Method for detecting chiral chloropropanol ester by using SFC-MS / MS

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for detecting chiral chloropropanol ester through SFC-MS / MS. Chromatographic detection conditions are as follows: a filler type of a chromatographic column is a polysaccharide derivative chiral stationary phase, a basic skeleton is amylose, and a functional group is 3, 5-dimethyl phenyl carbamate; the mobile phase is composed of a mobile phase A, a mobile phase B and a compensation mobile phase, and the mobile phase A is carbon dioxide; the mobile phase B is a methanol solution of 0.01 wt%-0.1 wt% formic acid; the compensation mobile phase comprises methanol and / or isopropanol; the chiral chloropropanol ester comprises chiral 3-chloro-1, 2-propylene glycol palmitic acid diester, and the chiral chloropropanol ester comprises chiral 3-chloro-1, 2-propylene glycol palmitic acid diester. The invention provides a supercritical fluid chromatography-tandem mass spectrometry separation analysis method for detecting the chiral chloropropanol ester by an external standard method, does not need to add an expensive internal standard, is good in separation effect and high in precision and accuracy, and is suitable for various complex matrixes such as edible oil and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, in particular to a method for detecting chiral chloropropanol esters by SFC-MS / MS. Background Art

[0002] Chloropropanol esters are a class of compounds formed by the esterification of chloropropanols and fatty acids. They can also be formed by the substitution of chlorine atoms for acyl groups on triacylglycerols. They are contaminants generated during food processing, primarily during the refining and deodorization of oils and fats. Therefore, refined vegetable oils are the most severely contaminated. In recent years, trichloropropanol esters have been detected in a variety of heat-processed foods, such as baked goods and fried foods. Trichloropropanol esters have been reported to induce various toxic effects in vivo, including nephrotoxicity, reproductive toxicity, neurotoxicity, and carcinogenicity in rodents. Among these, chiral 3-chloro-1,2-propanediol palmitate and achiral 2-chloro-1,3-propanediol stearate are the most studied. However, there are currently no reports on their chiral enantiomers, and the configurations and properties of both enantiomers remain uncertain.

[0003] Currently, analytical methods for chloropropanol esters fall into two main categories: direct and indirect. Indirect methods involve hydrolyzing chloropropanol esters into free chloropropanol and the corresponding fatty acids, which are then derivatized for determination. Hydrolysis methods include acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Alkaline hydrolysis is the most widely used hydrolysis method. However, theoretically, indirect analytical methods also present potential challenges. The free trichloropropanol obtained through hydrolysis may degrade during continued hydrolysis, and the use of chloride salts in the salting-out step may result in the formation of additional trichloropropanol. Indirect methods can only determine the total amount of chloropropanol esters and do not provide useful information on the specific fatty acids present in the trichloropropanol esters or the presence of mono- or diesters.

[0004] Direct determination is relatively simple. LC-MS, a commonly used analytical technique in food testing, eliminates the need for derivatization and provides structural information on chloropropanol esters. Therefore, it has long been used to analyze chloropropanol esters. However, this method has limitations in its ability to separate certain chiral compounds, particularly in complex matrices, where the separation of chiral isomers is incomplete.

[0005] Therefore, how to provide a rapid, accurate, convenient, safe and environmentally friendly method for detecting trichloropropane esters has become a technical problem that urgently needs to be solved in this field.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] In order to solve the above problems in the prior art, the present invention provides a method for detecting chiral chloropropanol esters by SFC-MS / MS.

[0008] Supercritical fluid chromatography (SFC) has been widely used to separate compounds in complex matrices. In recent years, SFC has been particularly popular for the separation of chiral substances. Using CO2 as the primary mobile phase, SFC offers advantages over high-performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLC-MS / MS) in terms of the use of organic reagents, lower costs, and safer, more environmentally friendly procedures.

[0009] Currently, methods used domestically and internationally require the use of expensive isotope internal standards, have retention times of 20–30 minutes, and detection limits of approximately 10–50 μg / kg. This innovative method enables rapid trace detection of two high-risk chiral chloropropanol esters within 4 minutes, extending the linear range and applicability. Specifically, the method utilizes an external standard method, significantly reducing costs and detection workflow, shortening detection time by more than 70%, and achieving sensitivity 50–250 times greater than other methods.

[0010] Based on this, the present invention has the following technical solutions: In a first aspect, the present invention provides a method for detecting chiral chloropropane esters by SFC-MS / MS, and the chromatographic detection conditions include: the filler type of the chromatographic column is a polysaccharide derivative chiral stationary phase, the basic skeleton is amylose, and the functional group is 3,5-dimethylphenylcarbamate; the mobile phase is composed of mobile phase A, mobile phase B and a compensation mobile phase, wherein the mobile phase A is carbon dioxide; the mobile phase B is a methanol solution of 0.01wt% to 0.1wt% formic acid; the compensation mobile phase includes methanol and / or isopropanol; the chiral chloropropane ester includes chiral 3-chloro-1,2-propylene glycol palmitic acid diester (hereinafter also referred to as "3-MCPDE").

[0011] In the present invention, the chiral 3-chloro-1,2-propanediol palmitic acid diester includes enantiomer 1 and enantiomer 2.

[0012] The present invention establishes for the first time a supercritical fluid chromatography-tandem mass spectrometry method for separating and analyzing chiral trichloropropane diesters using an external standard method. The method has good separation effect, high precision and accuracy, can use only carbon dioxide and methanol as the mobile phase without adding any salts, uses a small amount of organic solvent, and requires only 0.5 mL of methanol for a single injection of the organic phase. The method is safe and environmentally friendly, has a short detection time, and peaks can be detected before 4 minutes. The detection limit can reach 0.2 μg / kg. The pretreatment operation is simple, and a small amount of solvent is used for treatment. The method is applicable to complex matrices such as various edible oils.

[0013] According to the method for detecting chiral chloropropanol esters by SFC-MS / MS provided by the present invention, the chromatographic column is CHIRALPAK IA; preferably, the chromatographic column specifications are 5 μm and 4.6×250 mm.

[0014] According to a method for detecting chiral chloropropanol esters by SFC-MS / MS provided by the present invention, isocratic elution is adopted, wherein the mobile phase A accounts for 88% to 92%, and the mobile phase B accounts for 8% to 12%.

[0015] According to a method for detecting chiral chloropropanol esters by SFC-MS / MS provided by the present invention, the volume content of the mobile phase A is 85% to 95% of the volume content of the compensation mobile phase.

[0016] The present invention has discovered that by controlling the ratio of the mobile phase A and the compensating mobile phase, it is possible to better adapt to diverse analytical needs. By adjusting the ratio of CO2 to the compensating mobile phase, the solubility and separation performance of the product can be regulated. Variations in the amount of CO2 added can change the elution capacity of the mobile phase, thereby having a certain impact on the retention time and selectivity of the sample. By adjusting the CO2 ratio, it is possible to separate target substances in complex systems. A high CO2 content can cause the mobile phase in the mass spectrometry ion source to volatilize, thereby reducing the ionization effect. On this basis, further research is conducted to study the influence of CO2 concentration on chromatographic separation performance, achieve effective regulation of CO2 content during chromatographic separation, and achieve rapid and stable chromatographic analysis during chromatographic separation. Changes in CO2 content will have a certain impact on the system backpressure, thereby having a significant impact on column efficiency and separation efficiency. Rational selection of the CO2 ratio is conducive to maintaining the stability and reproducibility of the system. Substances of different polarity and molecular weights require different CO2 ratios. By selecting the CO2 content, it can better meet different needs.

[0017] Because the mobile phase that UPC2 liquid chromatography uses is supercritical fluid CO , it is a kind of weak polar solvent, the separation effect to medium polarity and strong polarity substance is not good, it is necessary to enhance the separation performance of supercritical carbon dioxide by adding modifier, and enhance its analysis range.In addition, because the character of various modifiers themselves is different, therefore various modifiers are also different to the separating action of target object. In the present invention, for enhancing CO 2 to the elution and solubility of compound, organic solvents such as methanol, isopropanol are added in mobile phase as modifier, simultaneously, can enhance the hydrogen bonding between free silicon dioxide hydroxyl group and chloropropanol ester group, enhance chromatographic selectivity.

[0018] According to the method for detecting chiral chloropropanol esters by SFC-MS / MS provided by the present invention, the compensating mobile phase is methanol.

[0019] According to a method for detecting chiral chloropropanol esters by SFC-MS / MS provided by the present invention, the flow rates of the mobile phase A and the mobile phase B are 1.2-1.5 mL / min.

[0020] According to a method for detecting chiral chloropropanol esters by SFC-MS / MS provided by the present invention, the flow rate of the compensating mobile phase is 0.1-0.5 mL / min.

[0021] According to a method for detecting chiral chloropropanol esters by SFC-MS / MS provided by the present invention, the mass spectrometry conditions include: Using a triple quadrupole mass spectrometer; preferably a Waters Xevo TQ-XS; Ionization mode: ESI+; Scan mode: multiple reaction monitoring (dMRM); Capillary voltage: 4000 V and 3500 V; Nebulizer pressure: 15-20 psi; Drying gas temperature: 280~320℃; Drying air flow rate: 12~17 L / min; Sheath gas temperature: 250~270℃; Sheath gas flow rate: 10~12 L / min.

[0022] According to a method for detecting chiral chloropropanol esters using SFC-MS / MS provided by the present invention, the mass spectrometry parameters are as follows: .

[0023] According to the present invention, a method for detecting chiral chloropropanol esters by SFC-MS / MS is provided, comprising: dissolving a sample to be tested in acetone to obtain a dissolved solution with a concentration of 0.04-0.06 g / mL, then diluting the solution to a constant volume with methanol to obtain a diluted solution with a concentration of 4-6 mg / mL; vortexing the diluted solution, and taking the supernatant for detection on the instrument.

[0024] Domestic and foreign methods are unable to achieve rapid direct extraction, and pretreatment requires the addition of large amounts of organic solvent, typically 17 to 25 mL. However, the present invention innovatively implements a direct one-step extraction method, featuring a highly efficient, rapid, low-carbon, and environmentally friendly pretreatment technique with less than 2 mL of organic solvent, a reduction of 88 to 92%. Pretreatment techniques used domestically and internationally are time-consuming, typically 30 to 60 minutes, while the present invention only requires 1 minute, shortening the process by 30 to 60 times and making it suitable for large-scale analysis and determination.

[0025] According to the present invention, a method for detecting chiral chloropropane esters by SFC-MS / MS is provided, wherein the sample to be tested includes chiral 3-chloro-1,2-propylene glycol palmitate diester or an edible oil containing chiral 3-chloro-1,2-propylene glycol palmitate diester.

[0026] In the present invention, the edible oil can be corn oil, soybean oil, peanut oil, sunflower oil, rapeseed oil, palm oil, etc., which is not limited here, and those skilled in the art can choose according to actual conditions.

[0027] Based on this, the technical solution of the present invention has the following beneficial effects: The present invention provides a supercritical fluid chromatography tandem mass spectrometry separation and analysis method for two chiral chloropropanol esters by providing an external standard method for detecting chiral chloropropanol esters without adding an expensive internal standard, having good separation effect, high precision and accuracy, being applicable to complex matrices such as various edible oils, having simple pretreatment operations, using a small amount of solvent for treatment, achieving a detection limit of 0.5 μg / L, and being able to produce a peak before 4 minutes, with a short detection time, and having a high proportion of carbon dioxide, which is environmentally friendly and saves solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is a chromatogram of chiral 3-chloro-1,2-propylene glycol palmitate diester in palm oil matrix in Example 1 provided by the present invention.

[0030] Figure 2 This is a chromatogram of chiral 3-chloro-1,2-propylene glycol palmitate diester in corn oil matrix in Example 1 provided by the present invention.

[0031] Figure 3 This is a chromatogram of chiral 3-chloro-1,2-propylene glycol palmitate diester in peanut oil matrix in Example 1 provided by the present invention.

[0032] Figure 4 This is a chromatogram of chiral 3-chloro-1,2-propylene glycol palmitate diester in rapeseed oil matrix in Example 1 provided by the present invention.

[0033] Figure 5 This is a chromatogram of chiral 3-chloro-1,2-propylene glycol palmitate diester in sunflower oil matrix in Example 1 provided by the present invention.

[0034] Figure 6 This is a chromatogram of chiral 3-chloro-1,2-propylene glycol palmitate diester in soybean oil matrix in Example 1 provided by the present invention.

[0035] Figure 7 The chromatograms of Example 1 and Example 2 provided by the present invention are compared.

[0036] Figure 8 The chromatograms of Example 1 and Example 3 provided by the present invention are compared.

[0037] Figure 9 The figures are the comparison results of the chromatographic column tests of Example 1 and Comparative Example 1 provided by the present invention. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, the various raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0040] Example 1 This embodiment provides a method for detecting chiral chloropropanol esters by SFC-MS / MS, comprising: 1. The chromatographic conditions used are as follows: The chromatographic column used was a CHIRALPAK IA column (5 μm 4.6 × 250 mm); The detector used was a triple quadrupole mass spectrometer; The mobile phase includes mobile phase A, mobile phase B and compensation mobile phase; mobile phase A is carbon dioxide; B is a methanol solution of 0.05% formic acid; the compensation mobile phase is methanol; Isocratic elution was performed according to the following procedure: mobile phase A accounted for 90% and mobile phase B accounted for 10%; the ratio of mobile phase A to compensation mobile phase was 95:100; the total program run time was 10 min.

[0041] The flow rate was 1.4 mL / min, the compensation mobile phase flow rate was 0.3 mL / min; the system back pressure was 2200 psi; the column temperature was 30°C; and the injection volume was 2 μL.

[0042] 2. The mass spectrometry conditions used are as follows: Triple quadrupole mass spectrometer: Waters Xevo TQ-XS; Ionization mode: ESI+; Scan mode: multiple reaction monitoring (dMRM); Capillary voltage: 4000 V and 3500 V; Nebulizer pressure: 15 psi; Drying gas temperature: 300℃; Drying gas flow rate: 14 L / min; Sheath gas temperature: 260°C; Sheath gas flow rate: 11 L / min.

[0043] The mass spectrometry parameters are shown in Table 1: Table 1

[0044] 3. Standard curve: A 5 μg / mL standard solution of two chiral 3-chloro-1,2-propanediol palmitate diesters was prepared in mass spectrometry-grade isopropanol and stored at -20°C. A 0.1 g sample (accurate to 0.01 g) of soybean oil, corn oil, sunflower oil, rapeseed oil, and palm oil was weighed into a 5 mL centrifuge tube. 2 mL of acetone was added and vortexed. 0.2 mL of the solution was transferred to a 5 mL centrifuge tube and the volume was adjusted to 2 mL with methanol. Vortexed for 1 minute, the supernatant was aspirated and filtered through a 0.22 μm organic microporous filter to obtain solvent A. Solvent A was used to dilute the 5 μg / mL standard mixture to 5 μg / L, 10 μg / L, 20 μg / L, 40 μg / L, 80 μg / L, 100 μg / L, 200 μg / L, and 500 μg / L concentrations as matrix standard working solutions.

[0045] The standard curve is shown in Table 2 below: Table 2

[0046] The chromatograms of chiral 3-chloro-1,2-propanediol palmitic acid diester in different matrices are as follows: Figures 1 to 6 shown.

[0047] 4. Recovery rate The recoveries in five edible oil samples, including soybean oil, corn oil, sunflower oil, rapeseed oil, and palm oil, are as follows: Experimental Method: Weigh 0.1g of each of soybean oil, corn oil, sunflower oil, rapeseed oil, and palm oil (accurate to 0.01g) into a 5mL centrifuge tube. Add the appropriate amount of standard solution to prepare spiked samples with component concentrations of 40, 100, and 500 μg / kg. Add 2mL of acetone and vortex mix thoroughly. Pipette 0.2mL of the solution into a 5mL centrifuge tube, dilute to 2mL with methanol, and vortex mix thoroughly for 1 minute. The supernatant is then filtered through a 0.22μm organic microporous filter membrane and analyzed by a spectrophotometer. The recovery rates are shown in Table 3: Table 3

[0048] Example 2 This embodiment provides a method for detecting chiral chloropropanol esters by SFC-MS / MS, which differs from Example 1 in that the volume content of the mobile phase A is 80%, 85%, 90% or 100% of the volume content of the compensating mobile phase.

[0049] The chromatogram comparison results of Example 2 and Example 1 are as follows: Figure 7 shown.

[0050] according to Figure 7 It can be seen that when 100% supercritical CO2 is used, no substance can be eluted, the chromatogram shows a straight line, and the response value is 0. As the supercritical CO2 gradually decreases, the retention time of the target gradually shifts forward, but the response value gradually decreases, affecting the detection limit, and the separation degree also decreases with the gradual decrease of supercritical CO2. Under 80% reference conditions, the separation degree is only 0.78, which is less than 1.5, and baseline separation is not achieved. Under 95% reference conditions, the separation degree reaches 1.63, and the chiral monomers are completely separated, showing better peak shape and separation effect. The reason may be that a high proportion of CO2 is more likely to volatilize when entering the mass spectrometer detector, reducing interference with the ion source and improving ionization efficiency. A high proportion of CO2 can improve separation efficiency, improve peak shape and resolution, enhance mass spectrometry compatibility, reduce system back pressure, and has environmental and economic advantages.

[0051] Example 3 This example provides a method for detecting chiral chloropropanol esters using SFC-MS / MS. The difference from Example 1 is that the compensating mobile phase is replaced in equal amounts with methanol:isopropanol in a volume ratio of 3:1, methanol:isopropanol in a volume ratio of 1:1, methanol:isopropanol in a volume ratio of 1:3, or 100% isopropanol.

[0052] The chromatogram comparison results of Example 3 and Example 1 are as follows: Figure 8 shown.

[0053] Depend on Figure 8 It can be seen that the retention times of 3-MCPD esters by different modifiers are similar, but as the proportion of methanol decreases, the separation degree of chiral 3-MCPD ester gradually decreases. Under 100% methanol conditions, the separation effect is the best, and the separation degree R=2.24 reaches baseline separation.

[0054] Comparative Example 1 This comparative example provides a method for detecting chiral chloropropanol esters by SFC-MS / MS, which differs from Example 1 in that the chromatographic columns are replaced with CHIRALPAK® AD-RH (5 μm 4.6×150 mm), Venusil CA (5 μm 4.6×150 mm), and Trefoil AMY-1 (2.5 μm 2.1×250 mm), respectively.

[0055] The comparison results of the chromatographic column of comparative example 1 and the chromatographic column of embodiment 1 are as follows Figure 9 shown.

[0056] from Figure 9 It can be seen that the AD-RH column has a poor separation effect on the two chiral monomers of 3-chloro-1,2-propylene glycol palmitate diester, and there is interference from miscellaneous peaks; the CA and AMY-1 columns have average separation effects on the two chiral monomers, the second monomer cannot be completely eluted, and the chromatographic peaks are not smooth, with split peaks; while the IA column can retain the target substance very well, and the chromatographic peaks are smooth, without split peaks, and the separation degree is moderate, achieving complete separation.

[0057] Finally, 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting chiral chloropropanol esters by SFC-MS / MS, characterized in that: Chromatographic testing conditions include: The chromatographic column is filled with a polysaccharide derivative chiral stationary phase, the basic skeleton is amylose, and the functional group is 3,5-dimethylphenylcarbamate; the mobile phase consists of mobile phase A, mobile phase B, and a compensation mobile phase, wherein the mobile phase A is carbon dioxide; the mobile phase B is a methanol solution containing 0.01wt% to 0.1wt% formic acid; and the compensation mobile phase includes methanol and / or isopropanol. The chiral chloropropanol ester includes chiral 3-chloro-1,2-propylene glycol palmitate diester.

2. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to claim 1, characterized in that: The chromatographic column is CHIRALPAK IA; preferably, the chromatographic column specification is 5 μm, 4.6×250 mm.

3. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to claim 1 or 2, characterized in that: Isocratic elution was used, wherein the mobile phase A accounted for 88% to 92% and the mobile phase B accounted for 8% to 12%.

4. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to any one of claims 1 to 3, characterized in that: The volume content of the mobile phase A is 85% to 95% of the volume content of the compensating mobile phase.

5. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to any one of claims 1 to 4, characterized in that: The compensating mobile phase is methanol.

6. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to any one of claims 1 to 5, characterized in that: The flow rate of the mobile phase A and the mobile phase B is 1.2-1.5 mL / min; And / or, the flow rate of the compensating mobile phase is 0.1-0.5 mL / min.

7. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to any one of claims 1 to 6, characterized in that: Mass spectrometry conditions included: Using a triple quadrupole mass spectrometer; preferably a Waters Xevo TQ-XS; Ionization mode: ESI+; Scan mode: multiple reaction monitoring (dMRM); Capillary voltage: 4000 V and 3500 V; Nebulizer pressure: 15-20 psi; Drying gas temperature: 280~320℃; Drying air flow rate: 12~17 L / min; Sheath gas temperature: 250~270℃; Sheath gas flow rate: 10~12 L / min.

8. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to claim 7, characterized in that: The mass spectrometry parameters are as follows: 。 9. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to any one of claims 1 to 8, characterized in that: include: The sample to be tested was dissolved in acetone to obtain a solution with a concentration of 0.04-0.06 g / mL, and then diluted with methanol to a constant volume to obtain a dilution with a concentration of 4-6 mg / mL; the dilution was vortexed and the supernatant was taken for detection on the instrument.

10. The method for detecting chiral chloropropanol esters by SFC-MS / MS according to claim 9, characterized in that: The sample to be tested includes chiral 3-chloro-1,2-propylene glycol palmitic acid diester or edible oil containing chiral 3-chloro-1,2-propylene glycol palmitic acid diester.

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