Method for rapidly detecting vitamin B12 and biotin in nutrient product

Abstract

The invention discloses a method for rapidly detecting vitamin B12 and biotin in a nutrient product, and belongs to the technical field of analysis and detection.The method comprises the steps that an ammonium thiocyanate solution and a phosphoric acid solution are mixed, and an extracting agent is prepared; adding the nutrient product into an extracting agent for treatment to obtain filtrate; and carrying out two-dimensional liquid chromatography treatment on the filtrate. The specific extraction solvent is used for pre-treating the product, so that the detection rate of vitamin B12 and biotin in the product and the solution stability are improved; under the combination of a specific chromatographic column and a mobile phase, matrix interference can be eliminated and an accurate result can be obtained by utilizing orthogonality and peak capacity characteristics of the two-dimensional liquid chromatography without performing complicated impurity removal and concentration processes on a sample.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a method for quickly detecting vitamin B12 and biotin in nutrient products. Background Art

[0002] Vitamin B12, also known as glutamine, is the only water-soluble vitamin in the human body that contains metal elements. Its main physiological function is to participate in the production of bone marrow red blood cells, prevent diseases such as Alzheimer's disease and depression, and plays an important role in maintaining human health. Biotin, also known as vitamin B7, is a necessary substance for the synthesis of vitamin C. As a coenzyme of carboxylase, carboxyltransferase, and decarboxylase, it participates in the body's gluconeogenesis, lipase and amino acid metabolism, and energy transfer processes. It is an important micronutrient for the human body to maintain normal physiological functions.

[0003] Among the current standards, GB 5009.259-2016 "National Food Safety Standard for the Determination of Biotin in Foods" adopts microbiological methods for detection, which has a long detection cycle and low accuracy; other national standards are mostly liquid phase methods, but they are all applicable to raw materials and have difficulties in analyzing and detecting complex products. GB 5009.285-2022 "National Food Safety Standard for the Determination of Vitamin B12 in Foods" has microbiological methods and chromatography methods. Chromatography is a method in which the sample is treated with enzymatic hydrolysis, purified by immunoaffinity column method, and analyzed by liquid phase or liquid-mass method. GB / T5009.217-2008 "Determination of Vitamin B12 in Health Foods", the sample is purified by solid phase extraction or immunoaffinity column, and then analyzed by liquid phase.

[0004] At present, the existing nutrient products have complex formulas, contain a large amount of vitamins and minerals, and have low VB12 and biotin contents, with the lowest VB12 at 0.05μg / g and the lowest biotin at 1μg / g, which have a great impact on daily testing. Products usually require complex pre-purification treatment, including protein removal, solid phase extraction column or immunoaffinity column purification, enrichment, etc. At present, these methods are difficult to operate, time-consuming, and require high personnel and consumables, so low recovery rates, poor repeatability and accuracy of results are often observed.

[0005] The detection methods usually used are microbiological method, high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry, etc. Among them, the microbiological method has a long analysis time and large deviation; the detection limit and chromatographic separation ability of the high performance chromatography method are sometimes not enough to meet the sample detection. Although high performance liquid chromatography tandem mass spectrometry combines the dual functions of chromatographic separation and mass spectrometry identification, the requirements for sample separation are slightly lower, but it is easy to produce matrix effects, resulting in ion suppression or ion enhancement, which has a serious impact on accuracy and precision. It is usually necessary to purify the sample, chromatographic separation, add internal standards or blank matrix correction to optimize and reduce the impact of the matrix effect, but there will be problems such as expensive purification columns, difficult and long operation time, expensive internal standards, and difficult to obtain blank matrices.

[0006] Therefore, there is an urgent need in this field for a detection method with simple pre-treatment and satisfactory analysis process. Summary of the invention

[0007] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0008] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0009] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for rapidly detecting vitamin B12 and biotin in nutrient products.

[0010] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for rapidly detecting vitamin B12 and biotin in nutrient products, comprising:

[0011] The ammonium thiocyanate solution and the phosphoric acid solution are mixed to prepare an extractant;

[0012] adding an extractant to the nutrient product for treatment to obtain a filtrate;

[0013] The filtrate was subjected to two-dimensional liquid chromatography.

[0014] As a preferred embodiment of the method of the present invention, the extractant, wherein the concentration of the ammonium thiocyanate solution is 0.1% to 0.25% by mass volume ratio, and the concentration of the phosphoric acid solution is 0.1% to 0.5% by volume ratio. For example, the concentration of the ammonium thiocyanate solution is 0.1%, 0.15%, 0.2% or 0.25% by mass volume ratio. For example, the concentration of the phosphoric acid solution is 0.1%, 0.2%, 0.3%, 0.4 or 0.5% by volume ratio.

[0015] As a preferred embodiment of the method of the present invention, the nutrient products include soft candies, tablets and soft capsules.

[0016] As a preferred embodiment of the method of the present invention, the filtrate includes the filtrate of soft candies, the filtrate of tablets and the filtrate of soft capsules.

[0017] As a preferred embodiment of the method of the present invention, the method for preparing the filtrate of the soft candy comprises:

[0018] Take the soft candy, cut it into pieces, mix it evenly, put it in a centrifuge tube, add the extractant, bathe it in water until it is completely dissolved, cool it, centrifuge it in a centrifuge, take the supernatant and filter it to obtain the soft candy filtrate.

[0019] As a preferred embodiment of the method of the present invention, the method for preparing the filtrate of the soft candy comprises:

[0020] Take the soft candy, cut it into pieces, mix it evenly, put it in a centrifuge tube, add the extractant, and place it in a 50-70℃ water bath until it is completely dissolved. Cool it, centrifuge it in a centrifuge at 8000-9500r / min for 5-10min, take the supernatant and filter it to obtain the soft candy filtrate.

[0021] As a preferred embodiment of the method of the present invention, the method for preparing the filtrate of the tablets comprises:

[0022] The tablets are taken, powdered and mixed evenly, and then an extractant is quantitatively added, ultrasonicated in a water bath, cooled, centrifuged, and the supernatant is filtered to obtain the filtrate of the tablets.

[0023] As a preferred embodiment of the method of the present invention, the method for preparing the filtrate of the tablets comprises:

[0024] Take the tablets, grind them into powder, mix them evenly, add the extractant quantitatively, perform water bath ultrasound at 50-70°C, cool them, centrifuge them at 8000-9500 r / min for 5-10 minutes, take the supernatant and filter it to obtain the filtrate of the tablets.

[0025] As a preferred embodiment of the method of the present invention, the method for preparing the filtrate of the soft capsule comprises:

[0026] Take the soft capsule, cut the rubber skin, take out the content and mix evenly, add petroleum ether, vortex for 1-2 minutes, ultrasonic for 3-8 minutes, quantitatively add the extractant, mix evenly, take out by ultrasonic, cool, centrifuge, take out the supernatant and filter to obtain the filtrate of the soft capsule.

[0027] As a preferred embodiment of the method of the present invention, the method for preparing the filtrate of the soft capsule comprises:

[0028] Take the soft capsule, cut the rubber, take out the contents and mix them evenly, add petroleum ether, vortex for 1-2 minutes, ultrasonicate for 3-8 minutes, quantitatively add the extractant, mix evenly, ultrasonicate at 50-70°C, take out and cool, centrifuge at 8000-9500r / min for 5-10 minutes, take out the supernatant and filter to obtain the filtrate of the soft capsule.

[0029] As a preferred embodiment of the method of the present invention, the two-dimensional liquid chromatography treatment uses an octadecyl silica gel chromatography column in the first dimension and a perfluorophenyl silica gel column in the second dimension.

[0030] As a preferred embodiment of the method of the present invention, the octadecyl silica gel chromatographic column is an octadecyl silica gel chromatographic column with the specifications of 4.6×100 mm and 2.7 μm;

[0031] The perfluorophenyl silica gel column is a perfluorophenyl silica gel column, and its specifications are: 4.6x250mm, 3μm.

[0032] As a preferred embodiment of the method of the present invention, the mobile phase of the first dimension includes mobile phase A and mobile phase B, wherein mobile phase A is 0.04-0.06% trifluoroacetic acid solution with a pH of 2-3, and mobile phase B is acetonitrile;

[0033] The second dimension mobile phase includes mobile phase C and mobile phase D, wherein mobile phase C is 0.08-0.12% phosphoric acid with a pH of 3.5-4, and mobile phase D is acetonitrile.

[0034] Beneficial effects of the present invention:

[0035] The present invention provides a method for rapidly detecting vitamin B12 and biotin in nutrient products. The product is pre-treated by a specific extraction solvent to improve the detection rate of vitamin B12 and biotin in the product and the stability of the solution. By using a specific chromatographic column and mobile phase combination and utilizing the orthogonality and high peak capacity characteristics of two-dimensional liquid chromatography, the sample does not need to undergo a complicated impurity removal and concentration process, and matrix interference can be eliminated to obtain accurate results. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0037] Figure 1 This is a valve switching diagram for the first, third and fifth stages of analyzing biotin and vitamin B12 in an embodiment of the present invention.

[0038] Figure 2 This is a diagram of the second and fourth stage valve switching for analyzing biotin and vitamin B12 in the embodiments of the present invention.

[0039] Figure 3 This is a chromatogram of the determination of biotin in the chromatographic column 1 in Example 2 of the present invention.

[0040] Figure 4 This is a chromatogram of measuring VB12 in chromatographic column 1 in Example 2 of the present invention.

[0041] Figure 5 This is a chromatogram of the determination of biotin in the chromatographic column 2 in Example 2 of the present invention.

[0042] Figure 6 This is a chromatogram of measuring VB12 in chromatographic column 2 in Example 2 of the present invention.

[0043] Figure 7 This is a chromatogram of the determination of biotin in the chromatographic column 3 in Example 2 of the present invention.

[0044] Figure 8 This is a chromatogram of measuring VB12 in chromatographic column 3 in Example 2 of the present invention.

[0045] Fig. 9 This is a chromatogram of the determination of biotin in the chromatographic column 4 in Example 2 of the present invention.

[0046] Fig.10 This is a chromatogram of measuring VB12 in chromatographic column 4 in Example 2 of the present invention.

[0047] Fig.11 This is a chromatogram of the determination of biotin in the chromatographic column 5 in Example 2 of the present invention.

[0048] Fig.12 This is a chromatogram of measuring VB12 in chromatographic column 5 in Example 2 of the present invention.

[0049] Fig.13 This is a chromatogram of measuring VB12 in chromatographic column 6 in Example 2 of the present invention.

[0050] Fig.14 This is a chromatogram of the determination of biotin in the chromatographic column 7 in Example 2 of the present invention.

[0051] Fig.15 This is a chromatogram of measuring VB12 in chromatographic column 7 in Example 2 of the present invention.

[0052] Fig.16 This is a chromatogram of the determination of biotin in the chromatographic column 8 in Example 2 of the present invention.

[0053] Fig.17This is a chromatogram of measuring VB12 in chromatographic column 8 in Example 2 of the present invention. DETAILED DESCRIPTION

[0054] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0055] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0056] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0057] Pretreatment steps (summarizing the pretreatment conditions that will affect the technical effects of different dosage forms): The pretreatment uses 0.1%-0.25% ammonium thiocyanate solution and 0.1%-0.5% phosphoric acid solution (that is, 1.0g-2.5g ammonium thiocyanate and 1mL-5mL phosphoric acid per 1000mL water) as an extractant to extract VB12 and biotin from nutrient gummies, tablets, and soft capsules, thereby improving the detection rate and sample solution stability.

[0058] Two-dimensional liquid chromatography: A two-dimensional liquid chromatography method was developed, using the designed interface and capture column to connect two specific chromatographic columns (one-dimensional: octadecyl silica gel column 4.6×100mm, 2.7μm, two-dimensional: perfluorophenyl silica gel column 4.6x250mm, 3μm), and using a specific mobile phase (one-dimensional: 0.04~0.06% trifluoroacetic acid solution, pH2.0-3.0+acetonitrile; two-dimensional: 0.08~0.12% phosphoric acid% pH3.5-4.0)+acetonitrile to achieve separation and enrichment effects, and to achieve simultaneous analysis of vitamin B12 and biotin.

[0059] The method has a low detection limit, and the separation conditions meet the requirements for the detection of products with different contents. The pretreatment operation is simple and does not require a complicated purification process.

[0060] Example 1

[0061] 1. Materials and sources:

[0062] Phosphoric acid, methanol, acetonitrile, ammonium thiocyanate;

[0063] Extraction agent: 0.1%-0.25% (m / V) ammonium thiocyanate, 0.1%-0.5% phosphoric acid solution (V / V); that is, every 1000mL of water contains 1.0g-2.5g of ammonium thiocyanate and 1mL-5mL of phosphoric acid.

[0064] 2. Preparation method

[0065] 2.1 Preparation of standard solution:

[0066] 2.1.1 Biotin:

[0067] Biotin standard stock solution: Accurately weigh 10 mg of biotin standard, place in a 100 mL volumetric flask, add water at 80°C to dissolve, dilute to the mark, shake well, and use as the standard stock solution.

[0068] 2.1.2 Vitamin B12:

[0069] Vitamin B12 standard stock solution: Accurately weigh 10 mg of vitamin B12 standard, place it in a 100 mL volumetric flask, dissolve it to the scale, shake well, and use it as the standard stock solution.

[0070] 2.1.3 Biotin and vitamin B12 mixed standard solution:

[0071] As needed, take 0.5mL-10mL of biotin standard stock solution and 0.1mL-1mL of vitamin B12 standard stock solution into the same 100mL volumetric flask, add water to the scale, and shake well as the standard solution for the machine.

[0072] 2.1.4 Biotin and VB12 positioning standard solution:

[0073] Take 1 mL of the standard stock solution in 2.1.1 and 1 mL of the standard stock solution in 2.1.2 and place them in the same injection vial for use as positioning standard solutions.

[0074] 2.2 Sample preparation

[0075] 2.2.1 Soft candy:

[0076] Take the soft candy, cut it into pieces, mix it evenly, take 1g-5g into a centrifuge tube, add the extractant to make the total volume reach 25mL, bathe in 60℃ water until completely dissolved, cool, centrifuge at 9000r / min in a centrifuge for 5-10min, take the supernatant and filter to obtain the filtrate.

[0077] 2.2.2 Tablets:

[0078] Take the tablets, grind into powder, mix well, take 1-5g into a 50mL volumetric flask, quantitatively add about 30mL of extractant, ultrasonicate in a 50℃ water bath for 20min, cool, use the extractant to make up to the volume, shake well, centrifuge at 9000r / min for 5-10min, take the supernatant and filter it onto the machine.

[0079] 2.2.3 Soft capsules:

[0080] Take the soft capsule, cut the rubber, take out the contents and mix them evenly, take 1-5g into a 50mL centrifuge tube, add 10mL petroleum ether (30℃-60℃), vortex for 1min, ultrasonic for 5min, quantitatively add 40mL extractant, mix evenly, ultrasonic at 50℃ for 30min, take out and cool, centrifuge at 9000r / min for 5-10min, take the supernatant and filter it.

[0081] 2.3 Test method:

[0082] Instrument used: Agilent 1260infinity II liquid chromatography system;

[0083] Chromatographic column: 1D HALOAQ-C18 4.6×100mm, 2.7μm;

[0084] 2D YMC Triart PFP 4.6x250mm, 3μm;

[0085] Trap column ZORBAX RR Eclipse Plus C18 4.6 x 30 mm, 3.5 μm.

[0086] The chromatographic conditions are shown in Table 1.

[0087] Table 1 Chromatographic conditions

[0088]

[0089] Figure 1-2 It is a valve switching diagram from the first stage to the fifth stage for simultaneously analyzing biotin and vitamin B12 by the column switching method of center cutting; wherein, R is a six-way valve, 1, 2, 3, 4, 5, 6 are valve positions, wherein 5 is a closed state; 7 is a one-dimensional mobile phase from a one-dimensional pump; 8 is an automatic sampler; 9 is a one-dimensional chromatographic column; 10 is a one-dimensional UV / diode array detector; 11 is a two-dimensional mobile phase from a two-dimensional pump; 12 is a three-way valve; 13 is a capture column; 14 is a two-dimensional chromatographic column; 15 is a two-dimensional diode array detector.

[0090] The analysis of biotin and vitamin B12 using this method consists of five stages:

[0091] The first stage: sample injection, the first-dimensional mobile phase carries the sample solution through the first-dimensional chromatographic column, at this time the valve position is 1 to 2 (see Figure 1 ), when biotin is about to be detected (15min), enter the second stage;

[0092] The second stage: The one-dimensional mobile phase carries the sample solution through the one-dimensional chromatographic column for separation, and biotin is captured on the capture column. At this time, the valve position is 1 to 6 (see Figure 2 ), when biotin is completely captured (17min), it enters the third stage;

[0093] Phase 3: The first dimension continues to detect vitamin B12 in the sample solution, and the second dimension separates and detects biotin in the capture column. At this time, the valve position is 1 to 2 (see Figure 1 ), when the first dimension is about to detect vitamin B12 (36.50min), it enters the fourth stage;

[0094] Stage 4: The one-dimensional mobile phase carries the sample solution through the one-dimensional chromatographic column for separation, and vitamin B12 is captured on the capture column. At this time, the valve position is 1 to 6 (see Figure 2 ), when vitamin B12 is completely captured (39.50min), it enters the fifth stage;

[0095] Stage 5: Second dimension separation and detection of vitamin B12, at this time the valve position is 1 to 2 (see Figure 1 ).

[0096] 3. Calculation of parameters

[0097] The content (%) of vitamin B12 or biotin was calculated using the external standard method.

[0098]

[0099] In the formula,

[0100] A1——peak area of ​​vitamin B12 or biotin in the sample solution;

[0101] A0——peak area of ​​vitamin B12 or biotin in standard solution;

[0102] W1——the amount of sample weighed, mg;

[0103] W0——weighing amount of vitamin B12 or biotin standard, mg;

[0104] V1——volume of sample solution, mL;

[0105] V0——volume of standard solution, mL;

[0106] K: Purity of the standard.

[0107] Evaluation criteria:

[0108] A. Biotin or vitamin B12 detection rate 90%-105%

[0109] Note: Biotin or vitamin B12 detection rate (%) = (measured value of biotin or vitamin B12 in the sample ÷ theoretical value of biotin or vitamin B12 in the sample) × 100%

[0110] Theoretical value of biotin or vitamin B12 in the sample (%) = the proportion of biotin or vitamin B12 raw materials in the sample (%) × the measured value of biotin or vitamin B12 in the biotin or vitamin B12 raw materials (%))

[0111] B. Stability: Degradation rate ≤ 5%

[0112] Note: Degradation rate (%) = 0 hour detection rate - 12 hour detection rate

[0113] C. Separation effect: Separation degree ≥1.5

[0114] Note: Separation degree is in accordance with Part IV of the Chinese Pharmacopoeia <0512> The calculation formula for calculating the separation degree described by high performance liquid chromatography is:

[0115]

[0116] Where:

[0117] tR2 is the retention time of the latter of two adjacent chromatographic peaks;

[0118] tR1 is the retention time of the previous peak between two adjacent chromatographic peaks;

[0119] W1 and W2 are the peak widths of the two adjacent chromatographic peaks.

[0120] 4. Test Results

[0121] The detection rates of biotin and vitamin B12 and the stability of biotin and vitamin B12 in sample solutions within 12 hours were investigated when the samples were extracted with different extraction solvents.

[0122] The results are shown in Tables 2 and 3: The detection rate and stability of the mixed extractant of 0.1%-0.25% (m / V) ammonium thiocyanate and 0.1%-0.5% phosphoric acid solution (V / V) meet the requirements.

[0123] Table 2 Tablets

[0124]

[0125]

[0126] The data of soft candies and soft capsules are shown in Table 3.

[0127] Table 3 Soft candies and soft capsules

[0128]

[0129] Example 2

[0130] The effects of different chromatographic column combinations were investigated and the results are shown in Table 4.

[0131] When the one-dimensional (1-D) column is AQ-C18 4.6×100 mm, 2.7 μm, and the two-dimensional column is PFP column 4.6x250 mm, 3 μm, the best separation effect of biotin and vitamin B12 is obtained.

[0132] Table 4 Chromatographic column investigation

[0133]

[0134]

[0135]

[0136]

[0137] Example 3

[0138] The effects of different combinations of mobile phase components were investigated, and the results are shown in Table 5;

[0139] The mobile phase of the first dimension (1-D) was 0.05% trifluoroacetic acid solution (pH 2-3), acetonitrile,

[0140] When the two-dimensional (2-D) mobile phase is 0.1% phosphoric acid solution (pH 3.5-4) and acetonitrile, the separation effect of biotin and impurity peaks in the sample is excellent, and vitamin B12 has no interference.

[0141] Table 5 Mobile phase investigation

[0142]

[0143]

[0144]

[0145] Example 4

[0146] The detection rates of the two-dimensional liquid chromatography and liquid chromatography-mass spectrometry methods of the present invention were compared, and the results are shown in Table 6.

[0147] The LC-MS method is prone to matrix effects. The matrix effect of low-content products is obvious, which will lead to different degrees of instability in the results. The detection rates of biotin and vitamin B12 are too high or too low, especially for low-content products. The detection rate of two-dimensional liquid chromatography is in line with expectations (90%-105%).

[0148] Table 6

[0149]

[0150]

[0151] Among them, the specific process of liquid mass method is:

[0152] 1. VB12

[0153] 1. Sample pretreatment:

[0154] Take an appropriate amount of sample (containing about 10-100 mg of vitamin B12), add 100 μL of vitamin B12-[ 13 C7] internal standard aqueous solution (1 μg / mL), add water for ultrasonic extraction, adjust the pH to 5-7 with NaOH solution, make up to volume, centrifuge, and take the supernatant for later use;

[0155] Add all the filtrate to the activated immunoaffinity column, open the device valve to allow the sample solution to flow out through the immunoaffinity column at a certain flow rate (flow rate 2-3mL / min), and after the sample solution has completely passed through the column, rinse the immunoaffinity column with 10mL of water and drain;

[0156] Place a 20mL brown sample bottle under the immunoaffinity column, elute with 3mL methanol three times, collect all the eluent, blow to dryness with nitrogen below 60°C, accurately add 1.0mL of ammonium acetate solution, cover, vortex, and ultrasonically dissolve the residue, filter through a 0.22μm microporous membrane, and wait for testing. (The entire preparation process must be protected from light).

[0157] 2. Standard working fluid

[0158] Take 5 brown injection vials, transfer 10μL, 25μL, 50μL, 125μL, 250μL, and 500μL of vitamin B12 standard solution (0.2μ / mL) respectively, accurately add 100μL of vitamin B12-[13C7] internal standard aqueous solution (1μg / mL) to each, and then add 890μL, 875μL, 850μL, 775μL, 650μL, and 400μL of pure water respectively, mix thoroughly to obtain a series of standard working solutions containing the internal standard. The concentrations of vitamin B12 in this series of working solutions are 2ng / mL, 5ng / mL, 10ng / mL, 25ng / mL, 50ng / mL, and 100ng / mL, respectively.

[0159] 3. Chromatographic conditions

[0160] Chromatographic column: Waters ACQUITY HSS T3 column, 100×2.1 mm, 1.8 μm or equivalent performance column;

[0161] Column temperature: 40°C;

[0162] Flow rate: 0.3 mL / min;

[0163] Injection volume: 10 μL;

[0164] Mobile phase: Mobile phase A: 2.5 mmol / L ammonium acetate solution; Mobile phase B: 90% acetonitrile. Mobile phase gradient elution is shown in Table 7.

[0165] Table 7 Mobile phase elution gradient

[0166] Time min Mobile phase A% Mobile phase B% 0.00 93 7 0.53 93 7 2.12 85 15 2.65 10 90 3.18 10 90 3.71 93 7 8.00 93 7

[0167] Mass spectrometry reference conditions:

[0168] Ionization method: ESI + ;

[0169] Air curtain airflow: 35psi;

[0170] Collision air flow: 7psi;

[0171] Spray voltage: 5500V;

[0172] Atomization temperature: 600℃;

[0173] Atomizing gas: 55psi;

[0174] Auxiliary gas: 60psi;

[0175] The declustering voltage and collision energy are shown in Table 8.

[0176] Table 8 Vitamin B12 mass spectrometry reference conditions

[0177]

[0178] *Quantitative ion pairs.

[0179] 2. Biotin

[0180] 1. Sample pretreatment

[0181] Accurately weigh an appropriate amount of the mixed sample (equivalent to about 10 μg of biotin) in a 50 mL centrifuge tube, add 3.0 mL of biotin-d2 internal standard aqueous solution (10 μg / mL), add 25 mL of water, heat in a water bath at 80°C, transfer to a 50 mL volumetric flask with about 10 mL of water, and ultrasonicate for 30 minutes. After the ultrasonication is completed, cool and dilute to the mark with water. Mix well and filter through a 0.22 μm filter membrane.

[0182] 2. Standard working fluid

[0183] Biotin series working solution: Accurately pipette 0.2mL, 0.6mL, 1.0mL, 3.0mL, and 4.0mL of biotin standard aqueous solution (2μg / mL) into 10mL volumetric flasks, add 0.6mL of biotin-d2 internal standard aqueous solution (10μg / mL) to each, dilute to the mark with water, and mix well. The standard curve solutions with biotin concentrations of 40ng / mL, 120ng / mL, 200ng / mL, 600ng / mL, and 800ng / mL are prepared.

[0184] 3. Chromatographic conditions:

[0185] Chromatographic column: ACQUITY UPLC HSS T3, 100×2.1mm, 1.8μm or HSS C18 column, 100×2.1mm, 1.8μm; chromatographic column with equivalent performance.

[0186] Mobile phase: A: 0.1% formic acid methanol solution; B: 0.1% formic acid solution.

[0187] See Table 9 for the elution gradient.

[0188] Table 9

[0189] Time (min) A(％) B(％) 0.0 10 90 2.0 10 90 4.0 55 45 7.0 90 10 11.0 100 0 12.5 100 0 13.5 10 90 15.0 10 90

[0190] Column temperature: 30°C;

[0191] Flow rate: 0.3 mL / min;

[0192] Injection volume: 1 μL;

[0193] Mass spectrometry reference conditions:

[0194] Ionization method: ESI + ;

[0195] Air curtain air pressure: 40psi;

[0196] Collision gas pressure: 7psi;

[0197] Spray voltage: 5500V;

[0198] Atomization temperature: 550℃;

[0199] Atomizing air pressure 1: 45psi;

[0200] Atomizing air pressure 2: 45psi.

[0201] See Table 10 for ion pair information.

[0202] Table 10

[0203]

[0204] *Quantitative ion pairs.

[0205] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for rapid detection of vitamin B12 and biotin in nutrient products, characterized in that: include, The ammonium thiocyanate solution and the phosphoric acid solution are mixed to prepare an extractant; adding an extractant to the nutrient product for treatment to obtain a filtrate; The filtrate was subjected to two-dimensional liquid chromatography.

2. The method according to claim 1, characterized in that: The extractant, wherein the concentration of the ammonium thiocyanate solution is 0.1% to 0.25% by mass to volume ratio, and the concentration of the phosphoric acid solution is 0.1% to 0.5% by volume ratio.

3. The method according to claim 1 or 2, characterized in that: The nutrient products include gummies, tablets and soft capsules.

4. The method according to claim 3, characterized in that: The filtrate includes the filtrate of soft candy, the filtrate of tablets and the filtrate of soft capsules.

5. The method according to claim 4, characterized in that: The method for preparing the filtrate of the soft candy comprises: Take the soft candy, cut it into pieces, mix it evenly, put it in a centrifuge tube, add the extractant, bathe it in water until it is completely dissolved, cool it, centrifuge it in a centrifuge, take the supernatant and filter it to obtain the soft candy filtrate.

6. The method according to claim 4, characterized in that: The method for preparing the filtrate of the tablet comprises: The tablets are taken, powdered and mixed evenly, and then an extractant is quantitatively added, ultrasonicated in a water bath, cooled, centrifuged, and the supernatant is filtered to obtain the filtrate of the tablets.

7. The method according to claim 4, characterized in that: The method for preparing the filtrate of the soft capsule comprises: Take the soft capsule, cut the rubber skin, take out the content and mix evenly, add petroleum ether, vortex for 1-2 minutes, ultrasonic for 3-8 minutes, quantitatively add the extractant, mix evenly, take out by ultrasonic, cool, centrifuge, take out the supernatant and filter to obtain the filtrate of the soft capsule.

8. The method according to any one of claims 1, 2, 4 to 7, characterized in that: The two-dimensional liquid chromatography treatment uses an octadecyl silica gel chromatography column in the first dimension and a perfluorophenyl silica gel column in the second dimension.

9. The method according to claim 8, characterized in that: The octadecyl silica gel chromatographic column is an octadecyl silica gel chromatographic column, and its specifications are: 4.6×100mm, 2.7μm; The perfluorophenyl silica gel column is a perfluorophenyl silica gel column, and its specifications are: 4.6x250mm, 3μm.

10. The method according to claim 8, characterized in that: The mobile phase of the first dimension includes mobile phase A and mobile phase B, wherein mobile phase A is 0.04-0.06% trifluoroacetic acid solution with a pH of 2-3, and mobile phase B is acetonitrile; The second dimension mobile phase includes mobile phase C and mobile phase D, wherein mobile phase C is 0.08-0.12% phosphoric acid with a pH of 3.5-4, and mobile phase D is acetonitrile.

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