A method for detecting 3-chloro-1,2-propanediol ester and 2-chloro-1,3-propanediol ester in aquatic oils by liquid chromatography-tandem mass spectrometry.

Abstract

This invention discloses a method for detecting 3-chloro-1,2-propanediol esters and 2-chloro-1,3-propanediol esters in aquatic oils using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method employs porcine pancreatic lipase to specifically enzymatically hydrolyze esterified contaminants in krill oil, releasing free 3-chloro-1,2-propanediol and 2-chloro-1,3-propanediol. Derivatization is then performed using quinalyl chloride as a derivatizing agent. Combined with LC-MS / MS technology, this achieves high sensitivity and specificity for detection and effectively distinguishes the chromatographic behavior of these two isomers. By using mild and highly specific enzymatic hydrolysis instead of harsh chemical hydrolysis, and combining it with LC-MS / MS detection, the destruction of target analytes and side reactions during pretreatment are effectively reduced. The optimized pretreatment process improves the stability, accuracy, and detection efficiency of the method in complex lipid matrices, providing a reliable analytical tool for risk assessment and quality control of chloropropanol ester contaminants in aquatic oils and related oil products.

Description

Technical Field

[0001] This invention relates to the field of food contaminant detection technology, specifically to a method for detecting 3-chloro-1,2-propanediol ester and 2-chloro-1,3-propanediol ester in aquatic oils by liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Antarctic krill oil is a high-value marine resource rich in Omega-3 polyunsaturated fatty acids (mainly EPA and DHA) and astaxanthin, making it highly sought after in dietary supplements and functional foods. However, similar to other animal and vegetable oils, it inevitably forms a potential chemical contaminant—chloropropanol fatty acid esters—during its refining, processing, and storage. Common types include 2-chloro-1,3-propanediol esters (2-MCPD esters) and 3-chloro-1,2-propanediol esters (3-MCPD esters). Similarly, fish oil, another important aquatic oil, is also rich in EPA and DHA, and its processing also generates the same chloropropanol ester contaminants. These contaminants are relatively stable, but they can be hydrolyzed by lipases in the human gastrointestinal tract, releasing free 2-MCPD and 3-MCPD, which have potential nephrotoxicity and reproductive toxicity. Therefore, accurate monitoring of their content in oils is crucial for ensuring food safety. However, since these pollutants do not exist in a free form, but are covalently bound to the glycerol backbone through ester bonds and embedded in complex molecular networks of triglycerides, phospholipids, etc., they cannot be directly detected and must be released from the ester bonds through pretreatment.

[0003] Currently, national standard methods and other official detection methods rely on vigorous chemical hydrolysis, specifically concentrated sulfuric acid hydrolysis or sodium methoxide alkaline hydrolysis. These methods use strong acid and strong base reagents at high temperatures to treat oil samples, indiscriminately breaking all ester bonds and converting bound MCPDEs into detectable free monomers MCPD. These reaction conditions are extremely harsh, easily leading to further dehydration, cyclization, and other degradation reactions in some of the released free MCPD, resulting in the loss of the target analyte. Simultaneously, other components in the oil matrix, such as triglycerides, may undergo complex reactions under strong acid or strong base conditions, potentially interfering with subsequent analysis. Furthermore, gas chromatography-mass spectrometry (GC-MS) is the most commonly used analytical method for detecting MCPD esters. This involves determining the content of free MCPD released after hydrolysis and using derivatizing reagents such as phenylboronic acid or heptafluorobutyryl imidazole to improve detection sensitivity. The separation mechanism based on gas chromatography relies on the difference in boiling point and polarity of compounds, making it difficult to simultaneously distinguish and detect the two isomers 2-MCPD and 3-MCPD. Furthermore, this step requires advanced technical skills and is time-consuming, and the derivatization reagents can easily contaminate the instrument.

[0004] Therefore, how to construct a direct, efficient and accurate analytical detection scheme for the bound state of chloropropanol esters in oils is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for detecting 3-chloro-1,2-propanediol esters and 2-chloro-1,3-propanediol esters in aquatic oils using liquid chromatography-tandem mass spectrometry. This method replaces traditional, vigorous chemical hydrolysis with a mild and highly efficient enzymatic digestion technique, accurately converting bound chloropropanol esters into free chloropropanol. This provides a milder, more specific, and more sensitive pretreatment solution for the detection of chloropropanol esters in oils.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for detecting 3-chloro-1,2-propanediol ester and 2-chloro-1,3-propanediol ester in aquatic oils by liquid chromatography-tandem mass spectrometry, comprising the following steps: S1. Sample enzymatic hydrolysis: Add a buffer solution containing surfactant and lipase to the oil sample and carry out an isothermal enzymatic hydrolysis reaction to hydrolyze the bound 3-MCPDE and 2-MCPDE into free 3-MCPD and 2-MCPD; after the reaction is completed, heat treatment is performed to inactivate the enzyme. S2. Sample purification and extraction: After centrifuging the reaction solution obtained in step S1, the supernatant is purified by passing it through a solid phase extraction column, the eluent is collected, and liquid-liquid extraction is performed with an organic solvent. The organic phases are combined and concentrated. S3. Dehydration and derivatization: A dehydrating agent is added to the organic phase concentrated in step S2 to remove water, followed by the addition of quinalyl chloride and a catalyst to carry out the derivatization reaction; S4. LC-MS / MS detection: The product derivatized in step S3 was analyzed by liquid chromatography-tandem mass spectrometry, and qualitative and quantitative analysis was performed based on retention time and characteristic ion pairs.

[0007] Preferably, the lipase in step S1 is porcine pancreatic lipase.

[0008] Preferably, the buffer solution in step S1 is a McIlvaine buffer solution with a pH of 6.5-7.5.

[0009] Preferably, the concentration of the lipase in step S1 is 1000-2000 U / mL; the temperature of the isothermal enzymatic hydrolysis is 35-40℃, and the time is 6-10 h.

[0010] Preferably, the surfactant in step S1 is Tween 80, and its final volume concentration in the reaction system is 0.1%-1.0%.

[0011] Preferably, in step S2, the solid-phase extraction column is a dephospholipid PPR column; and the organic solvent is ethyl acetate.

[0012] Preferably, the dehydrating agent in step S3 is 3A molecular sieve; and the catalyst is 4-dimethylaminopyridine.

[0013] Preferably, in step S4, the liquid chromatography uses a C18 column, with an aqueous solution containing formic acid and ammonium acetate as mobile phase A and a methanol solution containing formic acid as mobile phase B for gradient elution; the mass spectrometry uses electrospray positive ion mode and multiple reaction monitoring scanning. Preferably, during the processing in step S1, isotopic internal standards of 3-MCPD-d5 and 2-MCPD-d5 are added.

[0014] Preferably, the oil sample is Antarctic krill oil or fish oil.

[0015] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for detecting 3-chloro-1,2-propanediol ester and 2-chloro-1,3-propanediol ester in aquatic oils by liquid chromatography-tandem mass spectrometry, which has the following beneficial effects: This invention utilizes porcine pancreatic lipase to efficiently and gently hydrolyze bound 2-MCPDE and 3-MCPDE in aquatic oils into free 2-MCPD and 3-MCPD. A novel derivatizing agent, quinalyl chloride, is used to distinguish and simultaneously quantify 2-MCPD and 3-MCPD in Antarctic krill oil. A PPR dephospholipid column is used to efficiently purify phospholipids, fats, and other impurities in the aquatic oils to obtain a clear solution for subsequent experiments. Multiple extractions with large volumes of ethyl acetate are used to transfer the target 2-MCPD and 3-MCPD from the aqueous phase to the organic phase. Finally, a porous... The high-specific-surface-area dehydrating agent 3A molecular sieve, with its efficient dehydration, facilitates the effective subsequent derivatization reaction. A UHPLC-MS / MS method developed using isotope internal standard for the hydrolysis of bound 2-MCPDE and 3-MCPDE by BCL lipase exhibits advantages such as high hydrolysis efficiency, stable and rapid derivatization, good linearity, high sensitivity, and high recovery. This method provides mild and efficient pretreatment hydrolysis conditions, excellent derivatization effect, and novel detection techniques. It has significant reference value for the detection of 3-chloro-1,2-propanediol esters and 2-chloro-1,3-propanediol esters in dietary supplements such as Antarctic krill oil. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 This is a flowchart of the pretreatment and derivatization process for aquatic oil samples in Example 1; Figure 2 A schematic diagram of the hydrolysis reaction mechanism of 2-MCPD and its isotope internal standard; Figure 3 A schematic diagram of the hydrolysis reaction mechanism of 3-MCPD and its isotope internal standard; Figure 4 A schematic diagram of the reaction mechanism between 2-MCPD and its isotopic internal standard with quinalyl chloride; Figure 5 A schematic diagram of the reaction mechanism between 3-MCPD and its isotopic internal standard with quinalyl chloride; Figure 6 Extraction ion chromatograms of 2-MCPD and 3-MCPD standard derivatives; Figure 7 Extraction ion chromatograms of 2-MCPD-d5 and 3-MCPD-d5 isotope internal standard derivatives; Figure 8 A bar chart showing the effect of different lipase concentrations on enzymatic hydrolysis. Figure 9 A bar chart showing the effect of buffer solutions with different pH values ​​on the hydrolysis efficiency of lipase; Figure 10 A bar chart showing the effect of different enzymatic hydrolysis temperatures on the hydrolysis efficiency; Figure 11 A bar chart showing the effect of different enzymatic hydrolysis times on the hydrolysis efficiency; Figure 12 A bar chart showing the effect of different amounts of Tween 80 on the hydrolysis effect; Figure 13 For linear range; Figure 14 The extracted ion chromatograms show the extraction concentrations of 0.1 ppb 2-MCPDE and 3-MCPDE in a 0.1 g Antarctic krill oil sample. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1 I. Main Reagents and Instruments Reagents: 3-MCPD, 2-MCPD, 3-MCPDE, 2-MCPDE and their corresponding deuterated isotope internal standards (-d5); porcine pancreatic lipase (BCL); quinalyl chloride (Q-Cl); 4-dimethylaminopyridine (DMAP); Tween 80; disodium hydrogen phosphate, citric acid (for preparing McIlvaine buffer solution); sodium bromide; ethyl acetate, acetonitrile (chromatographic grade); 3A molecular sieve; dephospholipid PPR solid-phase extraction column (500 mg / 6 mL).

[0020] Instruments: Ultra-high performance liquid chromatography-tandem high resolution mass spectrometry (UHPLC-MS / MS); constant temperature shaker; centrifuge; solid phase extraction device; rotary evaporator; vortex mixer, etc.

[0021] II. Solution Preparation McIlvaine buffer solution (pH=7.0): Mix 0.2 mol / L Na2HPO4 solution and 0.1 mol / L citric acid solution in a certain proportion; Lipase solution (1500 U / mL): Prepare fresh using the McIlvaine buffer solution at pH 7.0 as described above; Sodium bromide solution (70%, w / v).

[0022] Series of standard working solutions: Dilute each standard stock solution stepwise with a suitable solvent to prepare a series of standard working solutions containing different concentrations of 3-MCPDE and 2-MCPDE (calculated as alcohol) and a fixed concentration of isotope internal standard (e.g., 10 ng / mL).

[0023] Sample pretreatment steps (see) Figure 1 ) Sample weighing and spiking: Take 0.1 mL of Antarctic krill oil sample into a 15 mL plastic centrifuge tube, add appropriate amounts of 3-MCPDE-d5 and 2-MCPDE-d5 isotope internal standard working solutions (200 μL each, 1 μg / mL), and vortex to mix. (2) Enzymatic hydrolysis: Add 15 μL of Tween 80 and 3 mL of freshly prepared lipase solution (1500 U / mL) to the sample, vortex mix, and place in a constant temperature shaker at 37℃ for 8 h of shaking reaction. (3) Termination of reaction and purification: After the reaction is completed, add 1 mL of 70% NaBr solution, vortex to mix, heat in an 80℃ water bath for 10 minutes to inactivate the enzyme, then cool in an ice bath, centrifuge at 6000 rpm for 10 minutes, take the lower layer of clear liquid, pass it through a PPR column pre-activated with 4 mL of water, and collect the filtrate. (4) Extraction and concentration: Take 2 mL of filtrate and extract twice with 10 mL of ethyl acetate, shaking for 15 minutes each time. Combine the ethyl acetate layers and concentrate them to about 1 mL by rotary evaporation at 30 °C. Transfer the concentrate to a glass test tube. (5) Dehydration: Add about 200 mg of activated 3A molecular sieve to the test tube, vortex until no bubbles are generated, and remove water completely; (6) Derivatization: After removing the molecular sieve, add 8 mg of quinalyl chloride and 8 mg of DMAP to the test tube in sequence. After each addition of a reagent, vortex and mix well. React at room temperature in the dark for 30 minutes. After the reaction, blow dry with mild nitrogen gas. (7) Reconstitution: Dissolve the residue in 1 mL of acetonitrile, filter through a 0.22 μm organic phase membrane, transfer to a sample vial for LC-MS / MS analysis. The LC-MS / MS analysis conditions are as follows: Chromatographic analysis conditions: EclipsePlus C18 RRHD (3.0 mm × 150 mm, 1.8 µm); flow rate: 0.5 mL / min; column temperature: 35 ℃; injection volume: 4 µL; mobile phase A was water (containing 0.2% formic acid and 5 mM ammonium acetate), mobile phase B was 0.2% formic acid in methanol; elution gradient: 0–1.9 min, 55% B; 1.9–2 min, 55%–75% B; 2–4.5 min, 75% B; 4.5–5 min, 75%–100% B; 5–6.8 min, 100% B; 6.8–7 min, 100%–55% B; 7–8 min, 55% B.

[0024] Mass spectrometry analysis conditions: HESI ion source, spray voltage: 3200V (+), 2800V (-); sheath gas: 50 arb; auxiliary gas: 15 arb; purge gas: 1 arb; capillary temperature: 350℃; auxiliary gas heating temperature: 400℃; mass spectrometry data acquisition mode: PRM scan.

[0025] Monitoring ions (m / z): Qualitative ions, quantitative ions, and retention times are shown in Table 1. Table 1 Monitoring Information for 3 / 2-MCPD and 3 / 2-MCPD-d5 Compound Name Retention time (min) Parent ion (m / z) Daughter ions (m / z) 3-MCPD 7.27 421.09496 <![CDATA[248.04711 a 146.06010]]> 2-MCPD 7.15 421.09496 <![CDATA[248.04711 a 146.06010]]> 3-MCPD-d5 7.26 426.12634 <![CDATA[253.07886 a ]]> 2-MCPD-d5 7.12 426.12634 <![CDATA[253.07886 a ]]> a This indicates that the ion is used for the quantification of analytes.

[0026] Qualitative and quantitative analysis: Qualitative analysis: The sample solution is analyzed under the same instrument conditions as the standard stock solution. If the retention time of the chromatographic peak of the test sample corresponds to the retention time of the chromatographic peaks of 3-MCPD and 2-MCPD, and the allowable deviation is less than ±0.05 min, and the relative abundance of the qualitative ions in the sample after background subtraction is compared with the relative abundance of the corresponding qualitative ions in a mixed standard working solution with similar concentrations, and the deviation is within the range specified in Table 2, then the corresponding 3-MCPD and 2-MCPD can be determined to be present in the sample.

[0027] Table 2 Maximum permissible deviation of relative ion abundance during qualitative analysis Relative ion abundance ＞50% ＞20-50% ＞10-20% ≤10% Maximum allowable deviation ±20% ±25% ±30% ±50% Quantitative analysis: The internal standard method was used. A standard curve was plotted against the concentration (x) by the peak area ratio (y) of the target analyte to the corresponding internal standard. The peak area ratio of the target analyte to the internal standard in the sample was substituted into the standard curve to calculate the content of 3-MCPDE and 2-MCPDE in the sample (calculated as 3-MCPD or 2-MCPD).

[0028] Example 2 Experiment on optimization of pretreatment conditions Key pretreatment parameters were optimized through single-factor experiments, and the results are as follows: Figure 8-12 As shown.

[0029] Enzyme concentration optimization ( Figure 8 The response of the target compound at different lipase concentrations was compared. The response reached a plateau at 1500 U / mL, so 1500 U / mL was selected as the optimal concentration.

[0030] pH optimization Figure 9 The effects of different pH (5.0-8.0) McIlvaine buffer solutions were investigated. The highest response of the target analyte was observed at pH 7.0, indicating that the lipase activity was optimal at this pH.

[0031] Temperature optimization ( Figure 10 The effects of different enzymatic hydrolysis temperatures (4-60℃) were investigated. The target analyte showed the strongest response signal at 37℃, which was determined to be the optimal temperature.

[0032] Time optimization ( Figure 11 The effects of different enzymatic hydrolysis times (0.5-16 h) were investigated. The response reached a stable state after 8 hours of reaction, so 8 hours was selected as the optimal enzymatic hydrolysis time. Tween 80 dosage optimization ( Figure 12 The effect of Tween 80 addition (0-1.5%, v / v) was investigated. Adding 0.5% Tween 80 resulted in a stable increase in the target analyte response, which is beneficial for oil phase dispersion; therefore, 0.5% was selected as the optimal addition amount.

[0033] Example 3 The method established in Implementation Case 1 was validated.

[0034] Linear range and detection limit, as follows Figure 13-14 As shown; The accuracy and precision are shown in Table 3-5 below: Table 3. Inter-batch spiked recoveries of 2-MCPDE and 3-MCPDE in Antarctic krill oil (n=6)

[0035] Table 4. Detection results of 3-MCPDE and 2-MCPDE in actual krill oil samples (n=3) Sample number 3-MCPDE content (μg / kg) 2-MCPDE content (μg / kg) K1 11.9 119.8 K2 ND ND K3 ND ND K4 489.1 225.6 K5 ND ND K6 127 174.8 K7 ND 25.8 K8 ND 6.1 K9 73.9 126.9 K10 ND ND Table 5. Detection results of 3-MCPDE and 2-MCPDE in actual fish oil samples (n=3) Sample number 3-MCPDE content (μg / kg) 2-MCPDE content (μg / kg) F1 152.2 188.5 F2 ND ND F3 153.0 211 F4 ND 23.8 F5 1746.1 199.6 F6 590.3 88.9 F7 80.3 53.0 F8 18.4 72.2 F9 73.8 13.7 F10 18.7 46.3 Note: ND indicates that the detection limit is below the detection limit and the substance was not detected.

[0036] In summary, this invention provides a method for the detection of chloropropanol esters in oils and fats based on lipase hydrolysis-quinalyl chloride derivatization-LC-MS / MS. This method features mild pretreatment, high specificity, and can effectively separate and accurately quantify 3-MCPDE and 2-MCPDE. It also exhibits high sensitivity and good repeatability, providing an excellent technical means for the safety and quality control of oil and fat products.

[0037] The various embodiments described in this specification are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting 3-chloro-1,2-propanediol ester and 2-chloro-1,3-propanediol ester in aquatic oils by liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: S1. Sample enzymatic hydrolysis: Add a buffer solution containing surfactant and lipase to the oil sample and carry out an isothermal enzymatic hydrolysis reaction to hydrolyze the bound 3-MCPDE and 2-MCPDE into free 3-MCPD and 2-MCPD; after the reaction is completed, heat treatment is performed to inactivate the enzyme. S2. Sample purification and extraction: After centrifuging the reaction solution obtained in step S1, the supernatant is purified by passing it through a solid phase extraction column, the eluent is collected, and liquid-liquid extraction is performed with an organic solvent. The organic phases are combined and concentrated. S3. Dehydration and derivatization: A dehydrating agent is added to the organic phase concentrated in step S2 to remove water, followed by the addition of quinalyl chloride and a catalyst to carry out the derivatization reaction; S4. LC-MS / MS detection: The product derivatized in step S3 was analyzed by liquid chromatography-tandem mass spectrometry, and qualitative and quantitative analysis was performed based on retention time and characteristic ion pairs.

2. The detection method according to claim 1, characterized in that, The lipase mentioned in step S1 is porcine pancreatic lipase.

3. The detection method according to claim 1, characterized in that, The buffer solution mentioned in step S1 is a McIlvaine buffer solution with a pH of 6.5-7.

5.

4. The detection method according to claim 1, characterized in that, The concentration of lipase in step S1 is 1000-2000 U / mL; the isothermal enzymatic hydrolysis temperature is 35-40℃, and the time is 6-10 h.

5. The detection method according to claim 1, characterized in that, The surfactant mentioned in step S1 is Tween 80, and its final volume concentration in the reaction system is 0.1%-1.0%.

6. The detection method according to claim 1, characterized in that, In step S2, the solid-phase extraction column is a dephospholipid PPR column; the organic solvent is ethyl acetate.

7. The detection method according to claim 1, characterized in that, The dehydrating agent in step S3 is 3A molecular sieve; the catalyst is 4-dimethylaminopyridine.

8. The detection method according to claim 1, characterized in that, In step S4, liquid chromatography uses a C18 column, with an aqueous solution containing formic acid and ammonium acetate as mobile phase A and a methanol solution containing formic acid as mobile phase B for gradient elution; mass spectrometry uses electrospray positive ion mode and multiple reaction monitoring scanning.

9. The detection method according to any one of claims 1-8, characterized in that, During the processing in step S1, isotopic internal standards of 3-MCPD-d5 and 2-MCPD-d5 are added.

10. The detection method according to any one of claims 1-8, characterized in that, The oil samples were Antarctic krill oil and fish oil.

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