the method further comprises:

[0022] Obtaining a linear equation of the standard working solution of the water-soluble vitamin; and

[0023] According to the linear equation of the water-soluble vitamins, the content of the water-soluble vitamins in the human assisted reproductive culture medium is calculated.

[0024] Optionally, the linear equation of the standard working solution of the water-soluble vitamin is obtained in the following manner:

[0025] Prepare standard stock solutions of thiamine hydrochloride, riboflavin, calcium pantothenate, and pyridoxal hydrochloride respectively;

[0026] Using the standard stock solution to prepare a corresponding standard working solution of a predetermined concentration;

[0027] Pre-treating the standard working solution to obtain a standard test sample;

[0028] Pre-treating a blank matrix to obtain a first comparative test sample, wherein the blank matrix does not include thiamine hydrochloride, riboflavin, calcium pantothenate and pyridoxal hydrochloride compared to the human assisted reproductive culture medium;

[0029] Pre-treating the water to obtain a second comparative test sample;

[0030] The standard test sample, the first comparative test sample and the second comparative test sample are subjected to liquid chromatography analysis under the liquid chromatography analysis conditions. Compared with the prior art, the method for simultaneously detecting multiple aqueous solution vitamins in human assisted reproductive culture medium of the present invention has the following advantages:

[0031] The aforementioned method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium includes the following steps: first, pre-treating the human assisted reproductive culture medium to obtain a sample working solution, and then performing liquid chromatography analysis on the sample working solution, wherein the liquid chromatography analysis conditions are: chromatographic column: C18 chromatographic column, column temperature is 25°C to 45°C; mobile phase: sodium acetate buffer-methanol, flow rate is 0.8mL / min to 1.5mL / min; elution conditions: gradient elution; detector: ultraviolet detector or diode array detector; wherein the gradient elution includes continuous The first elution stage, the second elution stage, the third elution stage and the fourth elution stage are successively performed; in the first elution stage, the volume ratio of sodium acetate buffer to methanol is constant, and the volume percentage of sodium acetate buffer is greater than the volume percentage of methanol; in the second elution stage, the volume percentage of the sodium acetate buffer decreases according to the first change rate; in the third elution stage, the volume percentage of the sodium acetate buffer decreases to zero according to the second change rate, and the second change rate is greater than the first change rate; in the fourth elution stage, the volume percentage of the sodium acetate buffer is constant at zero. By reasonably setting the chromatographic column, mobile phase, and elution conditions, the water-soluble vitamins and multiple vitamins in the human assisted reproductive culture medium can be separated simultaneously, and are not interfered by other components in the human assisted reproductive culture medium, and have the advantages of high detection sensitivity and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings are used to better understand the present invention and do not constitute an improper limitation of the present invention.

[0033] Figure 1The spectrum is obtained when the standard working solution is detected by the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium provided by one embodiment of the present invention, wherein the upper figure is the spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is the spectrum obtained when the detection light with a wavelength of 266nm is used;

[0034] Figure 2 The spectrum is obtained when the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium provided by one embodiment of the present invention is used to detect human assisted reproductive culture medium without adding B vitamins, wherein the upper figure is the spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is the spectrum obtained when the detection light with a wavelength of 266nm is used;

[0035] Figure 3 The spectrum obtained when water is detected by the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium provided by one embodiment of the present invention, wherein the upper figure is the spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is the spectrum obtained when the detection light with a wavelength of 266nm is used;

[0036] Figure 4 1 is a liquid chromatogram obtained in Example 1, wherein the upper figure is a spectrum obtained when the detection light with a wavelength of 215 nm is used, and the lower figure is a spectrum obtained when the detection light with a wavelength of 266 nm is used;

[0037] Figure 5 The liquid chromatogram obtained in Comparative Example 1, wherein the upper figure is the spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is the spectrum obtained when the detection light with a wavelength of 266nm is used;

[0038] Figure 6 The liquid chromatogram obtained in Comparative Example 2, wherein the upper figure is the spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is the spectrum obtained when the detection light with a wavelength of 266nm is used;

[0039] Figure 7 It is a liquid chromatogram of the No. 4 standard working solution in Comparative Example 3, wherein the upper figure is a spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is a spectrum obtained when the detection light with a wavelength of 266nm is used;

[0040] Figure 8 It is a liquid chromatogram of the No. 4 standard working solution in Comparative Example 4, wherein the upper figure is a spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is a spectrum obtained when the detection light with a wavelength of 266nm is used;

[0041] Fig. 9It is a liquid chromatogram of the No. 4 standard working solution in Comparative Example 5, wherein the upper figure is a spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is a spectrum obtained when the detection light with a wavelength of 266nm is used;

[0042] Fig.10 It is a liquid chromatogram of the No. 4 standard working solution in Comparative Example 6, wherein the upper figure is a spectrum obtained when the detection light with a wavelength of 215nm is used, and the lower figure is a spectrum obtained when the detection light with a wavelength of 266nm is used;

[0043] Fig.11 It is the liquid chromatogram of the No. 4 standard working solution in Example 2, wherein the upper figure is the spectrum obtained when the detection light with a wavelength of 215 nm is used, and the lower figure is the spectrum obtained when the detection light with a wavelength of 266 nm is used. DETAILED DESCRIPTION

[0044] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the present embodiment only illustrate the basic concept of the present invention in a schematic manner, and only the components related to the present invention are shown in the figure instead of being drawn according to the number, shape and size of the components in the actual implementation. The type, quantity and proportion of each component in the actual implementation can be a random change, and the component layout type may also be more complicated.

[0045] In addition, each embodiment of the following description has one or more technical features, but this does not mean that the user of the present invention must implement all the technical features in any embodiment at the same time, or can only implement part or all of the technical features in different embodiments separately. In other words, under the premise that implementation is possible, those skilled in the art can selectively implement part or all of the technical features in any embodiment according to the disclosure of the present invention and according to the design specifications or implementation requirements, or selectively implement a combination of part or all of the technical features in multiple embodiments, thereby increasing the flexibility of the implementation of the present invention.

[0046] As used in this specification, the singular forms "a", "an", and "the" include plural objects, and the plural form "a plurality" includes more than two objects, unless the context clearly indicates otherwise. As used in this specification, the term "or" is generally used to include the meaning of "and / or", unless the context clearly indicates otherwise. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0047] In order to make the purpose, advantages and features of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings. It should be noted that the drawings are all in very simplified form and in non-precise proportions, and are only used to conveniently and clearly assist in explaining the purpose of the embodiments of the present invention. The same or similar reference numerals in the drawings represent the same or similar components.

[0048] The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium provided by the embodiment of the present invention includes:

[0049] Step S10: pre-treat the human assisted reproductive culture medium to obtain a sample working solution.

[0050] Step S20: Perform liquid chromatography analysis on the sample working solution. The conditions of the liquid chromatography analysis include:

[0051] Chromatographic column: C18 column.

[0052] Mobile phase: sodium acetate buffer-methanol, flow rate: 0.8ml / min~1.5ml / min.

[0053] Elution condition: gradient elution. Specifically, the gradient elution includes a first elution stage, a second elution stage, a third elution stage and a fourth elution stage which are performed continuously. In the first elution stage, the volume ratio of the sodium acetate buffer to methanol remains constant, and the volume percentage of the sodium acetate buffer is greater than the volume percentage of methanol. In the second elution stage, the volume percentage of the sodium acetate buffer solution decreases according to the first change rate, while the volume percentage of methanol increases according to the first change rate. In the third elution stage, the volume percentage of the sodium acetate buffer decreases according to the second change rate until the volume percentage of sodium acetate becomes zero, while the volume percentage of methanol increases according to the second change rate until the volume percentage of methanol becomes 100. In the fourth elution stage, the volume percentage of the sodium acetate buffer is constant at zero.

[0054] Detector: UV detector or diode array detector.

[0055] Through the reasonable setting of the chromatographic column, mobile phase and elution conditions, this method can effectively separate multiple water-soluble vitamins when applied to the simultaneous detection of multiple water-soluble vitamins in human assisted reproductive culture medium, and can be accurately detected without being interfered by other substances in the human assisted reproductive culture medium. It has the characteristics of high detection sensitivity and high accuracy.

[0056] The water-soluble vitamins in the embodiment of the present invention include at least vitamin B1, vitamin B2, vitamin B5 and vitamin B6. In other words, the detection method of the present application can simultaneously detect the content of at least the following four vitamins: vitamin B1, vitamin B2, vitamin B5 and vitamin B6. The component of vitamin B1 is thiamine hydrochloride, the component of vitamin B2 is riboflavin, and the component of vitamin B5 is calcium pantothenate. Those skilled in the art know that vitamin B6 can have three forms in nature: pyridoxal hydrochloride, pyridoxine hydrochloride and pyridoxamine hydrochloride, but pyridoxal hydrochloride is generally used in human assisted reproductive culture fluid, and therefore, the vitamin B6 involved in the embodiment of the present invention actually refers to pyridoxal hydrochloride. The detection wavelength applicable to vitamin B1 and vitamin B2 is 210nm~215nm, the detection wavelength applicable to vitamin B5 is 260nm~270nm, and the detection wavelength applicable to vitamin B6 is 210nm~215nm or 260nm~270nm.

[0057] In addition, in the following text, thiamine hydrochloride is directly referred to as vitamin B1, riboflavin is directly referred to as vitamin B2, calcium pantothenate is directly referred to as vitamin B5, and pyridoxal hydrochloride is directly referred to as vitamin B6.

[0058] In an optional embodiment, the specific model of the chromatographic column can be C18 (4.6×250 mm, 5 μm).

[0059] The optional flow rate of the mobile phase includes but is not limited to 0.8 ml / min, 1.0 ml / min, 1.2 ml / min, 1.3 ml / min, 1.5 ml / min, etc.

[0060] During gradient elution, the duration of the first elution stage is, for example, 4 min to 5 min. In the first elution stage, the volume ratio of sodium acetate buffer to methanol is 98:2 to 95:5. The duration of the second elution stage is 8 min to 10 min. At the end of the second elution stage, the volume ratio of sodium acetate buffer to methanol is 40:60, so the first change rate is 5.8 to 7.25. The duration of the third elution stage is 2 min to 5 min, so the second change rate is 8 to 20. The duration of the fourth elution stage is 2 min to 5 min. In a specific embodiment, the duration of the first elution stage is 4 min. In the first elution stage, the volume ratio of sodium acetate buffer to methanol is 95:5. The duration of the second elution stage is 9 min, so the first change rate is 6.11. The duration of the third elution stage is 4 min, so the second change rate is 10.

[0061] Sodium acetate buffer can be prepared by the following method: weigh sodium acetate trihydrate according to the ratio. Then dissolve sodium acetate trihydrate in water. Afterwards, determine whether it is necessary to add glacial acetic acid to the sodium acetate aqueous solution to adjust the pH according to the target pH. Optionally, the concentration of the sodium acetate buffer is 0.05mol / L to 0.25mol / L, and its pH at 25°C is 6.5-7.5. In a specific embodiment, the concentration of the sodium acetate buffer can be 0.1mol / L, and its pH can be adjusted by glacial acetic acid during the preparation process so that its pH at 25°C is 7 (i.e., the sodium acetate buffer solution is a neutral solution). In another specific embodiment, after the sodium acetate is dissolved, glacial acetic acid is not used to adjust the pH, and its pH at 25°C is 7.32.

[0062] It should be noted that when performing liquid chromatography analysis, the injection volume each time is determined according to needs. For example, for a liquid chromatography analyzer with a maximum injection volume of 50 μL, the injection volume each time does not exceed 50 μL, and can generally be injected according to the maximum injection volume.

[0063] The step S10 specifically includes: placing a first predetermined amount of human assisted reproductive culture fluid in an ultrafiltration tube, then centrifuging, and taking the target filtrate. The target filtrate is filtered using a water filter membrane to filter out impurities to obtain the sample working fluid. The specification of the ultrafiltration tube selected in the embodiment of the present invention is 10K to intercept human albumin. The centrifugal conditions are: 6000r / min~10000r / min, and the time is 5min~10min. The specification of the water filter membrane is 0.22μm (that is, the pore size of the filter pores on the filter membrane is 0.22μm) or 0.45μm (that is, the pore size of the filter pores on the filter membrane is 0.45μm), preferably a 0.22μm water filter membrane to filter out more impurities. Through pretreatment, the human assisted reproductive culture fluid is purified to remove some substances that may interfere, eliminate the adverse effects of these substances on the detection, and improve the detection accuracy.

[0064] Preferably, before executing step S10, step S00 is also executed, and step S00 includes adding a second predetermined amount of human assisted reproductive culture fluid to the ultrafiltration tube, and discarding the filtrate after centrifugation. In this step, the centrifugal speed of the ultrafiltration tube can be 6000r / min~10000r / min, and the time can be 5min~10min. In a specific embodiment, step S00 includes taking 1mL of human assisted reproduction and placing it in the ultrafiltration tube, and then centrifuging it at a speed of 9500r / min for 5min, and then discarding the filtrate. The execution of step S00 can reduce the influence of the substances present in the ultrafiltration tube on the test results and improve the detection accuracy.

[0065] Furthermore, the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture fluid also includes calculating the content of the water-soluble vitamins in the human assisted reproductive culture fluid according to the linear equation of the standard working solution of the water-soluble vitamins. Furthermore, the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture fluid also includes the step of obtaining the linear equation of the standard working solution of the water-soluble vitamins.

[0066] Next, the effectiveness of the pretreatment method and liquid chromatography conditions provided by the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium is explained.

[0067] <1> Preparation of standard stock solution of the water-soluble vitamins

[0068] The standard stock solution is used to prepare the standard working solution. The preparation method of the standard stock solution includes: weighing 12.71 mg of thiamine hydrochloride, dissolving it with water and diluting it to a 10 ml volumetric flask, and obtaining a thiamine hydrochloride standard stock solution with a concentration of 1000 mg / L. Weigh 10 mg of riboflavin, dissolve it with 0.1 mol / L sodium hydroxide solution, transfer it to a 10 ml volumetric flask, and then dilute it with water to obtain a riboflavin standard stock solution with a concentration of 1000 mg / L. Weigh 10 mg of calcium pantothenate, dissolve it with water and dilute it to a 10 ml volumetric flask, and obtain a calcium pantothenate standard stock solution with a concentration of 1000 mg / L. And, weigh 12.18 mg of pyridoxal hydrochloride, dissolve it with water and dilute it to a 10 ml volumetric flask, and obtain a pyridoxal hydrochloride standard stock solution with a concentration of 1000 mg / L.

[0069] <2> Comparative test of standard working solution and comparative example

[0070] First, accurately measure appropriate amounts of thiamine hydrochloride standard stock solution, riboflavin standard stock solution, calcium pantothenate standard stock solution and pyridoxal hydrochloride standard stock solution, dilute and fix the volume to a volumetric flask to obtain a standard working solution of a predetermined concentration. The amount of thiamine hydrochloride standard stock solution, riboflavin standard stock solution, calcium pantothenate standard stock solution and pyridoxal hydrochloride standard stock solution is related to the predetermined concentration and the volume of the selected volumetric flask.

[0071] Then, take an appropriate amount of the standard working solution, and pre-treat the standard working solution (i.e., perform step S10) to obtain a standard test sample. Pre-treat an appropriate amount of blank matrix to obtain a first comparative test sample. And pre-treat an appropriate amount of water to obtain a second comparative test sample. The difference between the blank matrix and the human assisted reproductive culture medium is that the blank matrix does not include thiamine hydrochloride, riboflavin, calcium pantothenate and pyridoxal hydrochloride.

[0072] Then, the standard test sample, the first comparative test sample and the second comparative test sample were subjected to liquid chromatography analysis respectively to obtain the following Figures 1 to 3 Spectrum shown.

[0073] The pre-treatment methods and parameters of the standard working solution, the blank matrix and water are exactly the same, specifically, the substance to be treated is placed in a 10K ultrafiltration tube, centrifuged and then passed through a water filter membrane, the speed of the centrifugation is 9500r / min, the time is 5min, and the specification of the water filter membrane is 0.22μm. It should be understood that before pre-treating each of the standard working solutions, each of the standard working solutions is also used to perform step S00, before pre-treating the blank matrix, the blank matrix is ​​used to perform step S00, and before pre-treating water, water is used to perform step S00.

[0074] The chromatographic conditions for liquid chromatography analysis of the standard working solution, the blank matrix and water are all condition 1. Condition 1 is specifically as follows:

[0075] Chromatographic column: C18 (4.6×250 mm, 5 μm).

[0076] Column temperature: 30℃.

[0077] Mobile phase: sodium acetate buffer-methanol, wherein the concentration of sodium acetate buffer is 0.1 mol / L, and the pH is 7.0 at 25°C. The flow rate is 1.0 mL / min.

[0078] Injection volume: 50uL.

[0079] Detector: UV detector.

[0080] Detection wavelengths: 215nm and 266nm.

[0081] Gradient elution conditions: The duration of the first elution stage is 4 minutes, and the volume ratio of sodium acetate buffer to methanol is 95:5. The duration of the second elution stage is 9 minutes. The duration of the third elution stage is 4 minutes. The duration of the third elution stage is 2 minutes.

[0082] Depend on Figure 1 It can be seen that the peak at 6.460 is the characteristic peak of calcium pantothenate, the peak at 7.686 is the characteristic peak of pyridoxal hydrochloride, the peak at 11.706 is the characteristic peak of thiamine hydrochloride, and the peak at 17.106 is the characteristic peak of riboflavin. This shows that the liquid chromatography conditions provided by the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium can achieve effective separation of four B vitamins. Figure 2 and Figure 3The peaks are at 6.460, 7.686, 11.706 and 17.106, which indicates that the other components and water in the human assisted reproductive culture medium are subjected to liquid chromatography analysis under such chromatographic conditions, and no peaks overlapped with any of calcium pantothenate, pyridoxal hydrochloride, thiamine hydrochloride and riboflavin hydrochloride are produced, and thus no interference is caused to the liquid chromatography detection of calcium pantothenate, pyridoxal hydrochloride, thiamine hydrochloride and riboflavin hydrochloride under the chromatographic conditions. In other words, the chromatographic conditions provided in the embodiment of the present invention can be used for liquid chromatography analysis of human assisted reproductive culture medium to achieve simultaneous detection of calcium pantothenate, pyridoxal hydrochloride, thiamine hydrochloride and riboflavin.

[0083] It should be noted that when the same liquid chromatograph is used to perform liquid chromatography analysis multiple times in a row, a homogenization elution should be performed between the fourth elution stage of the previous analysis and the first elution stage of the next analysis, so that the chromatographic column is restored to the same state as the first elution stage. The duration of the homogenization elution is 10 minutes to 15 minutes, and the ratio of sodium acetate buffer to methanol is 98:2 to 95:5. In a typical embodiment, the duration of the homogenization elution is 12 minutes, and the ratio of sodium acetate buffer to methanol is 95:5.

[0084] The pre-treatment mentioned in the following text refers to placing the material to be treated (such as the No. 1 to No. 5 standard working solutions involved in the linear equation determination test, the No. 6 standard working solution involved in the instrument precision test, the No. 1 to No. 11 sample working solutions, etc.) in a 10K ultrafiltration tube that has been subjected to the step S00 with the corresponding material to be treated, and passing through a water filter membrane after centrifugation. The speed of the centrifugation is 9500r / min, the time is 5min, and the specification of the water filter membrane is 0.22μm. And, the chromatographic conditions used in the liquid chromatography analysis involved in the following text are all condition 1.

[0085] <III> Linear equation determination test

[0086] Prepare the standard working solution used in the linear equation determination test: first take 100uL thiamine hydrochloride standard stock solution, 100uL pyridoxal hydrochloride standard stock solution, 10uL riboflavin standard stock solution and 100uL calcium pantothenate standard stock solution to a 1mL volumetric flask, dilute with water and make up to volume, and shake up the primary mixed solution. Then use the primary mixed solution as the mother liquor to prepare 5 portions of standard working solutions, respectively, and the 5 portions of standard working solutions are standard working solution No. 1, standard working solution No. 2, standard working solution No. 3, standard working solution No. 4 and standard working solution No. 5. In the standard working solution No. 1, the concentration of thiamine hydrochloride is 0.3mg / L, the concentration of pyridoxal hydrochloride is 0.3mg / L, the concentration of riboflavin is 0.03mg / L, and the concentration of calcium pantothenate is 0.3mg / L. In the No. 2 standard working solution, the concentration of thiamine hydrochloride is 0.5 mg / L, the concentration of pyridoxal hydrochloride is 0.5 mg / L, the concentration of riboflavin is 0.05 mg / L, and the concentration of calcium pantothenate is 0.5 mg / L. In the No. 3 standard working solution, the concentration of thiamine hydrochloride is 1 mg / L, the concentration of pyridoxal hydrochloride is 1 mg / L, the concentration of riboflavin is 0.1 mg / L, and the concentration of calcium pantothenate is 1 mg / L. In the No. 4 standard working solution, the concentration of thiamine hydrochloride is 3 mg / L, the concentration of pyridoxal hydrochloride is 3 mg / L, the concentration of riboflavin is 0.3 mg / L, and the concentration of calcium pantothenate is 3 mg / L. In the No. 5 standard working solution, the concentration of thiamine hydrochloride is 5 mg / L, the concentration of pyridoxal hydrochloride is 5 mg / L, the concentration of riboflavin is 0.5 mg / L, and the concentration of calcium pantothenate is 5 mg / L.

[0087] The No. 1 standard working solution, the No. 2 standard working solution, the No. 3 standard working solution, the No. 4 standard working solution and the No. 5 standard working solution were pre-treated and then subjected to liquid chromatography analysis to obtain a chromatogram (not shown in the figure), and the peak areas of the characteristic peaks of thiamine hydrochloride, pyridoxal hydrochloride, riboflavin and calcium pantothenate in each chromatogram were recorded. According to the peak area and concentration of each substance, the linear equation corresponding to each substance was obtained, as shown in Table 1 below:

[0088] Table 1

[0089]

[0090]

[0091] As shown in Table 1, the correlation coefficients of the linear equations of the four vitamins all reached 0.9999, meeting the linear requirements of the quantitative test. Therefore, after pre-treatment and liquid chromatography analysis of the human assisted reproductive culture medium to obtain the peak area of ​​the characteristic peak of each target vitamin (i.e., at least one of thiamine hydrochloride, pyridoxal hydrochloride, riboflavin and calcium pantothenate), the corresponding linear equation can be combined to obtain the content of the corresponding vitamin in the human assisted reproductive culture medium, thereby achieving quantitative analysis of water-soluble vitamins in the human assisted reproductive culture medium.

[0092] <III> Instrument precision test

[0093] Accurately pipette 100uL of thiamine hydrochloride standard stock solution, 100uL of pyridoxal hydrochloride stock solution, 10uL of riboflavin standard stock solution and 100uL of calcium pantothenate stock solution into a 100mL volumetric flask, dilute with water, make up to volume, and shake well to obtain No. 6 standard working solution, in which the concentration of thiamine hydrochloride is 1mg / L, the concentration of pyridoxal hydrochloride is 1mg / L, the concentration of riboflavin is 0.1mg / L, and the concentration of calcium pantothenate is 1mg / L.

[0094] Take an appropriate amount of the No. 6 standard working solution, perform liquid chromatography analysis six times in succession after pretreatment, record the peak retention time and corresponding peak area of ​​each analysis, and calculate the relative standard deviation of the retention time and the relative standard deviation of the peak area of ​​each vitamin. The results are shown in Table 2 below.

[0095] Table 2

[0096]

[0097]

[0098]

[0099] As shown in Table 2, the same standard working solution was subjected to six consecutive liquid chromatography analyses using the same liquid chromatograph, and the relative standard deviation of the peak retention time was less than 0.05%, and the relative standard deviation of the peak area was less than 2%, indicating that the stability and precision of the test instrument meet the requirements, and therefore the test results are highly reliable.

[0100] <IV> Repeatability test

[0101] Prepare sample working solution: Take 2 mL of human assisted reproductive culture medium and pre-treat it to obtain sample working solution. Use the same method to prepare six sample working solutions, numbered as sample working solution No. 1, sample working solution No. 2, sample working solution No. 3, sample working solution No. 4, sample working solution No. 5 and sample working solution No. 6.

[0102] Then, the six sample working solutions were subjected to liquid chromatography analysis, and the peak areas of various vitamins were recorded, and the relative standard deviation of the peak areas of various vitamins was calculated. The results are shown in Table 3 below.

[0103] Table 3

[0104]

[0105] It can be seen from Table 3 that the relative standard deviation of each vitamin is much less than 10%, which proves that the pretreatment method provided by the embodiment of the present invention has good stability and repeatability.

[0106] <V> Accuracy test

[0107] Using 1000 mg / mL thiamine standard stock solution as the mother solution, prepare 100 mg / L thiamine standard solution; using 1000 mg / mL pyridoxal hydrochloride standard stock solution as the mother solution, prepare 100 mg / L pyridoxal hydrochloride standard solution; using 1000 mg / mL riboflavin standard stock solution as the mother solution, prepare 10 mg / L riboflavin standard solution; and using 1000 mg / mL calcium pantothenate standard stock solution as the mother solution, prepare 100 mg / mL calcium pantothenate standard solution.

[0108] Low concentration spike: Accurately pipette 1965 μL of the blank matrix, 10 μL of the thiamine hydrochloride standard solution, 5 μL of the pyridoxal hydrochloride standard solution, 10 μL of the riboflavin standard solution, and 10 uL of the calcium pantothenate standard solution, mix them evenly, and obtain a human assisted reproductive culture medium. Among them, the spiked amount of thiamine hydrochloride is 0.500 mg / L, the spiked amount of pyridoxal hydrochloride is 0.250 mg / L, the spiked amount of riboflavin is 0.050 mg / L, and the spiked amount of calcium pantothenate is 0.500 mg / L. That is, the content of thiamine hydrochloride in the human assisted reproductive culture medium is about 0.5ppm, the content of pyridoxal hydrochloride is about 0.25ppm, the content of riboflavin is about 0.05ppm, and the content of calcium pantothenate is about 0.5ppm.

[0109] The human assisted reproductive culture fluid obtained by low-concentration spike-in was pre-treated to obtain a sample working solution, which was called sample working solution No. 7.

[0110] Medium concentration spike: Accurately pipette 1930 μL of the blank matrix, 20 μL of the thiamine hydrochloride standard solution, 10 μL of the pyridoxal hydrochloride standard solution, 20 μL of the riboflavin standard solution, and 20 μL of the calcium pantothenate standard solution, mix evenly, and obtain a human assisted reproductive culture solution. Among them, the spiked amount of thiamine hydrochloride is 1.000 mg / L, the spiked amount of pyridoxal hydrochloride is 0.500 mg / L, the spiked amount of riboflavin is 0.100 mg / L, and the spiked amount of calcium pantothenate is 1.000 mg / L. In other words, the content of thiamine hydrochloride in the human assisted reproductive culture solution is about 1 ppm, the content of pyridoxal hydrochloride is about 0.5 ppm, the content of riboflavin is about 0.1 ppm, and the content of calcium pantothenate is about 1 ppm.

[0111] The human assisted reproductive culture fluid obtained by adding medium concentration was pre-treated to obtain a sample working solution, which was called sample working solution No. 8.

[0112] High concentration spike-in: Accurately pipette 1800 μL of the blank matrix, 60 μL of the thiamine hydrochloride standard solution, 40 μL of the pyridoxal hydrochloride standard solution, 40 μL of the riboflavin standard solution, and 60 μL of the calcium pantothenate standard solution, mix evenly, and obtain a human assisted reproductive culture solution. Among them, the spiked amount of thiamine hydrochloride is 3.000 mg / L, the spiked amount of pyridoxal hydrochloride is 2.000 mg / L, the spiked amount of riboflavin is 0.200 mg / L, and the spiked amount of calcium pantothenate is 3.000 mg / L. In other words, the content of thiamine hydrochloride in the human assisted reproductive culture solution is about 3 ppm, the content of pyridoxal hydrochloride is about 2 ppm, the content of riboflavin is about 0.2 ppm, and the content of calcium pantothenate is about 3 ppm.

[0113] The human assisted reproductive culture fluid obtained by high-concentration spike-in was pre-treated to obtain a sample working solution, which was called sample working solution No. 9.

[0114] The No. 7 sample working solution, the No. 8 sample working solution, and the No. 9 sample working solution were subjected to liquid chromatography analysis, and each sample working solution was subjected to liquid chromatography analysis three times. The spectra of each sample working solution were recorded and the detection values ​​of each vitamin were calculated in combination with the linear equation of each vitamin, and the recovery rate was calculated based on the detection results and the spiked amount. The results are shown in 4 below.

[0115] Table 4

[0116]

[0117]

[0118]

[0119] According to the relevant provisions of the "Chinese Pharmacopoeia 2020", when the content of the component to be determined is below 10ppm and above 1ppm, the recovery limit of the component to be determined is 80% to 115%, and when the content of the component to be determined is below 1ppm and above 10ppb, the recovery limit of the component to be determined is 75% to 120%. As shown in Table 4, the recovery rates of each vitamin in the No. 7 sample working solution, the recovery rates of each vitamin in the No. 8 sample working solution, and the recovery rates of each vitamin in the No. 9 sample working solution are all within the corresponding recovery limits, indicating that the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium is effective.

[0120] <VI> Sample working fluid stability test

[0121] Take the same batch of human assisted reproductive culture fluid, and prepare a sample working fluid according to the method in the repeatability test, which is called sample working fluid No. 10. After the preparation of sample working fluid No. 10 is completed, take an appropriate amount of sample working fluid No. 10 immediately for liquid chromatography analysis (i.e., perform liquid chromatography analysis on sample working fluid No. 10 at 0h after the preparation of sample working fluid No. 10), and record the peak area of ​​the characteristic peak of each vitamin. Then store the sample working fluid No. 10 at room temperature (about 25°C), and take an appropriate amount of the sample working fluid No. 10 for liquid chromatography analysis at 7h and 16.5h, respectively, record the peak area of ​​each vitamin, and calculate the relative standard deviation. The results are shown in Table 5 below.

[0122] Table 5

[0123]

[0124]

[0125] Deviation 1 in Table 5 refers to the relative standard deviation of the peak area of ​​the characteristic peak of each vitamin at the 7th hour relative to the peak area at the 0th hour, and Deviation 2 refers to the relative standard deviation of the peak area of ​​the characteristic peak of each vitamin at the 16.5th hour relative to the peak area at the 0th hour. It can be seen from Table 5 that as the storage time of the No. 10 sample working solution increases, the deviation of the peak area of ​​the characteristic peak of each vitamin increases, and the accuracy of the test result decreases accordingly. Therefore, when the method provided in the embodiment of the present invention is used to simultaneously detect multiple water-soluble vitamins in the human assisted reproductive culture medium, it is advisable to perform the test immediately after the pretreatment of the human assisted reproductive culture medium.

[0126] Next, the advantages of the method for simultaneously detecting multiple vitamins in human assisted reproductive culture medium provided by the embodiment of the present invention are explained.

[0127] Embodiment 1

[0128] A human assisted reproductive culture medium was prepared, and based on the amount of materials added during preparation, the concentrations of calcium pantothenate, pyridoxal, riboflavin, and thiamine in the human assisted reproductive culture medium were calculated to be 1.0 mg / L, 1.0 mg / L, 0.1 mg / L, and 1.0 mg / L.

[0129] Take 2 ml of the above human assisted reproductive culture medium and perform step S10 on it to obtain sample working solution No. 11. Then perform liquid chromatography analysis on the sample working solution No. 11 to obtain the following: Figure 4 The chromatogram shown. Figure 4 and the linear equations determined in the previous text, it was obtained that the concentration of calcium pantothenate in the working solution of sample No. 11 was 1.035 mg / L, the concentration of pyridoxal was 0.867 mg / L, the concentration of riboflavin was 0.0916 mg / L, and the concentration of thiamine was 0.876 mg / L, which were basically consistent with the dosage, indicating that the method provided in the embodiment of the present invention can effectively simultaneously detect the contents of multiple water-soluble vitamins in human assisted reproductive culture medium.

[0130] Comparative Example 1

[0131] The difference between this comparative example and Example 1 is that step S1 is performed on 2 ml of human assisted reproductive culture medium, but step S10 is not performed (i.e., step S1 is used to replace step S10). Step S1 includes: mixing 4% by mass sulfosalicylic acid solution with human assisted reproductive culture medium, precipitating, centrifuging and taking the supernatant as the No. 1 comparative sample working solution; wherein the volume ratio of the sulfosalicylic acid solution to the human assisted reproductive culture medium is 1:1.

[0132] The No. 1 comparative sample working solution was subjected to liquid chromatography analysis under the chromatographic conditions of condition 1, and the following was obtained: Figure 5 The spectrum shown. Figure 5 It can be seen that there is an interference peak near the characteristic peak of pyridoxal, which is not conducive to the detection of pyridoxal.

[0133] Comparative Example 2

[0134] The difference between this comparative example and Example 1 is that step S1' is performed on 2 ml of human assisted reproductive culture medium, but step S10 is not performed (i.e., step S1' replaces step S10). Step S1' comprises: mixing acetonitrile with the human assisted reproductive culture medium, precipitating, centrifuging and taking the supernatant as the No. 2 comparative sample working solution; wherein the volume ratio of the sulfosalicylic acid solution to the human assisted reproductive culture medium is 1:1.

[0135] The No. 2 comparative sample working solution was subjected to liquid chromatography analysis under the chromatographic conditions of condition 1, and the following was obtained: Figure 6In the spectrum shown, the characteristic peak of calcium pantothenate does not appear in the spectrum, and the characteristic peak of pyridoxal is not significant, that is, calcium pantothenate was not successfully separated in Comparative Example 2, and the separation effect of pyridoxal was poor.

[0136] Comparative Example 3

[0137] Prepare the standard working solutions No. 1 to No. 5 as described above. Perform liquid chromatography analysis on the standard working solutions No. 1 to No. 5 under condition 2, wherein the difference between condition 2 and condition 1 is that isocratic elution is used instead of gradient elution, and the volume ratio of sodium acetate buffer to methanol is 80:20. Figure 7 The liquid chromatogram of standard working solution No. 4 is shown. The liquid chromatograms of other standard working solutions are Figure 7 They are basically the same, only the peak area is different, so it is not given here. Figure 7 It can be seen that the characteristic peak of riboflavin does not appear, that is, riboflavin cannot be separated by condition 2.

[0138] Comparative Example 4

[0139] Prepare the standard working solutions No. 1 to No. 5 as described above. Perform liquid chromatography analysis on the standard working solutions No. 1 to No. 5 under condition 3, wherein the difference between condition 3 and condition 1 is that isocratic elution is used instead of gradient elution, and the volume ratio of sodium acetate buffer to methanol is 90:10. Figure 8 The liquid chromatogram of standard working solution No. 4 is shown. The liquid chromatograms of other standard working solutions are Figure 8 They are basically the same, only the peak area is different, so it is not given here. Figure 8 It can be seen that the characteristic peak of riboflavin does not appear, that is, riboflavin cannot be separated by condition 3.

[0140] Comparative Example 5

[0141] Prepare the standard working solutions No. 1 to No. 5 as described above. Perform liquid chromatography analysis on the standard working solutions No. 1 to No. 5 under condition 4, wherein the difference between condition 4 and condition 1 is that the pH of the sodium acetate buffer is 5. Fig. 9 The liquid chromatogram of standard working solution No. 4 is shown. The liquid chromatograms of other standard working solutions are Fig. 9 They are basically the same, only the peak area is different, so it is not given here. Fig. 9 It can be seen that the characteristic peak of calcium pantothenate is not present, that is, calcium pantothenate cannot be separated by condition 5.

[0142] Comparative Example 6

[0143] Prepare the standard working solutions No. 1 to No. 5 as described above. Perform liquid chromatography analysis on the standard working solutions No. 1 to No. 5 under condition 5, wherein the difference between condition 5 and condition 1 is that the pH of the sodium acetate buffer is 8.5. Fig.10 The liquid chromatogram of standard working solution No. 4 is shown. The liquid chromatograms of other standard working solutions are Fig.10 They are basically the same, only the peak area is different, so it is not given here. Fig.10 It can be seen that although the characteristic peaks of calcium pantothenate, thiamine, pyridoxal, and riboflavin appear at the same time, the response values ​​of the peaks are all low, and the baseline of thiamine is poor, which is not conducive to accurate quantification.

[0144] Embodiment 2

[0145] Prepare the aforementioned standard working solutions No. 1 to No. 5. Perform liquid chromatography analysis on the standard working solutions No. 1 to No. 5 under condition 5, wherein the difference between condition 5 and condition 1 is that the pH of the sodium acetate buffer is 7.32. Fig.11 The liquid chromatogram of standard working solution No. 4 is shown. The liquid chromatograms of other standard working solutions are Fig.11 They are basically the same, so they are not given here. Fig.11 It can be seen that condition 5 can achieve effective separation of calcium pantothenate, thiamine, pyridoxal, and riboflavin, and make each characteristic peak have a higher response value and a better baseline, which can achieve accurate quantification of calcium pantothenate, thiamine, pyridoxal, and riboflavin.

[0146] It can be seen from the above-mentioned Example 1, Example 2 and Comparative Examples 1 to 6 that the method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium provided by the embodiments of the present invention is effective.

[0147] Although the present invention is disclosed as above, it is not limited thereto. Those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for simultaneously detecting multiple water-soluble vitamins in a human assisted reproductive culture medium, wherein the water-soluble vitamins include at least vitamin B1, vitamin B2, vitamin B5 and vitamin B6, and the vitamin B6 includes pyridoxal hydrochloride, characterized in that: The method comprises the following steps: pre-treating the human assisted reproductive culture fluid to obtain a sample working fluid; and performing liquid chromatography analysis on the sample working fluid; the liquid chromatography analysis conditions are: Chromatographic column: C18 chromatographic column, column temperature 25℃~45℃; Mobile phase: sodium acetate buffer-methanol, flow rate 0.8mL / min~1.5mL / min; Elution conditions: gradient elution; Detector: UV detector or diode array detector; Wherein, the gradient elution includes a first elution stage, a second elution stage, a third elution stage and a fourth elution stage which are performed continuously; in the first elution stage, the volume ratio of sodium acetate buffer to methanol is constant, and the volume percentage of sodium acetate buffer is greater than the volume percentage of methanol; in the second elution stage, the volume percentage of sodium acetate buffer decreases according to a first change rate; in the third elution stage, the volume percentage of sodium acetate buffer decreases to zero according to a second change rate, and the second change rate is greater than the first change rate; in the fourth elution stage, the volume percentage of sodium acetate buffer is constant at zero.

2. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to claim 1, characterized in that: The first change rate is 5.8-7.25, and the second change rate is 8-20.

3. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to claim 1, characterized in that: The duration of the first elution stage is 4 minutes to 5 minutes, and the volume ratio of sodium acetate buffer to methanol is 98:2 to 95:5; the duration of the second elution stage is 8 minutes to 10 minutes, and at the end of the second elution stage, the volume ratio of sodium acetate buffer to methanol is 40:60; the duration of the third elution stage is 2 minutes to 5 minutes; the duration of the fourth elution stage is 2 minutes to 5 minutes.

4. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to claim 1, characterized in that: The preparation method of sodium acetate buffer is as follows: weigh sodium acetate trihydrate according to the ratio, dissolve the sodium acetate trihydrate in water to obtain a sodium acetate aqueous solution; or, The preparation method of sodium acetate buffer is as follows: sodium acetate trihydrate is weighed according to the ratio, sodium acetate trihydrate is dissolved in water to obtain a sodium acetate aqueous solution, and glacial acetic acid is added to the sodium acetate aqueous solution to adjust the pH; The concentration of the sodium acetate buffer is 0.05 mol / L to 0.25 mol / L, and the pH value at 25° C. is 6-7.

5.

5. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to claim 1, characterized in that: The pre-treatment step includes: placing a first predetermined amount of human assisted reproductive culture fluid in an ultrafiltration tube, centrifuging and taking a target filtrate, filtering the target filtrate through a water filter membrane to obtain the sample working fluid.

6. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to claim 5, characterized in that: The specification of the ultrafiltration tube is 10K, the centrifugal speed is 6000r / min to 10000r / min, and the centrifugal time is 5min to 10min; the pore size of the water filter membrane is 0.22μm or 0.45μm.

7. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to claim 5, characterized in that: Before placing the human assisted reproductive culture fluid in the ultrafiltration tube, the method also includes the following steps: adding a second predetermined amount of human assisted reproductive culture fluid into the ultrafiltration tube, centrifuging at 6000r / min to 10000r / min for 5min to 10min, and discarding the filtrate.

8. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to claim 1, characterized in that: The detection wavelength of vitamin B5 is 210nm~215nm, the detection wavelength of vitamin B1 and vitamin B2 is 260nm~270nm, and the detection wavelength of vitamin B6 is 210nm~215nm or 260nm~270nm.

9. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to any one of claims 1 to 8, characterized in that: The method further comprises: Obtaining a linear equation of the standard working solution of the water-soluble vitamin; and According to the linear equation of the water-soluble vitamins, the content of the water-soluble vitamins in the human assisted reproductive culture medium is calculated.

10. The method for simultaneously detecting multiple water-soluble vitamins in human assisted reproductive culture medium according to claim 9, characterized in that: The linear equation of the standard working solution of the water-soluble vitamin is obtained in the following manner: Prepare standard stock solutions of thiamine hydrochloride, riboflavin, calcium pantothenate, and pyridoxal hydrochloride respectively; Using the standard stock solution to prepare a standard working solution of a predetermined concentration; Pre-treating the standard working solution to obtain a standard test sample; Pre-treating a blank matrix to obtain a first comparative test sample, wherein the blank matrix does not include thiamine hydrochloride, riboflavin, calcium pantothenate and pyridoxal hydrochloride compared to the human assisted reproductive culture medium; Pre-treating the water to obtain a second comparative test sample; The standard test sample, the first comparative test sample and the second comparative test sample are subjected to liquid chromatography analysis under the liquid chromatography analysis conditions.

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