Method for detecting 2-chlorine-propylene glycol and 3-chlorine-propylene glycol

Abstract

The invention discloses a method for detecting 2-chlorine-propylene glycol and 3-chlorine-propylene glycol, and belongs to the technical field of food pollutant detection. The method for detecting 2-chlorine-propylene glycol and 3-chlorine-propylene glycol comprises the following steps: purifying a sample solution to be detected through a purification column to obtain a purified solution; removing water from the purified liquid, and adding a derivatization reagent for derivatization treatment to obtain a derivative liquid; the derivatization reagent is prepared from quinolaldehyde acyl chloride and 4-dimethylaminopyridine; and carrying out liquid chromatography-mass spectrometry detection on the derivative solution. The method provided by the invention can effectively distinguish two MCPD isomers, shorten derivatization time (20-30 min), increase hydrophobicity, promote chromatographic separation and improve electrospray ionization mass spectrometry (ESI-MS) sensitivity, and breaks through the limitation that a traditional method is limited to GC-MS.

Description

Technical Field

[0001] The invention relates to the technical field of food contaminant detection, in particular to a method for detecting 2-chloro-propylene glycol and 3-chloro-propylene glycol. Background Art

[0002] Chloropropanol compounds are formed by the replacement of the hydroxyl group of glycerol by one or two chlorine atoms. According to the number and position of chlorine substitution, they can be divided into monochloropropanediol (MCPD) and dichloropropanediol (DCP). MCPD includes 2-chloro-1,3-propanediol (2-MCPD) and 3-chloro-1,2-propanediol (3-MCPD); DCP includes 1,3-dichloro-2-propanol (1,3-DCP) and 2,3-dichloro-1-propanol (2,3-DCP). 2 / 3-chloro-propylene glycol esters and their free forms (2 / 3-chloro-propylene glycol, 2 / 3-MCPD) are chemical contaminants produced during processing and are present in various foods, including acid-hydrolyzed vegetable proteins, oil-containing products, etc. During the high-temperature acid hydrolysis of vegetable proteins, free chloropropanediols are produced when acid hydrolyzed with hydrogen chloride in the presence of glycerol or glycerophospholipid residues. 3-MCPD and its esters are produced during high-temperature processing of chlorinated fats or oils, and studies have shown that they are found in many food samples, including soy sauce, fish oil, and krill oil. The presence of 3-MCPD and its esters is of particular concern because they have potential health hazards to humans, including sperm inactivation, kidney damage, and possible carcinogenicity. Given these risks, accurate and effective methods are urgently needed to detect and quantify these contaminants in food.

[0003] At present, GC-MS / MS is generally used to determine MCPD in soy sauce. Derivatization with derivatization reagents such as phenylboronic acid or N-heptafluorobutylimidazole can achieve the detection of MCPD. This method has low derivatization efficiency, complex pretreatment, and high cross-contamination during instrument measurement. Recently, newly authorized patents and literature have reported the technology of determining MCPD by derivatization with p-dimethylaminophenol. This method makes 2-MCPD and 3-MCPD form the same derivatives, which cannot effectively distinguish the two, resulting in deviations in quantitative results. In addition, the derivatization process takes 4 to 6 hours, which affects the detection efficiency. MCPD often exists in the form of MCPD esters in fish oil and krill oil. Therefore, it is necessary to analyze and detect by hydrolyzing the MCPD ester binding state and calculating the free 2 / 3-MCPD level from the hydrolysis product. For example, the commonly used gas chromatography-mass spectrometry (GC-MS) method releases free MCPD after hydrolysis of MCPD esters, and then uses derivatization reagents such as phenylboronic acid or N-heptafluorobutylimidazole to achieve MCPD detection. However, the sample preparation procedure of this method is complicated, the derivatization conditions are relatively strict, and the analysis time required is long. For the detection of fish oil and krill oil matrices, the technology of determining MCPD by dimethylaminophenol derivatization cannot effectively distinguish 2-MCPD from 3-MCPD. The commonly used GC-MS / MS analysis method lacks alternative instruments, which limits the flexibility of many laboratories in the analysis of MCPD and its esters. The development of instrumental analysis methods based on liquid chromatography tandem mass spectrometry can effectively provide alternatives and flexible instrument selection.

[0004] Therefore, it is urgent to find a new method that can effectively distinguish 2-MCPD and 3-MCPD and realize the accurate detection of MCPD. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting 2-chloro-propylene glycol and 3-chloro-propylene glycol to solve the problems existing in the above-mentioned prior art. The method of the present invention can effectively distinguish the two MCPD isomers, shorten the derivatization time (20 to 30 minutes), and improve the sensitivity of electrospray ionization mass spectrometry (ESI-MS), breaking through the limitation of the traditional method being limited to GC-MS.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] A method for detecting 2-chloro-propylene glycol and 3-chloro-propylene glycol in a sample comprises the following steps:

[0008] Purify the sample solution to be tested through a purification column to obtain a purified solution;

[0009] After removing water from the purified liquid, a derivatization reagent is added to perform derivatization treatment to obtain a derivatized liquid; the derivatization reagent includes quinaloyl chloride (derivative agent) and 4-dimethylaminopyridine (catalyst);

[0010] The derivatized liquid is subjected to liquid chromatography-mass spectrometry detection.

[0011] Due to the different positions of hydroxyl groups in the structures of 2-chloro-propylene glycol and 3-chloro-propylene glycol, quinaloyl chloride and 4-dimethylaminopyridine react with them to produce two different diester compound isomers. The two newly derived compounds have good chromatographic retention properties and electrospray ionization activity, and are very suitable for separation and analysis by liquid chromatography.

[0012] Furthermore, when the sample solution to be tested is a liquid sample without oil or fat, the preparation method of the purified liquid specifically comprises: adding the sample solution to be tested into a purification column, standing it, and then eluting it with ethyl acetate to obtain a purified liquid;

[0013] The purification column is an activated carbon column.

[0014] Furthermore, the oil-free liquid sample includes soy sauce.

[0015] Furthermore, when the sample solution to be tested is a liquid sample containing oil, the method for preparing the purified liquid specifically includes:

[0016] A NaCl aqueous solution (10%, w / v) is added to the sample solution to be tested, and the mixture is shaken and centrifuged. Silica gel is added to the obtained emulsion, and the mixture is shaken and centrifuged again. The obtained solution is added to a purification column, allowed to stand, and then eluted with ethyl acetate to obtain a purified solution; the purification column is an activated carbon column.

[0017] NaCl aqueous solution (10%, w / v) can extract free 2-MCPD and 3-MCPD, and krill oil or fish oil is in a water-oil separation state after being extracted with NaCl aqueous solution, with obvious stratification.

[0018] Furthermore, the oil-containing liquid sample includes krill oil or fish oil.

[0019] Furthermore, the water removal specifically includes:

[0020] The purified liquid was concentrated to obtain a concentrated solution, 3A molecular sieves were added to the concentrated solution, and water was removed by shaking.

[0021] The chlorine atom in quinalcohol chloride has an electron-withdrawing effect, which enhances the electrophilicity of carbon and makes the acyl chloride more susceptible to attack by nucleophilic reagents. - It is also a good leaving group, so the activity of acyl chloride in nucleophilic acyl substitution reaction is strong, so acyl chloride will undergo hydrolysis reaction when it meets water: RCO-Cl+H-OH→RCOOH+HCl. Therefore, in order to achieve more accurate experimental results during sample pretreatment, 3A molecular sieve is used to fully remove water before derivatization with quinaloyl chloride.

[0022] Furthermore, the ratio of the amount of quinalcohol chloride to the content of MCPD is ≥4 mg:1 μg;

[0023] The ratio of the amount of 4-dimethylaminopyridine to the content of MCPD is ≥4mg:1μg.

[0024] The derivatization treatment time is 10 min to 6 h.

[0025] Furthermore, the derivatization treatment time is 20 to 30 minutes.

[0026] By adding 4-dimethylaminopyridine as a catalyst, the reaction is promoted to generate a stable derivative.

[0027] Furthermore, the liquid chromatography-mass spectrometry detection is as follows:

[0028] Chromatographic analysis conditions:

[0029] Column: Eclipse Plus C18 RRHD, 3.0 × 150 mm, 1.8 μm;

[0030] Flow rate: 0.5 mL / min;

[0031] Column temperature: 35 °C;

[0032] Injection volume: 4 μL;

[0033] Mobile phase: Phase A is water containing 0.2 vol.% formic acid and 5 mmol / L ammonium acetate, and phase B is acetonitrile;

[0034] Elution gradient: 0-1.9 min, 41% B; 1.9-2 min, 41-63% B; 2-5.5 min, 63-64% B; 5.5-6.8 min, 64-100% B; 6.8-8 min, 100-41% B;

[0035] Mass spectrometry analysis conditions:

[0036] Ion source: HESI;

[0037] Spray voltage: 3200 V in positive ion mode, 2800 V in negative ion mode;

[0038] Gas parameters: sheath gas 50arb, auxiliary gas 15arb, purge gas 1arb;

[0039] Temperature: capillary temperature 350°C, auxiliary gas heating temperature 400°C;

[0040] Scan Mode: PRM.

[0041] Furthermore, the method also includes adding an isotope internal standard to the sample solution to be tested for quantitative detection before purification.

[0042] Furthermore, the ratio of the amount of activated carbon in the activated carbon column to the content of MCPD is ≥1g:1μg.

[0043] Furthermore, the usage ratio of ethyl acetate (eluent) and activated carbon is ≥15mL:1.5g.

[0044] Furthermore, the elution also includes the step of vacuum drying the activated carbon column.

[0045] Furthermore, the method for concentrating the purified liquid includes rotary evaporation concentration; the temperature of the rotary evaporation concentration is ≤30°C.

[0046] Furthermore, after adding the sample solution to be tested into the purification column and before elution, the method also includes the step of rinsing the activated carbon column with pure water.

[0047] Eluting the activated carbon column with pure water can remove impurities without affecting the recovery rate of the target substance.

[0048] The present invention discloses the following technical effects:

[0049] (1) The method of the present invention can effectively distinguish two MCPD isomers, shorten the derivatization time (20 to 30 min), and improve the sensitivity and hydrophobicity of electrospray ionization mass spectrometry (ESI-MS) (the quinoline group attached to the ester bond after derivatization has good hydrophobicity and electrospray ionization activity), thus breaking through the limitation of traditional methods to GC-MS.

[0050] (2) The present invention uses quinaloyl chloride as a derivatization reagent to derivatize 2-chloro-propylene glycol (2-MCPD) and 3-chloro-propylene glycol (3-MCPD) in soy sauce, krill oil or fish oil, and obtains a liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection method. The method solves the problem of difficulty in fully removing water before the derivatization reaction (the ordinary sodium chloride, magnesium sulfate, and sodium sulfate dehydration cannot completely remove water. If the water is removed by evaporation, the target analyte will be taken away. The water content requirement is very high, and the reaction activity with the derivatization agent is high, which affects the reaction between the derivatization agent and the target substance). It breaks through the technical limitations of the current gas chromatography-mass spectrometry (GC-MS) detection of 2 / 3-MCPD, and breaks through the limitation that the previous liquid chromatography-mass spectrometry cannot distinguish 2 / 3-MCPD isomers under a single injection. The method improves the detection accuracy and simplifies the operation, and provides a more sensitive and effective method for the quantitative analysis of 2-MCPD and 3-MCPD in foods such as soy sauce.

[0051] (3) The present invention utilizes dispersed silica gel to efficiently demulsify krill oil or fish oil extracts, uses activated carbon to enrich the analytes, and finally uses quinaloyl chloride as a derivatization reagent to achieve the detection of 2-MCPD and 3-MCPD. The method of the present invention has the advantages of rapid derivatization, good linearity, high sensitivity and high recovery rate, and can be used as a feasible alternative to traditional GC-MS technology, opening up a new way for the direct and indirect determination of MCPD esters in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 paying creative work.

[0053] Figure 1 This is the flow chart of soy sauce sample pretreatment and derivatization;

[0054] Figure 2 It is the reaction diagram of 3-MCPD, 3-MCPD-d5 and quinaloyl chloride;

[0055] Figure 3 It is the reaction diagram of 2-MCPD, 2-MCPD-d5 and quinaloyl chloride;

[0056] Figure 4 The effect of different amounts of activated carbon on the loss rate of 3-MCPD-d5;

[0057] Figure 5 The effect of the amount of eluent and the state of the activated carbon column (vacuum extraction or not) on the recovery of MCPD (3-MCPD-d5 and 2-MCPD-d5);

[0058] Figure 6 The effect of different rotary evaporation temperatures on the recovery of MCPD (3-MCPD-d5 and 2-MCPD-d5);

[0059] Figure 7 The effect of rotary evaporation state on the recovery rate of the target compound;

[0060] Figure 8 The effect of water on the derivatization reaction and the effect of water removal by CaO and MgSO4;

[0061] Fig. 9 The effect of 3A molecular sieve on the derivatization reaction;

[0062] Fig.10 The effects of different derivatization reagent components and dosages on the response values ​​of the target substances;

[0063] Fig.11 The effect of different derivatization times on the response value of the target;

[0064] Fig.12 For the comparison of different demulsification methods;

[0065] Fig.13 Comparison of demulsified solutions obtained by different demulsification methods after centrifugation;

[0066] Fig.14 This is a flow chart of krill oil sample preparation and derivatization;

[0067] Fig.15 The effect of C18 and silica gel on the recovery rate of 3-MCPD-d5 under different conditions;

[0068] Fig.16 The effect of different silica gel usage on the recovery rate of 3-MCPD-d5;

[0069] Fig.17 The effects of different derivatization reagent components and dosages on the response values ​​of the target substances;

[0070] Fig.18 The effect of different derivatization times on the response value of the target. DETAILED DESCRIPTION

[0071] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0072] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0073] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0074] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0075] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0076] The preparation method of the experimental reagent adopted in the present invention is as follows:

[0077] (1) Preparation of standard stock solution:

[0078] 3-MCPD (3-chloro-1,2-propanediol, 1 mg / mL): Accurately weigh 10 mg of 3-MCPD standard (accurate to 0.01 mg, purity ≥ 99%), dissolve in ethyl acetate, dilute to 10 mL brown volumetric flask, and store at -4°C away from light.

[0079] 2-MCPD (2-chloro-1,3-propanediol, 1 mg / mL): Accurately weigh 10 mg of 2-MCPD standard (accurate to 0.01 mg, purity ≥99%), dissolve in ethyl acetate, dilute to 10 mL brown volumetric flask, and store at -4°C away from light.

[0080] 3-MCPD-d5 (3-chloro-1,2-propanediol-d5, 1 mg / mL): Accurately weigh 10 mg of 3-MCPD-d5 standard (accurate to 0.01 mg, purity ≥ 99%), dissolve and make up to volume, and store at -4°C away from light.

[0081] 2-MCPD-d5 (2-chloro-1,3-propanediol-d5, 1 mg / mL): Accurately weigh 10 mg of 2-MCPD-d5 standard (accurate to 0.01 mg, purity ≥ 99%), dissolve and make up to volume, and store at -4°C away from light.

[0082] Example 1

[0083] A method for detecting 2-chloro-propylene glycol and 3-chloro-propylene glycol:

[0084] (1) Preparation of standard working solution:

[0085] 3-MCPD (1 μg / mL): Pipette 1 mg / mL of 3-MCPD standard stock solution, dilute with water to a concentration of 1 μg / mL, and store at -4°C away from light.

[0086] 2-MCPD (1 μg / mL): Pipette 1 mg / mL of 2-MCPD standard stock solution, dilute with water to a concentration of 1 μg / mL, and store at -4°C away from light.

[0087] 3-MCPD-d5 (1 μg / mL): Pipette 1 mg / mL of 3-MCPD-d5 standard stock solution, dilute with water to a concentration of 1 μg / mL, and store at -4°C away from light.

[0088] 2-MCPD-d5 (1 μg / mL): Pipette 1 mg / mL of 2-MCPD-d5 standard stock solution, dilute with water to a concentration of 1 μg / mL, and store at -4°C away from light.

[0089] (2) Preparation of a series of standard working solutions

[0090] To 8 portions of ethyl acetate (1 mL each) were added 20 ng of 2-MCPD-d5 and 20 ng of 3-MCPD-d5, and 1 ng, 2 ng, 5 ng, 10 ng, 20 ng, 50 ng, 100 ng and 200 ng of 3-MCPD and 2-MCPD were added respectively to prepare a series of standard working solutions.

[0091] (3) Liquid chromatography-mass spectrometry conditions:

[0092] A. Chromatographic analysis conditions:

[0093] Chromatographic column: Eclipse Plus C18 RRHD (3.0 × 150 mm, 1.8 μm);

[0094] Flow rate: 0.5 mL / min;

[0095] Column temperature: 35 °C;

[0096] Injection volume: 4 μL;

[0097] Mobile phase: Phase A is pure water containing 0.2 vol.% formic acid and 5 mmol / L ammonium acetate, and phase B is acetonitrile;

[0098] Elution gradient: 0-1.9 min, 41% B; 1.9-2 min, 41-63% B; 2-5.5 min, 63-64% B; 5.5-6.8 min, 64-100% B; 6.8-8 min, 100-41% B.

[0099] B. Mass spectrometry analysis conditions:

[0100] Ion source: HESI;

[0101] Spray voltage: 3200 V in positive ion mode, 2800 V in negative ion mode;

[0102] Gas parameters: sheath gas 50arb, auxiliary gas 15arb, purge gas 1arb;

[0103] Temperature: capillary temperature 350°C, auxiliary gas heating temperature 400°C;

[0104] Scan Mode: PRM.

[0105] C. Monitoring ion information:

[0106] The qualitative ions, quantitative ions and residence times are shown in Table 1.

[0107] Table 13 / 2-MCPD and 3 / 2-MCPD-d5 monitoring information

[0108]

[0109] In Table 1, a Indicates that the ion is used for quantification of the analyte.

[0110] (4) Analysis methods

[0111] A. Qualitative analysis

[0112] The sample solution was measured under the same instrumental conditions as the standard working solution.

[0113] The sample solution is measured under the same instrument conditions as the standard stock solution. If the retention time of the chromatographic peak of the tested sample corresponds to the retention time of the chromatographic peak of 3-MCPD and 2-MCPD, and the allowable deviation is less than ±0.05min, after deducting the background, the relative abundance of the qualifier ion in the sample is compared with the relative abundance of the corresponding qualifier ion in the mixed standard working solution with a similar concentration. If the deviation is within the range specified in Table 2, it can be determined that the corresponding 3-MCPD and 2-MCPD exist in the sample.

[0114] Table 2 Maximum allowable deviation of relative ion abundance in qualitative analysis

[0115] Relative ion abundance ＞50％ 20～50％ 10～20％ ≤10％ Permissible deviation ±20％ ±25％ ±30％ ±50％

[0116] B. Quantitative Analysis

[0117] Record the quantitative ion peak areas of the target compound and internal standard in the sample solution and standard working solution.

[0118] The isotope internal standard method and instrument software were used for quantitative analysis.

[0119] (5) Sample preparation

[0120] A. Preparation of activated carbon solid phase extraction cartridges

[0121] Place a lower sieve plate (glass fiber filter paper) in a 12 mL affinity chromatography empty column, and press the bottom of the tube with a small polytetrafluoroethylene rod. Add 1.5 g (accurate to 0.01 g) of activated carbon and place it in the empty column with the lower sieve plate. After compacting, place an upper sieve plate (glass fiber filter paper) and compact it with a small polytetrafluoroethylene rod to make an activated carbon solid phase extraction column.

[0122] B. Sample Preparation

[0123] 2.00 mL of soy sauce sample was transferred into a 10 mL centrifuge tube, and 20 μL of 1 μg / mL 3-MCPD-d5 and 20 μL of 1 μg / mL 2-MCPD-d5 standard working solutions were added. The mixture was vortexed for 30 s to obtain a mixed solution.

[0124] On a vacuum solid phase extraction device, slowly pass the mixed liquid through an activated carbon solid phase extraction column (activated carbon column), discard the effluent, let it stand for 10 minutes, rinse the activated carbon column with 5 mL of pure water, and then elute the activated carbon column with 15 mL of ethyl acetate (EA), eluting once every 5 mL for a total of 3 times. Pump the activated carbon column to near dryness with a vacuum pump, and collect and combine the eluent.

[0125] The lower phase of the eluate was discarded, and the residual liquid was transferred to a pear-shaped bottle and rotary evaporated to about 0.5 mL (the solution was rotary evaporated to near dryness) at 30°C and 110 bar to obtain a concentrated solution.

[0126] The concentrate was transferred to a 5 mL centrifuge tube, and 200 mg of 3A molecular sieves were added. After sufficient shaking to remove water until no bubbles were generated, the derivatization reagent was added (8 mg of quinaloyl chloride and 8 mg of 4-dimethylaminopyridine were added in sequence, and each reagent was fully shaken), the mixture was shaken and mixed, and the solution was derivatized at room temperature for 30 min. Then, the solution was dried with gentle nitrogen gas, 1.00 mL of acetonitrile (ACN) was added for redissolution, and the sample solution was obtained by filtering through a 0.22 μm organic phase filter membrane, and the sample solution was analyzed by LC-MS / MS.

[0127] See the flowchart of soy sauce sample pretreatment and derivatization for details. Figure 1 The reaction diagram of 3-MCPD, 3-MCPD-d5 and quinaloyl chloride is shown in Figure 2 The reaction diagram of 2-MCPD, 2-MCPD-d5 and quinaloyl chloride is shown in Figure 3 .

[0128] Linear range and correlation coefficient: In the range of 1-200 ng / mL, the linear equation of 3-MCPD is Y=0.00169+0.0482X, R 2 =0.9970; 2-MCPD linear equation is Y = -0.0354 + 0.0547X, R 2 =0.9964.

[0129] Since the method of the present invention is analyzed in mass spectrometry, the magnitude of the matrix effect is evaluated mainly by the following method: the matrix effect can be evaluated by adding a standard to the blank sample solution after extraction. The matrix effect is determined by comparing the response ratio of the matrix standard solution and the pure standard solution.

[0130] Table 3 Matrix effects of 3-MCPD and 2-MCPD in soy sauce

[0131]

[0132] Calculation of matrix effect: The calculation formula of matrix effect (MF%) is MF% = B / A, where A represents the signal intensity of the pure solvent standard solution in the mass spectrometer, and B represents the signal intensity of the mass spectrometer of the solution with the same concentration as A prepared by using blank matrix extract. When MF = 1, it means there is no matrix effect, when MF>1, it means there is matrix enhancement, and when MF<1, it means there is matrix suppression.

[0133] The results showed that there was a matrix enhancement effect on both 2-MCPD and 3-MCPD.

[0134] Recovery and precision: When different concentrations of standard samples were added to the soy sauce matrix, the recovery was within a reasonable range and the method had good precision.

[0135] Limit of Detection (LOD) and Limit of Quantitation (LOQ):

[0136] Limit of detection (LOD): refers to the minimum concentration or amount of the substance to be tested that can be detected from a sample by a specific analytical method within a given confidence level. Limit of quantification (LOQ): refers to the minimum concentration or amount of the component to be tested in a sample that can be quantitatively determined by the analytical method, and the measurement result should have a certain accuracy and precision. Under the above-mentioned optimal derivatization conditions for 3-MCPD and 2-MCPD in soy sauce, the detection limit and quantification limit of the analytical method are determined when the peak height is approximately 3 times and 10 times the baseline noise, that is, S / N ≥ 3 and S / N ≥ 10, respectively. The detection limit that this method can achieve is 0.5ppb and the quantification limit is 1ppb.

[0137] Example 2

[0138] The same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, and the amounts of activated carbon used in the preparation of the activated carbon solid phase extraction cartridge were 0.5 g, 1.0 g, 1.5 g, 2.0 g and 2.5 g, respectively.

[0139] The content of MCPD (3-MCPD-d5) in the sample solutions obtained in Example 1 and Example 2 was determined and the MCPD loss rate was calculated. The results are shown in Figure 4 .

[0140] from Figure 4 It can be seen that when the amount of activated carbon is ≥1.5g, the loss rate of MCPD is less than 0.5%. Therefore, 1.5g of activated carbon is used to fill the solid phase extraction column, and the ratio of activated carbon to MCPD is ≥1g: 1μg or more to meet the needs of sufficient adsorption.

[0141] Example 3

[0142] The same as Example 1, except that the amount of ethyl acetate used during elution was 5 mL, 10 mL, 20 mL and 25 mL respectively.

[0143] Example 4

[0144] The same as Example 1, except that the activated carbon column was not evacuated to near dryness by a vacuum pump during elution; the amounts of ethyl acetate used were 5 mL, 10 mL, 15 mL, 20 mL and 25 mL respectively.

[0145] The MCPD (3-MCPD-d5 and 2-MCPD-d5) content in the sample solution prepared by taking 5 mL of eluent in Example 1 and Examples 3 to 4 in sequence was determined and the recovery rate of MCPD was calculated. The results are shown in Figure 5 .

[0146] Figure 5 In the dry-5, the vacuum pump was used to dry the mixture, and the amount of ethyl acetate was 5 mL; the dry-10 was used to dry the mixture, and the amount of ethyl acetate was 10 mL; the dry-15 was used to dry the mixture, and the amount of ethyl acetate was 15 mL (Example 1); the dry-20 was used to dry the mixture, and the amount of ethyl acetate was 20 mL; the dry-25 was used to dry the mixture, and the amount of ethyl acetate was 25 mL; the wet-5 was not dried by the vacuum pump, and the amount of ethyl acetate was 5 mL; the wet-10 was not dried by the vacuum pump, and the amount of ethyl acetate was 10 mL; the wet-15 was not dried by the vacuum pump, and the amount of ethyl acetate was 15 mL; the wet-20 was not dried by the vacuum pump, and the amount of ethyl acetate was 20 mL; the wet-25 was not dried by the vacuum pump, and the amount of ethyl acetate was 25 mL.

[0147] from Figure 5 It can be seen that 15 mL of ethyl acetate can fully elute the target compound, and the activated carbon column is vacuum-dryed after elution to reduce the loss of MCPD.

[0148] Example 5

[0149] The same as Example 1, except that the rotary evaporation temperatures are 20°C, 25°C, 35°C and 40°C respectively.

[0150] The MCPD (3-MCPD-d5 and 2-MCPD-d5) content in the sample solutions obtained in Example 1 and Example 5 was determined and the recovery rate of MCPD was calculated. The results are shown in Figure 6 .

[0151] from Figure 6 It can be seen that rotary evaporation at a temperature not higher than 30°C can avoid sample loss caused by excessive temperature.

[0152] Example 6

[0153] Same as Example 1, except that the solution was evaporated by rotary evaporation at 30° C. and 110 bar until it was solvent-free (dry).

[0154] The MCPD content in Example 1 and Example 6 was determined and the recovery rate of MCPD was calculated. The results are shown in Figure 7 .

[0155] from Figure 7 It can be seen that rotary evaporation of the solution to a nearly dry state (Wet) is more conducive to the recovery of MCPD.

[0156] Example 7

[0157] (1) Same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, 20 μL of 1 μg / mL 3-MCPD and 3-MCPD-d5 standard working solutions were added, 200 mg of 3A molecular sieve was not added, and the derivatization time was 20 min (QC);

[0158] (2) Same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, 20 μL of 1 μg / mL 3-MCPD and 3-MCPD-d5 standard working solutions were added, 200 mg 3A molecular sieve was replaced with 10 μL H2O, and the derivatization time was 20 min (10 μL H2O);

[0159] (3) Same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, 20 μL of 1 μg / mL 3-MCPD and 3-MCPD-d5 standard working solutions were added, 200 mg 3A molecular sieve was replaced with 200 mg CaO, and the derivatization time was 20 min (CaO);

[0160] (4) Same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, 20 μL of 1 μg / mL 3-MCPD and 3-MCPD-d5 standard working solutions were added, 200 mg 3A molecular sieve was replaced with 200 mg MgSO4, and the derivatization time was 20 min (MgSO4);

[0161] (5) Same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, 20 μL of 1 μg / mL 3-MCPD and 3-MCPD-d5 standard working solutions were added, 200 mg 3A molecular sieve was replaced with 10 μL H2O and 200 mg CaO, and the derivatization time was 20 min (CaO-H2O);

[0162] (6) Same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, 20 μL of 1 μg / mL 3-MCPD and 3-MCPD-d5 standard working solutions were added, 200 mg 3A molecular sieve was replaced with 10 μL H2O and 200 mg MgSO4, and the derivatization time was 20 min (MgSO4-H2O);

[0163] (7) The same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, 20 μL of 1 μg / mL 3-MCPD and 3-MCPD-d5 standard working solutions were added, and the derivatization time was 20 min (3A molecular sieve).

[0164] (8) Same as Example 1, except that 20 μL of 1 μg / mL 2-MCPD-d5 standard working solution was not added to the soy sauce sample, 20 μL of 1 μg / mL 3-MCPD and 3-MCPD-d5 standard working solutions were added, 200 mg 3A molecular sieve was replaced with 10 μL H2O and 200 mg 3A molecular sieve, and the derivatization time was 20 min (3A molecular sieve-H2O).

[0165] The signal ratio of the diester (m / z 426 / 421) and the monoester (m / z 271 / 266) in the sample solution obtained in this example was measured. The results are shown in Figure 8 and Fig. 9 .

[0166] contrast Figure 8 and Fig. 9 It can be seen from the data that the use of 3A molecular sieve for water removal is better than calcium oxide and magnesium sulfate, and has a promoting effect on the derivatization reaction.

[0167] Example 8

[0168] Same as Example 1, the only difference is that the derivatization reagent is different, see Table 4 for details.

[0169] Table 4 Optimization of derivatization reagent dosage

[0170] Serial number Triethylamine (μL) Quinaloyl chloride(mg) 4-Dimethylaminopyridine(mg) 1 15 8 8 2 (Example 1) 0 8 8 3 5 8 8 4 10 8 8 5 15 8 0 6 15 8 4 7 15 8 16 8 15 8 32 9 15 2 8 10 15 4 8 11 15 16 8 12 15 32 8

[0171] The response values ​​of the target substances in the sample solutions obtained in Example 1 and Example 8 were measured. The results are shown in Fig.10 .

[0172] from Fig.10 It can be seen that if the reaction is to be carried out efficiently, the optimal dosages of the derivatizing agent and the catalyst are: 8 mg quinaloyl chloride, 8 mg 4-dimethylaminopyridine, while other reaction conditions can obtain certain derivatized analytes, and under certain sensitivity conditions, they can be used in analytical detection derivatization conditions.

[0173] Example 9

[0174] The same as Example 1, except that 40 μL of 1 μg / mL 2-MCPD-d5 standard working solution and 40 μL of 1 μg / mL 3-MCPD-d5 standard working solution were added, and the derivatization times at room temperature were 10 min, 30 min, 1 h, 2 h, 4 h and 6 h, respectively.

[0175] The response value of the target substance in the sample solution obtained in this example was measured, and the results are shown in Fig.11 .

[0176] from Fig.11 It can be seen that the method of the present invention can obtain a higher response value when the derivatization time is short, and there is no obvious increase in the number of derivative product signals when the reaction time is increased, indicating that derivatization and detection of the target can be achieved in a shorter time.

[0177] Example 10

[0178] Detection of actual samples (using the method of Example 1):

[0179] Quality control samples were set up in each batch to examine the recovery results of added 3-MCPD-d5 and 2-MCPD-d5. The added concentration of 3-MCPD-d5 and 2-MCPD-d5 in the sample was 20 ng / mL. Under the premise that the recovery rates of 3-MCPD-d5 and 2-MCPD-d5 were between 60 and 110%, 7 soy sauce samples purchased on the market were tested. The results showed that the 3-MCPD content was between 4.21 and 11.7 ng / mL, and the 2-MCPD content was between 0.751 and 1.697 ng / mL.

[0180] Table 5 Test results of actual soy sauce samples

[0181]

[0182] Embodiment 11

[0183] Demulsifier selection:

[0184] (1) Use a syringe to puncture the krill oil capsule, draw 4 mL of krill oil sample into a 50 mL plastic centrifuge tube, add 16 mL of NaCl aqueous solution (10%, w / v), and shake vigorously for 10 min.

[0185] (2) Take out all the emulsions, divide them into 8 equal parts and place them in 5 mL centrifuge tubes. Add 500 mg of C18, PSA, C18 / PSA (1 / 1, w / w), neutral alumina, PEP, silica gel (100-200 mesh), and silica gel (200-300 mesh) to the emulsions No. 1 to 7, respectively. Shake vigorously for 10 minutes. After standing for 5 minutes, the phenomenon is seen. Fig.12 Then centrifuge it (10000rpm, 10min), the phenomenon is shown Fig.13 .

[0186] The emulsion No. 8 slowly passed through a 500 mg C18 solid phase extraction column, and the column liquid was collected and allowed to stand for 5 minutes. Fig.12 ; Then the column liquid was centrifuged (10000rpm, 10min), and the phenomenon was seen Fig.13 .

[0187] Fig.12 1 is a centrifuge tube with 500 mg of C18 added, 2 is a centrifuge tube with 500 mg of PSA added, 3 is a centrifuge tube with 500 mg of C18 / PSA (1 / 1, w / w) added, 4 is a centrifuge tube with 500 mg of neutral alumina added, 5 is a centrifuge tube with 500 mg of PEP added, 6 is a centrifuge tube with 500 mg of silica gel (100-200 mesh), 7 is a centrifuge tube with 500 mg of silica gel (200-300 mesh), and 8 is a centrifuge tube for collecting the column liquid after passing through a 500 mg C18 solid phase extraction column. Fig.13 This is a comparison of centrifuge tubes after centrifugation.

[0188] from Fig.12 and Fig.13 It can be seen that the use of silica gel (200-300 mesh) has a better effect and can purify and clarify the krill oil emulsion without centrifugation.

[0189] Example 12

[0190] The same as Example 1, the only difference is that the sample and the sample pretreatment method are different, as follows:

[0191] A. Use a syringe to puncture the krill oil capsule, draw 1 mL of krill oil sample into a 5 mL plastic centrifuge tube, add 50 μL of 3-MCPD-d5 (1 μg / mL) and 50 μL of 2-MCPD-d5 (1 μg / mL) standard working solutions, vortex (fully shake) for 30 seconds to mix, then add 4 mL of NaCl aqueous solution (10%, w / v), and shake vigorously for 10 minutes.

[0192] B. Pipette 5 mL of the emulsion into a new 5 mL plastic centrifuge tube, add 500 mg of silica gel (200-300 mesh, silica gel-DSPE), shake vigorously again for 10 min, and centrifuge at 4000 rpm for 10 min.

[0193] C. Take 2 mL of the upper purified liquid and slowly pass it through an activated carbon solid phase extraction column (1.5 g / 12 mL) on a vacuum solid phase extraction device. Discard the effluent and let it stand for 10 minutes. Rinse the activated carbon column with 5 mL of pure water, and then elute the activated carbon column with 15 mL of ethyl acetate (EA). Elute once every 5 mL for a total of 3 times. Pump the activated carbon column to near dryness with a vacuum pump and collect and combine the eluent.

[0194] D. Discard the lower phase of the eluate and transfer the residual liquid to a pear-shaped bottle. Rotary evaporate to about 0.5 mL (rotary evaporate the solution to near dryness) at 30°C and 110 bar to obtain a concentrated solution.

[0195] E. Transfer the concentrate to a 5 mL centrifuge tube, add 200 mg 3A molecular sieves, shake thoroughly to remove water until no bubbles are generated in the liquid, then add the derivatization reagent (8 mg quinaloyl chloride and 8 mg 4-dimethylaminopyridine are added in sequence, and each reagent needs to be shaken thoroughly), shake and mix, and derivatize for 30 minutes at room temperature. Then blow dry with gentle nitrogen, transfer 1.00 mL acetonitrile (ACN) for redissolution, filter through a 0.22 μm organic phase filter to obtain a sample solution (silicagel-DSPE), and perform LC-MS / MS analysis on the sample solution.

[0196] Krill oil sample pretreatment and derivatization flow chart is shown in Fig.14 .

[0197] Matrix Effects:

[0198] Since the method of the present invention is analyzed in mass spectrometry, the magnitude of the matrix effect is evaluated mainly by the following method: the matrix effect can be evaluated by adding a standard to the blank sample solution after extraction. The matrix effect is determined by comparing the response ratio of the matrix standard solution and the pure standard solution.

[0199] Table 6 Matrix effects of 3-MCPD and 2-MCPD in krill oil

[0200]

[0201] Calculation of matrix effect: The calculation formula of matrix effect (MF%) is MF% = B / A, where A represents the signal intensity of the pure solvent standard solution in the mass spectrometer, and B represents the signal intensity of the mass spectrometer of the solution with the same concentration as A prepared by using blank matrix extract. When MF = 1, it means there is no matrix effect, when MF>1, it means there is matrix enhancement, and when MF<1, it means there is matrix suppression.

[0202] Recovery and precision: When different concentrations of standard samples were added to the shrimp oil matrix, the recovery was within a reasonable range and the method had good precision.

[0203] Limit of Detection (LOD) and Limit of Quantitation (LOQ):

[0204] Limit of detection (LOD): refers to the minimum concentration or amount of the substance to be tested that can be detected from a sample by a specific analytical method within a given confidence level. Limit of quantification (LOQ): refers to the minimum concentration or amount of the component to be tested in a sample that can be quantitatively determined by the analytical method, and the measurement result should have a certain accuracy and precision. Under the above-mentioned optimal derivatization conditions for 3-MCPD and 2-MCPD in shrimp oil, the detection limit and quantification limit of the analytical method are determined when the peak height is approximately 3 times and 10 times the baseline noise, that is, S / N ≥ 3 and S / N ≥ 10, respectively. The detection limit that this method can achieve is 0.5ppb and the quantification limit is 1ppb.

[0205] Example 13

[0206] Same as Example 12, except that 500 mg of silica gel (200-300 mesh) is replaced with an equal mass of C18 (C18-DSPE).

[0207] Embodiment 14

[0208] The same as Example 12, except that step B is specifically as follows: the lower layer of emulsion is aspirated and placed in a new 5 mL plastic centrifuge tube, slowly passed through a 500 mg silica gel (200-300 mesh) solid phase extraction column, the column liquid is collected, and centrifuged at 4000 rpm for 10 min (silica gel-SPE).

[0209] Embodiment 15

[0210] The same as Example 12, except that 50 μL of 2-MCPD-d5 (1 μg / mL) standard working solution was not added, and step B was specifically as follows: the lower layer of emulsion was aspirated and placed in a new 5 mL plastic centrifuge tube, slowly passed through a C18 solid phase extraction column, the column liquid was collected, and centrifuged at 4000 rpm for 10 min (C18-SPE).

[0211] The MCPD (3-MCPD-d5) content in the sample solutions prepared in Examples 12 to 14 was determined and the MCPD recovery rate was calculated. The results are shown in Fig.15 .

[0212] from Fig.15 It can be seen that silica gel (200-300 mesh) and its dispersed form (DSPE) have little effect on MCPD. Its advantages are stable chemical properties, non-toxicity, strong adaptability, and can be used to purify impurities of different concentrations and types. At the same time, the purification process is simple, the cost is low, and its recovery rate is much higher than that of C18.

[0213] Embodiment 15

[0214] Same as Example 12, except that 50 μL of 2-MCPD-d5 (1 μg / mL) standard working solution was not added, and the amounts of silica gel (200-300 mesh) used were 200 mg, 300 mg, 400 mg, 500 mg and 600 mg, respectively.

[0215] The MCPD (3-MCPD-d5) content in the sample solutions prepared in Example 12 and Example 15 was determined and the MCPD recovery rate was calculated. The results are shown in Fig.16 .

[0216] Example 16

[0217] Same as Example 12, except that the derivatization reagent is different, see Table 7 for details.

[0218] Table 7 Optimization of derivatization reagent dosage

[0219]

[0220] The response values ​​of the target substances in the sample solutions obtained in Example 12 and Example 16 were measured. The results are shown in Fig.17 .

[0221] from Fig.17 It can be seen that the derivatization reaction must have a catalyst to proceed to the ideal state, and the effect of different amounts of derivatizing agents on 2-MCPD is not as great as that on 3-MCPD. After experiments, 8 mg of 4-dimethylaminopyridine was selected as the catalyst. The results showed that 8 mg of quinaloyl chloride was used as the derivatizing agent, and the derivatization efficiency could reach the optimal state without wasting the derivatizing agent.

[0222] Embodiment 17

[0223] The same as Example 12, except that 100 μL of 1 μg / mL 2-MCPD-d5 standard working solution and 100 μL of 1 μg / mL 3-MCPD-d5 standard working solution were added, and the derivatization time at room temperature was 10 min, 30 min, 1 h, 2 h, 4 h and 6 h, respectively.

[0224] The response values ​​of the target substances in the sample solutions obtained in Example 12 and Example 17 were measured. The results are shown in Fig.18 .

[0225] from Fig.18 It can be seen that 3-MCPD and 2-MCPD in the sample can reach the best derivatization state after the derivatization agent is added and fully shaken and mixed, and the derivatization efficiency does not change with time. However, in order to ensure the uniformity of derivatization, the derivatization time can be controlled to 20 to 30 minutes after adding the derivatization agent and shaking and mixing, in order to make the generated derivative product reach a stable state. Compared with the previous use of p-dimethylaminophenol hydrochloride as a derivatization agent, the use of quinalcohol chloride for derivatization greatly reduces the derivatization time, further improving the experimental efficiency.

[0226] Embodiment 18

[0227] Detection of actual samples (using the method of Example 12):

[0228] Six real shrimp oil samples were purchased randomly from the market, and three parallel samples were prepared for each sample. A total of 18 shrimp oil samples were quantitatively prepared by the internal standard method for pretreatment. At the same time, a derivatization reaction of a series of standard working solutions of 3-MCPD, 2-MCPD and its internal standard was performed. After the samples and the series of standard working solutions were derivatized at the same time, they were detected by LC-MS. Quality control samples were set in each batch to investigate the recovery results of adding 3-MCPD-d5 and 2-MCPD-d5. The concentration of 3-MCPD-d5 and 2-MCPD-d5 added to the sample was 20 ng / mL. The recovery rates of 3-MCPD-d5 and 2-MCPD-d5 were determined to be between 60% and 110%. The actual sample test results are shown in Table 8.

[0229] Table 8 Shrimp oil actual sample test results

[0230]

[0231] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting 2-chloro-propylene glycol and 3-chloro-propylene glycol in a sample, characterized in that: The following steps are involved: Purify the sample solution to be tested through a purification column to obtain a purified solution; After removing water from the purified liquid, a derivatization reagent is added to perform derivatization treatment to obtain a derivatized liquid; the derivatization reagent includes quinaloyl chloride and 4-dimethylaminopyridine; The derivatized liquid is subjected to liquid chromatography-mass spectrometry detection.

2. The method according to claim 1, characterized in that When the sample solution to be tested is a liquid sample without oil or fat, the preparation method of the purified liquid specifically includes: Add the sample solution to be tested into the purification column, let it stand and then elute with ethyl acetate to obtain a purified solution; The purification column is an activated carbon column.

3. The method according to claim 1, characterized in that When the sample solution to be tested is a liquid sample containing oil, the preparation method of the purified liquid specifically includes: A NaCl aqueous solution is added to the sample solution to be tested, and the mixture is shaken and centrifuged. Silica gel is added to the obtained emulsion, and the mixture is shaken and centrifuged again. The obtained solution is added to a purification column, and after standing, the solution is eluted with ethyl acetate to obtain a purified solution; the purification column is an activated carbon column.

4. The method according to claim 1, characterized in that: The dehydration specifically comprises: concentrating the purified liquid to obtain a concentrated liquid, adding 3A molecular sieves into the concentrated liquid, and oscillating to remove water.

5. The method according to claim 1, characterized in that The derivatization treatment time is 10 min to 6 h.

6. The method according to claim 1, characterized in that The liquid chromatography-mass spectrometry detection conditions are as follows: Chromatographic analysis conditions: Column: Eclipse Plus C18, 3.0×150 mm, 1.8 μm; Flow rate: 0.5 mL / min; Column temperature: 35°C; Injection volume: 4 μL; Mobile phase: Phase A is water containing 0.2 vol.% formic acid and 5 mmol / L ammonium acetate, and phase B is acetonitrile; Elution gradient: 0-1.9 min, 41% B; 1.9-2 min, 41-63% B; 2-5.5 min, 63-64% B; 5.5-6.8 min, 64-100% B; 6.8-8 min, 100-41% B; Mass spectrometry analysis conditions: Ion source: HESI; Spray voltage: 3200 V in positive ion mode, 2800 V in negative ion mode; Gas parameters: sheath gas 50arb, auxiliary gas 15arb, purge gas 1arb; Temperature: capillary temperature 350°C, auxiliary gas heating temperature 400°C; Scan Mode: PRM.

7. The method according to claim 1, characterized in that The method also includes adding an isotope internal standard to the sample solution to be tested for quantitative detection before purification.

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