Method for detecting total 25-hydroxyvitamin d in breast milk

Abstract

The application provides a detection method for the total amount of 25-hydroxy vitamin D in breast milk. The detection method specifically comprises the following steps: mixing a breast milk sample, an internal standard working solution and a first extracting agent, performing first extraction to obtain a precipitate and a first extracting solution; mixing the precipitate and a second extracting agent, performing second extraction to obtain a second extracting solution; mixing the first extracting solution and the second extracting solution, and then performing a derivatization reaction to obtain a sample to be tested; redissolving the sample to be tested to obtain a sample solution to be tested; detecting the sample solution to be tested by high performance liquid chromatography tandem mass spectrometry to obtain the total amount of 25-hydroxy vitamin D in the breast milk. The detection method can quickly and accurately detect the total amount of 25-hydroxy vitamin D and the content of metabolites 25-hydroxy vitamin D3, 25-hydroxy vitamin D2 and 1,25-dihydroxy vitamin D3.

Description

Technical Field

[0001] This invention relates to the field of vitamin analysis and detection technology, specifically to a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. Background Technology

[0002] Breast milk is the sole source of nutrition for infants during the breastfeeding period and is crucial in their growth and development. Since infants rely entirely on breast milk for nutrition, it is especially important whether the nutrient content of breast milk meets the needs of infants during the breastfeeding period.

[0003] Vitamin D plays a vital role in infant growth, brain development, and immune system development. Vitamin D deficiency in infants can lead to impaired calcium and phosphorus absorption and hinder bone and teeth development, thus affecting overall physical development. In severe cases, it can also impact the immune system, causing immune system diseases and rickets. In the womb, vitamin D primarily exists as vitamin D3 and D2; however, vitamin D3 is rapidly converted in the body into 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3.

[0004] The vitamin D content in breast milk varies with the seasons. Studies have shown that exposure to sunlight or UV-B can increase the level of 25-hydroxyvitamin D. Since 25-hydroxyvitamin D in breast milk reflects the mother's vitamin D reserves, this method is useful for reducing vitamin D deficiency in infants. It requires no complex blood tests and can quickly and easily measure the total amount of 25-hydroxyvitamin D in breast milk, allowing mothers to understand the vitamin D levels in their breast milk and make timely adjustments.

[0005] Current research rarely includes methods for detecting the total amount of 25-hydroxyvitamin D in breast milk; most methods focus on detecting 25-hydroxyvitamin D in serum. For example, CN108645942A discloses a high-performance liquid chromatography-tandem mass spectrometry method for detecting 25-hydroxyvitamin D in serum. In this method, only simple pretreatment and solid-liquid extraction are performed on the serum sample. Pretreatment involves adding internal standard working solution and sodium hydroxide solution to the serum sample sequentially. Solid-liquid extraction involves loading the pretreated sample onto an SLE 96-well plate, allowing it to stand for 5 minutes, and then adding 1-2 mL of n-hexane in four portions to the SLE 96-well plate for elution. This pretreatment method is suitable for extracting analytes from serum. However, because breast milk has a more complex composition, containing various proteins, carbohydrates, and lipids, directly converting it to breast milk is difficult to remove all these substances using this extraction method, resulting in a lack of clear analyte and a significant matrix effect.

[0006] Therefore, there is an urgent need to establish a method suitable for determining the total amount of 25-hydroxyvitamin D in breast milk.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The present invention aims to provide a method for detecting the total 25-hydroxyvitamin D in breast milk. The method specifically includes the following steps: mixing a breast milk sample, an internal standard working solution, and a first extraction agent for a first extraction to obtain a precipitate and a first extract; the first extraction agent includes an antioxidant, methanol, and acetonitrile; mixing the precipitate with a second extraction agent for a second extraction to obtain a second extract; the second extraction agent includes methyl tert-butyl ether, n-hexane, and ethyl acetate; mixing the first and second extracts and performing a derivatization reaction to obtain a sample to be tested; redissolving the sample to be tested to obtain a sample solution; and detecting the sample solution using high-performance liquid chromatography-tandem mass spectrometry to obtain the total 25-hydroxyvitamin D in the breast milk. This detection method, through the use of a specific extraction agent, better removes various proteins, carbohydrates, and lipid compounds, extracting and purifying the target compounds from the complex composition of the breast milk sample, thus improving the detection sensitivity and selectivity of the method and effectively reducing interference that may be introduced by complex matrices in the sample.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0010] In a first aspect, the present invention provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk, the method specifically comprising the following steps:

[0011] The breast milk sample, internal standard working solution, and first extraction reagent were mixed and subjected to first extraction to obtain precipitate and first extract; the first extraction reagent included antioxidant, methanol, and acetonitrile.

[0012] The precipitate and the second extractant are mixed and subjected to a second extraction to obtain a second extract; the second extractant includes methyl tert-butyl ether, n-hexane and ethyl acetate.

[0013] The first and second extracts were mixed and a derivatization reaction was carried out to obtain the sample to be tested.

[0014] The sample to be tested is reconstituted to obtain the sample solution.

[0015] The total amount of 25-hydroxyvitamin D in the breast milk was obtained by detecting the sample solution using high performance liquid chromatography-tandem mass spectrometry.

[0016] The purpose of this invention is to provide a method for rapidly determining the total amount of 25-hydroxyvitamin D in breast milk, which can rapidly detect the total amount of 25-hydroxyvitamin D and the content of its metabolites 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3.

[0017] In this invention, by employing specific extractants and extraction steps, various proteins, carbohydrates, and lipids are more effectively removed. This allows for the extraction and purification of the analyte from complex breast milk samples, significantly reducing matrix effects and improving the method's detection sensitivity and selectivity. Furthermore, derivatization imparts specific groups to the target compound, increasing its polarity and resulting in earlier peak elution, thus shortening the detection time for a single sample. It also improves ionization of the analyte, further enhancing sensitivity.

[0018] Preferably, the mass ratio of the breast milk sample, the internal standard working solution, and the first extraction agent is 1:(0.005-0.5):(0.5-5);

[0019] Among them, "0.005~0.5" can be, for example, 0.005, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, etc.;

[0020] Among them, "0.5 to 5" can be, for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0021] Preferably, the internal standard working solution includes 25-hydroxyvitamin D3-[d3], 25-hydroxyvitamin D2-[d6], and 1,25-dihydroxyvitamin D3-[d3].

[0022] Since vitamin D exists primarily in the mother's body in the forms of vitamin D3 and D2, and vitamin D3 is rapidly converted into 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 in vivo, the internal standard working solution of this invention includes 25-hydroxyvitamin D3-[d3], 25-hydroxyvitamin D2-[d6], and 1,25-dihydroxyvitamin D3-[d3], enabling rapid and accurate detection of the total amount of 25-hydroxyvitamin D and the content of its metabolites 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, and 1,25-dihydroxyvitamin D3.

[0023] The structural formula of 25-hydroxyvitamin D3 is shown below:

[0024]

[0025] The structural formula of 25-hydroxyvitamin D2 is shown below:

[0026]

[0027] Preferably, the internal standard working solution comprises, by mass concentration: 5-15 ng / mL of 25-hydroxyvitamin D3-[d3], 5-15 ng / mL of 25-hydroxyvitamin D2-[d6], and 5-15 ng / mL of 1,25-dihydroxyvitamin D3-[d3].

[0028] In the internal standard working solution, the content of 25-hydroxyvitamin D3-[d3] is 5 to 15 ng / mL, for example, it can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, etc.

[0029] In the internal standard working solution, the content of 25-hydroxyvitamin D-[d6] is 5 to 15 ng / mL, for example, it can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, etc.

[0030] In the internal standard working solution, the content of 1,25-dihydroxyvitamin D3-[d3] is 5 to 15 ng / mL, for example, it can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, etc.

[0031] Preferably, the solvent of the internal standard working solution is a methanol solution containing 0.05 to 0.5 wt% (e.g., 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%) of antioxidant.

[0032] Preferably, the antioxidant in the solvent of the internal standard working solution is 2,6-di-tert-butyl-p-cresol (BHT).

[0033] Preferably, the first extractant includes a methanol solution containing 0.05 to 0.5 wt% (e.g., 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%) of an antioxidant.

[0034] Preferably, in the first extractant, the mass ratio of methanol solution containing 0.05-0.5 wt% antioxidant to acetonitrile is 1:(5-15), for example, it can be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc.

[0035] Preferably, the antioxidant in the first extractant is 2,6-di-tert-butyl-p-cresol (BHT).

[0036] Preferably, the first extraction specifically includes the following steps:

[0037] First, the breast milk sample, internal standard working solution, and methanol solution containing 0.05-0.5 wt% antioxidant are mixed and vortexed; then acetonitrile is added to the system and ultrasonic extraction is performed; then centrifugation is performed to obtain precipitate and supernatant; wherein, the supernatant is the first extract.

[0038] Preferably, the rotational speed of the vortex is 500–3500 r / min, for example, it can be 500 r / min, 1000 r / min, 1500 r / min, 2000 r / min, 2500 r / min, 3000 r / min, 3500 r / min, etc., and the vortex duration is 20–40 s, for example, it can be 20 s, 22 s, 24 s, 26 s, 28 s, 30 s, 32 s, 34 s, 36 s, 38 s, 40 s, etc.

[0039] Preferably, the ultrasonic extraction power is 10-100W, for example, 10W, 20W, 30W, 40W, 50W, 60W, 70W, 80W, 90W, 100W, etc.; the ultrasonic extraction temperature is 1-10℃, for example, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, etc.; and the ultrasonic extraction time is 5-15min, for example, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.

[0040] Preferably, the centrifugation speed is 8000-12000 r / min, for example, 8000 r / min, 8500 r / min, 9000 r / min, 9500 r / min, 10000 r / min, 10500 r / min, 11000 r / min, 11500 r / min, 12000 r / min, etc., and the centrifugation time is 5-15 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.

[0041] Preferably, the mass ratio of the precipitate to the second extractant is 1:(5-15), for example, it can be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc.

[0042] Preferably, the second extractant is composed of methyl tert-butyl ether, n-hexane, and ethyl acetate in a volume ratio of (1-3):(0.5-2):(0.5-2);

[0043] Among them, "1 to 3" can be, for example, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc.;

[0044] The first "0.5~2" can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, etc.

[0045] The second "0.5~2" can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, etc.

[0046] In a preferred embodiment of the present invention, the volume ratio of the tert-butyl ether, n-hexane, and ethyl acetate is 2:1:1.

[0047] Preferably, the second extraction specifically includes the following steps:

[0048] First, the precipitate and the second extractant are mixed and subjected to ultrasonic extraction; then, centrifugation is performed to obtain the precipitate and the supernatant; wherein, the supernatant is the second extract.

[0049] Preferably, the ultrasonic extraction power is 10-100W, for example, 10W, 20W, 30W, 40W, 50W, 60W, 70W, 80W, 90W, 100W, etc.; the ultrasonic extraction temperature is 1-10℃, for example, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, etc.; and the ultrasonic extraction time is 5-15min, for example, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.

[0050] Preferably, the centrifugation speed is 8000-12000 r / min, for example, 8000 r / min, 8500 r / min, 9000 r / min, 9500 r / min, 10000 r / min, 10500 r / min, 11000 r / min, 11500 r / min, 12000 r / min, etc., and the centrifugation time is 5-15 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.

[0051] Preferably, the derivatization reaction is further subjected to a concentration treatment until the liquid content of the concentrate is below 0.5 wt%, for example, it can be 0.5 wt%, 0.45 wt%, 0.4 wt%, 0.35 wt%, 0.3 wt%, 0.25 wt%, 0.2 wt%, 0.15 wt%, 0.1 wt%, 0 wt%, etc.

[0052] Preferably, the concentration process is carried out using nitrogen blowing.

[0053] Preferably, the concentration process is carried out in a light-proof water bath nitrogen blowing apparatus.

[0054] Preferably, in the concentration process, the temperature of the water bath is 25-35°C, for example, 25°C, 26°C, 28°C, 30°C, 32°C, 34°C, 35°C, etc.

[0055] Preferably, in the concentration process, the nitrogen pressure is 0.05 to 0.5 MPa, for example, it can be 0.05 MPa, 0.1 MPa, 0.15 MPa, 0.2 MPa, 0.25 MPa, 0.3 MPa, 0.35 MPa, 0.4 MPa, 0.45 MPa, 0.5 MPa, etc.

[0056] Preferably, the derivatizing reagent used in the derivatization is 4-phenyl-1,2,4-triazolline-3,5-dione (PTAD).

[0057] The structural formula of PTAD is shown below:

[0058]

[0059] Preferably, the derivatization reaction specifically includes the following steps:

[0060] The first and second extracts were mixed and concentrated; the concentrate was then mixed with an acetonitrile solution containing PTAD and subjected to a derivatization reaction.

[0061] Preferably, the mass ratio of the concentrate to PTAD is (1-5):(1-5);

[0062] The first "1 to 5" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0063] The second "1 to 5" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0064] Preferably, the mass concentration of the acetonitrile solution containing PTAD is 40–60 μg / mL, for example, it can be 40 μg / mL, 42 μg / mL, 44 μg / mL, 46 μg / mL, 48 μg / mL, 50 μg / mL, 52 μg / mL, 54 μg / mL, 56 μg / mL, 58 μg / mL, 60 μg / mL, etc.

[0065] Preferably, the temperature of the derivatization reaction is 20-30°C, for example, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, etc., and the time of the derivatization reaction is 20-40 min, for example, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, etc.

[0066] Preferably, the derivatization reaction is followed by a concentration process until the liquid content of the concentrate is below 0.5 wt%, for example, it can be 0.5 wt%, 0.45 wt%, 0.4 wt%, 0.35 wt%, 0.3 wt%, 0.25 wt%, 0.2 wt%, 0.15 wt%, 0.1 wt%, 0 wt%, etc.; wherein, the concentrate is the sample to be tested.

[0067] The concentration process following the derivatization reaction can be performed using the same steps as the concentration process preceding the derivatization reaction.

[0068] Preferably, the mass ratio of the sample to be tested to the reconstitution solvent is 1:(0.1 to 1), for example, it can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, etc.

[0069] Preferably, the solvent used for redissolution includes an aqueous solution of formic acid and methanol.

[0070] Preferably, the volume ratio of the aqueous solution of formic acid to methanol is (5-7):(3-5);

[0071] Among them, "5 to 7" can be, for example, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, etc.;

[0072] Among them, "3 to 5" can be, for example, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, etc.

[0073] In a preferred embodiment of the present invention, the volume ratio of the aqueous solution of formic acid to methanol is 6:4.

[0074] Preferably, the formic acid content in the aqueous solution of formic acid is 0.05 to 0.2 wt%, for example, it can be 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.15 wt%, 0.2 wt%, etc.

[0075] Preferably, the resolution specifically includes the following steps:

[0076] The sample to be tested is dissolved in the reconstituted solvent and centrifuged to obtain a supernatant; wherein, the supernatant is the sample solution to be tested.

[0077] Preferably, the temperature of the resolution solvent is 1 to 10°C, for example, it can be 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, etc.

[0078] Preferably, the centrifugation speed is 8000-12000 r / min, for example, 8000 r / min, 8500 r / min, 9000 r / min, 9500 r / min, 10000 r / min, 10500 r / min, 11000 r / min, 11500 r / min, 12000 r / min, etc., and the centrifugation time is 5-15 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.

[0079] Preferably, in the detection, the chromatographic column is an ACQUITY UPLC HSS T3 column.

[0080] Preferably, the chromatographic column has the following specifications: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.

[0081] Preferably, the column temperature of the chromatographic column is 30-40℃, for example, it can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.

[0082] Preferably, in the detection, mobile phase A is an aqueous solution of ammonium formate, and mobile phase B is a methanol solution of formic acid.

[0083] Preferably, the pH of the mobile phase A is 5 to 5.5, for example, it can be 5, 5.1, 5.2, 5.3, 5.4, 5.5, etc.

[0084] Preferably, the concentration of ammonium formate in the mobile phase A is 8 to 12 mmol / L, for example, it can be 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L, etc.

[0085] Preferably, the content of formic acid in the mobile phase B is 0.05 to 0.2 wt%, for example, it can be 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.15 wt%, 0.2 wt%, etc.

[0086] Preferably, in the detection, the elution conditions of the chromatographic conditions are as follows:

[0087]

[0088]

[0089] Preferably, in the detection, the mass spectrometry parameters are as follows: MRM multiple reaction monitoring mode, ESI+ ion scanning, ion source temperature 115–125℃ (e.g., 115℃, 116℃, 118℃, 120℃, 122℃, 125℃, etc.), capillary voltage 2.5–3.5kV (e.g., 2.5kV, 2.6kV, 2.8kV, 3kV, 3.2kV, etc.). 3.4kV, 3.5kV, etc.), solvent removal temperature 350~450℃ (e.g., 350℃, 360℃, 380℃, 400℃, 420℃, 440℃, 450℃, etc.), solvent removal flow rate 750~850L / h (e.g., 750L / h, 760L / h, 780L / h, 800L / h, 820L / h, 840L / h, 850L / h, etc.).

[0090] As a preferred embodiment of the present invention, the method for detecting the total amount of 25-hydroxyvitamin D in breast milk specifically includes the following steps:

[0091] S1. First extraction: First, mix the breast milk sample, internal standard working solution, and methanol solution containing antioxidant, and vortex; then add acetonitrile to the system and perform ultrasonic extraction; then centrifuge to obtain precipitate and supernatant; wherein, the supernatant is the first extraction solution;

[0092] S2. Second extraction: First, the precipitate and the second extractant are mixed and subjected to ultrasonic extraction; then, centrifugation is performed to obtain the precipitate and the supernatant; wherein, the second extractant includes methyl tert-butyl ether, n-hexane and ethyl acetate; the supernatant is the second extract;

[0093] S3, Derivatization: The first extract and the second extract are mixed and concentrated for the first time; the concentrate obtained after concentration is then mixed with an acetonitrile solution containing PTAD and derivatization reaction is carried out. Finally, a second concentration is performed to obtain the sample to be tested.

[0094] S4. Resolution: The sample to be tested is dissolved in the resolution solvent and centrifuged to obtain a supernatant; wherein, the resolution solvent used in the resolution includes an aqueous solution of formic acid and methanol; the supernatant is the sample solution to be tested;

[0095] S5. Detection: The sample solution to be tested was detected by high performance liquid chromatography-tandem mass spectrometry to obtain the total amount of 25-hydroxyvitamin D in the breast milk.

[0096] Compared with the prior art, the present invention has the following beneficial effects:

[0097] The detection method described in this invention utilizes a specific extractant to better remove various proteins, carbohydrates, and lipids, extracting and purifying the analyte from complex breast milk samples. This improves the method's detection sensitivity and selectivity, effectively reducing interference that may be introduced by complex matrices in the sample. Furthermore, derivatization imparts specific groups to the target compound, increasing its polarity and resulting in an earlier peak time, thus shortening the detection time for a single sample. It also improves the ionization effect of the analyte, further enhancing sensitivity. Attached Figure Description

[0098] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0099] Figure 1 The total ion chromatogram of the compound provided in Example 1.

[0100] Figure 2 The 25-hydroxyvitamin D3-MRM multiple reaction monitoring chart provided in Example 1.

[0101] Figure 3 Abundance diagram of 25-hydroxyvitamin D3- ions provided in Example 1.

[0102] Figure 4 The 25-hydroxyvitamin D2-MRM multiple reaction monitoring chart provided in Example 1.

[0103] Figure 5 Abundance diagram of 25-hydroxyvitamin D2- ions provided in Example 1.

[0104] Figure 6 The 1,25-dihydroxyvitamin D3-MRM multiple reaction monitoring plot provided in Example 1.

[0105] Figure 7 Abundance diagram of 1,25-dihydroxyvitamin D3- ions provided in Example 1.

[0106] Figure 8 The total ion chromatogram of the compound provided in Example 14.

[0107] Figure 9 The total ion chromatogram of the compound provided in Example 15.

[0108] Figure 10 The total ion chromatogram of the compound provided in Example 16.

[0109] Figure 11 The total ion chromatogram of the compound provided in Example 17.

[0110] Figure 12 The total ion chromatogram of the compound provided in Example 18.

[0111] Figure 13 The total ion chromatogram of the compound provided in Example 19.

[0112] Figure 14 The total ion chromatogram of the compound provided in Example 20.

[0113] Figure 15 The total ion chromatogram of the compound provided in Example 21.

[0114] Figure 16 The total ion chromatogram of the compound provided in Example 22.

[0115] Figure 17 The total ion chromatogram of the compound provided in Example 23.

[0116] Figure 18 The total ion chromatogram of the compound provided in Example 24.

[0117] Figure 19 The total ion chromatogram of the compound provided in Example 25.

[0118] Figure 20 The total ion chromatogram of the compound provided in Example 26. Detailed Implementation

[0119] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0120] It should be noted that specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many ways other than those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0121] I. The sources and specifications of some of the raw materials used in the following examples are shown below (unless otherwise specified, all reagents used below are analytical grade reagents; water is Grade I water conforming to GB / T 6682):

[0122] I-1. Methanol, acetonitrile, n-hexane, ethyl acetate, methyl tert-butyl ether, formic acid (mass spectrometry grade, purchased from Fisher Scientific, USA);

[0123] I-2. Standards: 25-hydroxyvitamin D3 standard solution (100 μg / mL, 19356-17-3); 25-hydroxyvitamin D3-[d3] standard solution (100 μg / mL, 140710-94-7); 25-hydroxyvitamin D2 standard solution (100 μg / mL, 21343-40-8), 25-hydroxyvitamin D2-[d6] standard solution (100 μg / mL, 1262843-46-8), 1,25-dihydroxyvitamin D3 (100 μg / mL, 32222-06-3), 1,25-dihydroxyvitamin D3-[d3] (100 μg / mL, 128723-16-0) were purchased from Manhag (Shanghai) Biotechnology Co., Ltd.

[0124] I-3,4-Phenylacet-1,2,4-Triazoline-3,5-dione (PTAD, 4233-33-4) (Sigma-Aldrich, USA).

[0125] II. The models and manufacturers of the testing instruments and equipment used in the following examples are shown below:

[0126] ACQUITY UPLC TQD high-performance liquid chromatography-tandem mass spectrometry system (Waters Corporation, USA);

[0127] BP210S analytical balance (Sartorius GmbH, Germany);

[0128] HD-D12 water bath nitrogen evaporator (Shanghai Hengmei Electronic Technology Co., Ltd.);

[0129] 3-K-15 centrifuge (Sigma Corporation, USA);

[0130] SCI-FS MX-F Vortex Mixer (Shanghai Jinlan Instrument Manufacturing Co., Ltd.)

[0131] Note: To avoid photolysis, this experiment should be performed under light-protected conditions.

[0132] Example 1

[0133] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk, the method specifically including the following steps:

[0134] S1, First Extraction:

[0135] Weigh 0.1 g of BHT into a volumetric flask and dilute to 100 mL with methanol to obtain a 0.1 wt% BHT methanol solution.

[0136] Mixed internal standard working solution (10 μg / mL): Pipette 1.0 mL each of 100 μg / mL internal standard working solutions of 25-hydroxyvitamin D3-d3, 25-hydroxyvitamin D2-d6, and 1,25-dihydroxyvitamin D3-d3 into volumetric flasks, and dilute to 10.0 mL with 0.1 wt% BHT methanol solution to obtain the internal standard working solution;

[0137] Mixed internal standard working solution (10 ng / mL): Transfer 0.010 mL of the 10 μg / mL mixed internal standard working solution into a volumetric flask, and dilute to 10.0 mL with 0.1 wt% BHT methanol solution to obtain the internal standard working solution;

[0138] Weigh 1.0 g (accurate to 0.0001 g) of breast milk sample into a 10.0 mL polytetrafluoroethylene centrifuge tube, add 0.010 mL of the above internal standard working solution, then add 100 μL of 0.1 wt% BHT methanol solution, vortex at 3000 r / min for 30 s, add 1.0 mL of acetonitrile, and sonicate at 100 W and 4 °C for 10 min, and finally centrifuge at 10000 r / min for 10 min to obtain precipitate and supernatant. Transfer the supernatant to another centrifuge tube (first extraction solution).

[0139] S2, Second Extraction:

[0140] Weigh 10 mL of methyl tert-butyl ether, 5 mL of n-hexane and 5 mL of ethyl acetate, mix them evenly to obtain the second extractant;

[0141] Add 2.0 mL of the second extraction agent to the precipitate obtained in S1, and extract by ultrasonication at 100 W and 4 °C for 10 min. Finally, centrifuge at 10000 r / min for 10 min to obtain the precipitate and supernatant. Transfer the supernatant to another centrifuge tube (second extraction solution).

[0142] S3, Derivatization Process:

[0143] Weigh 0.1 g of PTAD and dissolve it in 100 mL of acetonitrile, mixing thoroughly. Then transfer 1.0 mL of the mixed solution and dilute it to 20.00 mL with acetonitrile to obtain a 50 μg / mL PTAD acetonitrile solution.

[0144] The first and second extracts were combined, and then the combined extracts were transferred to a light-proof water bath nitrogen blower. The water bath temperature was controlled at 30°C and the nitrogen flow rate was controlled within 0.1 MPa. The extracts were slowly blown to near dryness to obtain a concentrate.

[0145] Add 100 μL of PTAD acetonitrile solution with a concentration of 50 μg / mL to the above concentrate, and then perform derivatization at room temperature in the dark for 30 min. After derivatization, blow the solvent dry with nitrogen gas, and control the nitrogen flow rate to within 0.1 MPa to obtain the sample to be tested.

[0146] S4, Reconstitute:

[0147] Weigh 0.1 g of formic acid into a volumetric flask, and dilute to 100 mL with water to obtain a 0.1 wt% formic acid aqueous solution; then mix the formic acid aqueous solution with methanol in a volume ratio of 6:4 to obtain a reconstituted solvent;

[0148] The sample was dissolved in 0.10 mL of the above-mentioned reconstitution solvent, and the temperature of the reconstitution solvent was controlled at 4℃. Finally, it was centrifuged at 10000 r / min for 10 min to obtain the supernatant, which is the sample solution to be tested.

[0149] S5, Liquid Crystal Mass Detection

[0150] The supernatant was transferred to a sample vial, and the sample solution was detected by high performance liquid chromatography-tandem mass spectrometry.

[0151] The chromatographic conditions are as follows:

[0152]

[0153] Gradient elution is shown below:

[0154]

[0155]

[0156] The mass spectrometry conditions are as follows:

[0157] The MRM (Multi-Reaction Monitoring) mode, ESI+ ion scanning, ion source temperature 120℃, capillary voltage 3kV, desolventizing temperature 400℃, and desolventizing flow rate 800L / h were used.

[0158] The multiple reaction monitoring parameters for 25-hydroxyvitamin D are shown in Table 1 below:

[0159] Table 1

[0160]

[0161] The specific test results are shown in Table 2 below:

[0162] Table 2

[0163]

[0164] Example 2

[0165] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the acetonitrile in S1 is replaced with an equal volume of methanol, while the other steps are completely consistent with Example 1.

[0166] Example 3

[0167] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the acetonitrile in S1 is replaced with an equal volume of ethanol, while the other steps are completely consistent with Example 1.

[0168] Example 4

[0169] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the acetonitrile in S1 is replaced with an equal volume of acetone, while the other steps are completely consistent with Example 1.

[0170] Test Example 1

[0171] Optimization test of extraction solvent

[0172] Because breast milk samples have a very complex matrix, containing a large number of compounds such as proteins (whey protein, casein, mucin), lipids (glycerides, polar lipids, fatty acids), and carbohydrates (lactose, human milk oligosaccharides), these compounds can affect the detection process. Therefore, they need to be removed during pretreatment. This test example compares the removal of proteins, lipids, and carbohydrates from breast milk samples during pretreatment using different first extraction agents in Examples 1-4.

[0173] The specific test results are shown in Table 3 below:

[0174] Table 3

[0175]

[0176] As shown in Table 3 above, regarding protein precipitation, methanol, acetonitrile, acetone, and ethanol can all precipitate proteins to some extent, but the precipitation effect is acetonitrile > methanol > ethanol > acetone. Secondly, in removing lipids, ethanol and acetone are not very effective, while acetonitrile is more effective than methanol in removing lipids. Thirdly, in removing carbohydrates, only acetonitrile is the most effective, while methanol, acetone, and ethanol are less effective.

[0177] Example 5

[0178] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of methanol as a single solvent. All other steps are completely consistent with Example 1.

[0179] Example 6

[0180] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of a single solvent, acetonitrile. All other steps are completely consistent with Example 1.

[0181] Example 7

[0182] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of the single solvent n-hexane. All other steps are completely consistent with Example 1.

[0183] Example 8

[0184] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of ethyl acetate, a single solvent. All other steps are exactly the same as in Example 1.

[0185] Example 9

[0186] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of a single solvent, tert-butyl methyl ether. All other steps are completely consistent with Example 1.

[0187] Example 10

[0188] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of a mixture of methyl tert-butyl ether, n-hexane, and ethyl acetate in a volume ratio of 1:1:1. All other steps are completely consistent with Example 1.

[0189] Example 11

[0190] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of a mixture of methyl tert-butyl ether and n-hexane in a volume ratio of 1:1. All other steps are completely consistent with Example 1.

[0191] Example 12

[0192] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of a mixture of methyl tert-butyl ether and ethyl acetate in a volume ratio of 1:1. All other steps are completely consistent with Example 1.

[0193] Example 13

[0194] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the second extractant is replaced with an equal volume of a mixture of n-hexane and ethyl acetate in a volume ratio of 1:1. All other steps are completely consistent with Example 1.

[0195] Test Example 2

[0196] Optimization test of extraction solvent

[0197] This test example determines the type and amount of extraction solvent by spiked recovery test of the target analyte. The types of extraction solutions were investigated using the detection methods of Example 1 and Examples 5-13. Spiked recovery tests were performed on breast milk samples.

[0198] The specific test results are shown in Table 4 below:

[0199] Table 4

[0200]

[0201]

[0202] As shown in Table 4 above, the comparison between Examples 5 to 9 shows that when hexane, ethyl acetate, and methyl tert-butyl ether are used as extraction solvents, the spiked recovery results are higher than those when acetonitrile and methanol are used as extraction solvents, but the overall recovery rate is only 62% to 89%.

[0203] As shown in the comparison between Examples 1 and Examples 10-13, the extraction efficiency is higher when the extraction reagent contains methyl tert-butyl ether than when the mixed extraction reagent does not contain methyl tert-butyl ether. When using a combination solvent of ethyl acetate, n-hexane and methyl tert-butyl ether, the spiked recovery rate reaches more than 97%. In particular, when the ratio is 1:1:2, the extraction recovery rate reaches 100%.

[0204] Example 14

[0205] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the chromatographic column used is: ACQUITY UPLC BEH C. 18 (Column length 50mm, inner diameter 2.1mm, particle size 1.7μm), other steps are completely consistent with Example 1.

[0206] Example 15

[0207] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the chromatographic column used is: ACQUITY UPLC Peptide BEH C. 18 (Column length 50mm, inner diameter 2.1mm, particle size 1.7μm), other steps are completely consistent with Example 1.

[0208] Example 16

[0209] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the chromatographic column used is an X Bridge BEH Shield RP18 (column length 50 mm, inner diameter 2.1 mm, particle size 1.7 μm). All other steps are completely consistent with Example 1.

[0210] Example 17

[0211] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the chromatographic column used is an AccQ TAG ULTRA C18 (column length 50 mm, inner diameter 2.1 mm, particle size 1.7 μm). All other steps are completely consistent with Example 1.

[0212] Test Example 3

[0213] Column optimization testing

[0214] The effects of using different types of chromatographic columns on the resolution of compounds were compared.

[0215] The specific test results are as follows: Figure 1 ,as well as Figures 8-11 As shown.

[0216] like Figure 1 , Figures 8-11 The results showed that, under the same chromatographic conditions, the HSS T3 column (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size) with its end-sealing technology provided excellent retention for polar compounds, and its resolution met the requirements of the detection method. Other columns, lacking end-sealing technology, had lower overall resolution, and some compounds could not be separated at baseline. Therefore, this invention ultimately used the ACQUITY UPLC HSST3 column (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size) as the analytical column, achieving baseline separation of the target compounds and laying the foundation for accurate quantification in subsequent steps.

[0217] Example 18

[0218] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that mobile phase A is pure water and mobile phase B is methanol. All other steps are completely the same as in Example 1.

[0219] Example 19

[0220] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that mobile phase A is pure water and mobile phase B is acetonitrile. All other steps are completely consistent with Example 1.

[0221] Example 20

[0222] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that mobile phase A is 0.1 wt% formic acid aqueous solution; mobile phase B is methanol. The other steps are completely consistent with Example 1.

[0223] Example 21

[0224] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the mobile phase A is a 0.1 wt% formic acid aqueous solution and the mobile phase B is acetonitrile. The other steps are completely consistent with Example 1.

[0225] Example 22

[0226] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the mobile phase A is a 10 mmol / L ammonium formate aqueous solution with pH = 5.2; the mobile phase B is methanol. The other steps are completely consistent with Example 1.

[0227] Example 23

[0228] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the mobile phase A is a 10 mmol / L ammonium formate aqueous solution with pH = 5.2; the mobile phase B is acetonitrile. The other steps are completely consistent with Example 1.

[0229] Example 24

[0230] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that the mobile phase A is a 10 mmol / L ammonium formate aqueous solution with pH = 5.2; the mobile phase B is a 0.1 wt% formate acetonitrile solution. The other steps are completely consistent with Example 1.

[0231] Test Example 4

[0232] Optimization test of mobile phase

[0233] The effects of different flow rates on compound separation were compared.

[0234] The specific test results are as follows: Figure 1 , Figures 12-18 As shown.

[0235] Since 25-hydroxyvitamin D is difficult to ionize under ESI+ ionization conditions via electrospray ionization, PTAD is typically used to derivatize the target compound to improve its ionization effect. This involves adding nitrogen-containing groups to the original compound structure, making it easier for 25-hydroxyvitamin D to form a stable [M+H] group during ionization. + Ions enhance the ionization effect.

[0236] The results of Examples 18-20 show that the absence of acid in the mobile phase results in poor separation of the target substance and low response values. A mobile phase that can provide more H+ is needed. + To achieve better sensitivity, this paper selects a certain amount of formic acid solution as the mobile phase.

[0237] A comparison of Examples 1 and Examples 22-24 shows that the resolution, sensitivity, and peak shape of the target compounds are comparable. In Example 1, the mobile phase was determined as follows: Mobile phase A was a 10 mmol / L ammonium formate solution with a pH of 5.2; Mobile phase B was a 0.1 wt% formic acid methanol solution. Since the derivatized products have two peak shapes, 6S and 6R, baseline separation of all target compounds was achieved, and the sensitivity and peak shape were the best.

[0238] Example 25

[0239] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that a flow rate of 0.5 mL / min is used, while the other steps are completely consistent with Example 1.

[0240] Example 26

[0241] This embodiment provides a method for detecting the total amount of 25-hydroxyvitamin D in breast milk. The only difference from Example 1 is that a flow rate of 0.2 mL / min is used, while the other steps are completely consistent with Example 1.

[0242] Test Example 5

[0243] Flow rate optimization test

[0244] The effects of different flow rates on compound separation were compared.

[0245] The specific test results are as follows: Figure 1 , Figures 19-20 As shown.

[0246] A comparison of Examples 1 and 25-26 reveals differences in the resolution, sensitivity, and peak shape of the target compounds. The results show that using a flow rate of 0.3 mL / min with a mobile phase of 10 mmol / L ammonium formate solution and 0.1% formic acid in methanol resulted in baseline separation of all target compounds, with the best sensitivity and peak shape.

[0247] Test Example 6

[0248] Methodological establishment

[0249] Test method: The method for detecting the total amount of 25-hydroxyvitamin D in breast milk provided in Example 1;

[0250] Test items: (1) Linear range and sensitivity; (2) Accuracy and precision;

[0251] The specific test results are shown in Tables 5 and 6 below:

[0252] Table 5

[0253]

[0254] Table 6

[0255]

[0256]

[0257] As shown in Tables 5 and 6, the recovery rate and precision of the test samples were determined by performing recovery experiments at three concentrations (high, medium, and low) and in six parallel runs. The recoveries of the test samples ranged from 84.2% to 98.0%, and the RSDs ranged from 1.3% to 5.9%. The results indicate that this method has good accuracy and precision, meeting the detection requirements.

[0258] Application Example 1

[0259] Test method: The method for detecting the total amount of 25-hydroxyvitamin D in breast milk provided in Example 1;

[0260] Application scenario: To analyze the total amount of 25-hydroxyvitamin D and the content of its metabolites 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in breast milk from regions such as Beijing, Guangxi and Tibet;

[0261] The specific test results are shown in Table 7 below:

[0262] Table 7

[0263]

[0264] As shown in Table 7, the total 25-hydroxyvitamin D in breast milk was analyzed from 33 samples of colostrum, transitional milk, and mature milk from 11 mothers in Beijing, Guangxi, and Tibet, using a rapid method for determining the total 25-hydroxyvitamin D in breast milk. Among these, a large number of mothers in the early postpartum period (c) had low 25-hydroxyvitamin D levels in their breast milk, possibly due to significant depletion of 25-hydroxyvitamin D during the initial lactation period without effective replenishment. A large number of mothers in the transitional and mature postpartum periods had moderate 25-hydroxyvitamin D levels in their breast milk, which may be related to increased dietary intake and sun exposure during the later stages of lactation, leading to a higher level of stored 25-hydroxyvitamin D.

[0265] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the total amount of 25-hydroxyvitamin D in breast milk, characterized in that, The detection method is used to determine the total amount of 25-hydroxyvitamin D and the content of its metabolites 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3. The detection method specifically includes the following steps: The breast milk sample, internal standard working solution and first extraction reagent were mixed and subjected to first extraction to obtain precipitate and first extract; The precipitate and the second extractant are mixed for a second extraction to obtain a second extract. The first and second extracts were mixed and a derivatization reaction was carried out to obtain the sample to be tested. The sample to be tested is reconstituted to obtain the sample solution. The total amount of 25-hydroxyvitamin D in the breast milk was obtained by detecting the sample solution using high performance liquid chromatography-tandem mass spectrometry. The second extractant is composed of methyl tert-butyl ether, n-hexane, and ethyl acetate in a volume ratio of (1~3):(0.5~2):(0.5~2); The derivatization reaction specifically includes the following steps: mixing the first extract and the second extract and then concentrating them; then mixing the concentrate obtained after concentration with an acetonitrile solution containing PTAD and carrying out the derivatization reaction; In the aforementioned detection, an ACQUITY UPLC HSS T3 column was used; mobile phase A was an aqueous solution of ammonium formate with a pH of 5-5.5; mobile phase B was a methanol solution of formic acid. The elution conditions for the chromatographic conditions are as follows: 0~1 min 30 vol% mobile phase B, 70 vol% mobile phase A; 1~3 min 30~95 vol% mobile phase B, 70~5 vol% mobile phase A; 3-4 min 95 vol% mobile phase B, 5 vol% mobile phase A; 4~5 min 95~30 vol% mobile phase B, 5~70 vol% mobile phase A; 5~6 min 30 vol% mobile phase B, 70 vol% mobile phase A.

2. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The first extractant includes acetonitrile.

3. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The mass ratio of the breast milk sample, internal standard working solution, and first extraction reagent is 1:(0.005~0.5):(0.5~5).

4. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The internal standard working solution includes 25-hydroxyvitamin D3-[d3], 25-hydroxyvitamin D-[d6], and 1,25-dihydroxyvitamin D3-[d3].

5. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The internal standard working solution comprises, by mass concentration: 5-15 ng / mL of 25-hydroxyvitamin D3-[d3], 5-15 ng / mL of 25-hydroxyvitamin D2-[d6], and 5-15 ng / mL of 1,25-dihydroxyvitamin D3-[d3].

6. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The solvent of the internal standard working solution is a methanol solution containing 0.05~0.5 wt% antioxidant.

7. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The antioxidant in the solvent of the internal standard working solution is 2,6-di-tert-butyl-p-cresol.

8. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to any one of claims 1 to 7, characterized in that, The first extractant also includes a methanol solution containing 0.05~0.5 wt% antioxidant.

9. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, In the first extractant, the mass ratio of methanol solution containing 0.05~0.5 wt% antioxidant to acetonitrile is 1:(5~15).

10. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The antioxidant in the first extractant is 2,6-di-tert-butyl-p-cresol.

11. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The first extraction specifically includes the following steps: First, the breast milk sample, internal standard working solution, and methanol solution containing 0.05~0.5wt% antioxidant are mixed and vortexed; then acetonitrile is added and ultrasonic extraction is performed; then centrifugation is performed to obtain precipitate and supernatant; wherein, the supernatant is the first extract.

12. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 11, characterized in that, The rotational speed of the vortex is 500~3500 r / min, and the vortex duration is 20~40 s.

13. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 11, characterized in that, The ultrasonic extraction power is 10~100 W, the ultrasonic extraction temperature is 1~10℃, and the ultrasonic extraction time is 5~15 min.

14. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 11, characterized in that, The centrifugation speed is 8000~12000 r / min, and the centrifugation time is 5~15 min.

15. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The mass ratio of the precipitate to the second extractant is 1:(5~15).

16. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The second extraction specifically includes the following steps: First, the precipitate and the second extractant are mixed and subjected to ultrasonic extraction; then, centrifugation is performed to obtain the precipitate and the supernatant; wherein, the supernatant is the second extract.

17. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 16, characterized in that, The ultrasonic extraction power is 10~100 W, the ultrasonic extraction temperature is 1~10℃, and the ultrasonic extraction time is 5~15 min.

18. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 16, characterized in that, The centrifugation speed is 8000~12000 r / min, and the centrifugation time is 5~15 min.

19. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, Before the derivatization reaction, a concentration process is required, and the concentration process is carried out until the liquid content of the concentrate is below 0.5 wt%.

20. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 19, characterized in that, The concentration process is carried out using nitrogen blowing.

21. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 19, characterized in that, The concentration process was carried out in a light-proof water bath nitrogen blowing apparatus.

22. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 19, characterized in that, In the concentration process, the water bath temperature is 25~35℃.

23. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 19, characterized in that, During the concentration process, the nitrogen pressure is 0.05~0.5 MPa.

24. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 19, characterized in that, The mass ratio of the concentrate to PTAD is (1~5):(1~5).

25. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The mass concentration of the acetonitrile solution containing PTAD is 40~60 μg / mL.

26. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The derivatization reaction is carried out at a temperature of 20-30°C for 20-40 minutes.

27. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The derivatization reaction is followed by a concentration process until the liquid content of the concentrate is below 0.5 wt%; wherein the concentrate is the sample to be tested.

28. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The mass ratio of the sample to be tested to the reconstituted solvent is 1:(0.1~1).

29. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The solvents used for redissolution include an aqueous solution of formic acid and methanol.

30. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 29, characterized in that, The volume ratio of the aqueous solution of formic acid to methanol is (5~7):(3~5).

31. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 29, characterized in that, In the aqueous solution of formic acid, the content of formic acid is 0.05~0.2 wt%.

32. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The reconstitution specifically includes the following steps: The sample to be tested is dissolved in a reconstituted solvent and centrifuged to obtain a supernatant; wherein, the supernatant is the sample solution to be tested.

33. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 32, characterized in that, The temperature of the resolution solvent is 1~10℃.

34. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 32, characterized in that, The centrifugation speed is 8000~12000 r / min, and the centrifugation time is 5~15 min.

35. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, The specifications of the chromatographic column are as follows: column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm.

36. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, In the detection, the column temperature of the chromatographic column is 30~40℃.

37. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, In the mobile phase A, the concentration of ammonium formate is 8-12 mmol / L.

38. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, In the mobile phase B, the content of formic acid is 0.05~0.2 wt%.

39. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, In the detection, the flow rate is 0.1~0.5 mL / min.

40. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, In the detection, the injection volume is 5~15 μL.

41. The method for detecting the total amount of 25-hydroxyvitamin D in breast milk according to claim 1, characterized in that, In the detection, the mass spectrometry parameters are as follows: MRM multiple reaction monitoring mode, ESI+ ion scanning, ion source temperature 115~125℃, capillary voltage 2.5~3.5 kV, desolventizing temperature 350~450℃, and desolventizing flow rate 750~850 L / h.

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