CN114910581A - Method for rapidly determining chloropropanol ester content in milk powder by liquid chromatography-high resolution mass spectrometry - Google Patents
Method for rapidly determining chloropropanol ester content in milk powder by liquid chromatography-high resolution mass spectrometry Download PDFInfo
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- chloropropanol
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- 239000000843 powder Substances 0.000 title claims abstract description 88
- -1 chloropropanol ester Chemical class 0.000 title claims abstract description 87
- 235000013336 milk Nutrition 0.000 title claims abstract description 56
- 239000008267 milk Substances 0.000 title claims abstract description 56
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- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000013051 Liquid chromatography–high-resolution mass spectrometry Methods 0.000 title claims abstract description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 211
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 111
- 238000000605 extraction Methods 0.000 claims abstract description 69
- RZWHKKIXMPLQEM-UHFFFAOYSA-N 1-chloropropan-1-ol Chemical compound CCC(O)Cl RZWHKKIXMPLQEM-UHFFFAOYSA-N 0.000 claims abstract description 43
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims abstract description 23
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- KYVUJPJYTYQNGJ-UHFFFAOYSA-N oxiran-2-ylmethyl hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC1CO1 KYVUJPJYTYQNGJ-UHFFFAOYSA-N 0.000 description 7
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- 206010007269 Carcinogenicity Diseases 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a method for rapidly determining the content of chloropropanol ester in milk powder by liquid chromatography-high resolution mass spectrometry, wherein the chloropropanol ester comprises two glycidyl esters, two chloropropanol monoesters and three chloropropanol diesters; the method adopts a two-stage extraction method, wherein a first stage adopts methanol oscillation extraction and C18 solid phase extraction column purification to complete extraction and purification of glycidyl ester and partial chloropropanol monoester, a second stage adopts mixed solvent extraction with a volume ratio of dichloromethane to methanol of 8:2 to purify neutral alumina powder, extraction and purification of chloropropanol diester can be completed while grease interference is removed, the first and second extraction solutions are combined and detected by a high performance liquid chromatography-high resolution mass spectrometer, and high-throughput screening of chloropropanol ester in milk powder can be realized by adopting an external standard method for quantification, the detection limit of the method is 0.02mg/kg, the quantification limit is 0.05mg/kg, the recovery rate is more than or equal to 80.6%, and the standard deviation is less than 10%.
Description
Technical Field
The invention relates to a method for rapidly determining the content of chloropropanol ester in milk powder by liquid chromatography-high resolution mass spectrometry.
Background
Chloropropanol ester is a novel pollutant discovered in recent years and exists in infant formula milk powder, and chloropropanol which is a hydrolysis product of chloropropanol ester is proved to have carcinogenicity, genetic toxicity, renal toxicity and the like. At present, the detection object of chloropropanol ester in food is mainly concentrated in edible oil, and common pretreatment technologies comprise: the method mainly aims to effectively remove the interference of nonpolar compounds such as high-content grease-triglyceride and the like in the edible oil on the premise of ensuring the high extraction rate of chloropropanol ester compounds, and the compounds can generate strong ionization inhibition effect in subsequent mass spectrometry, so that the measurement result of the low-content chloropropanol ester is deviated or cannot be detected.
However, at present, the research on chloropropanol ester in infant formula milk powder is not deep enough, and especially, an extraction and purification method for a target compound in a complex milk powder matrix is not reported yet. The milk powder has various matrixes and complex components, and besides 20 percent of grease, other added nutritional compounds are extremely rich, and besides, some toxic and harmful compounds exist in the milk powder, including: microorganism, mycotoxin-aflatoxin and residual pesticide and animal medicine in milk powder matrix. Therefore, the establishment of a comprehensive and efficient pretreatment method is an important precondition for ensuring the acquisition of comprehensive chloropropanol ester structure information. How to realize the extraction and purification of chloropropanol ester in infant milk powder through one-time pretreatment is a great challenge.
Disclosure of Invention
The invention aims to provide a method for rapidly determining the content of chloropropanol ester in milk powder by utilizing liquid chromatography-high resolution mass spectrometry.
The technical scheme adopted by the invention is as follows:
the method for rapidly determining the content of chloropropanol ester in milk powder by liquid chromatography-high resolution mass spectrometry comprises the following steps of (1) quickly determining the content of chloropropanol ester in milk powder, wherein the chloropropanol ester is one or more of glycidol-palmitate, glycidol-stearate, 3-chloropropanol-2-stearate monoester, 3-chloropropanol-1-palmitate monoester, 3-chloropropanol-1, 2-palmitate diester, 2-chloropropanol-1, 3-stearate diester and 3-chloropropanol-1-palmitate-2-stearate diester; the method comprises the following steps:
(A) sample pretreatment
(1) Extracting a fat sample from a milk powder sample, extracting the fat sample with methanol, and centrifuging to obtain an organic phase A and residue A;
(2) purifying the organic phase A obtained in the step (1) by using a C18 solid phase extraction column, eluting by using methanol, eluting by using a mixed solvent of dichloromethane and methanol with the volume ratio of 8:2 as an eluent, blowing nitrogen to concentrate the obtained eluent to be nearly dry, and adding a methanol solution for redissolving;
(3) adding the residue A obtained in the step (1) into a mixed solvent of dichloromethane and methanol in a volume ratio of 8:2 for secondary extraction, and centrifuging to obtain an organic phase B and a residue B;
(4) taking the organic phase B in the step (3), carrying out nitrogen blowing concentration to be nearly dry, adding a methanol solution for redissolving, adding neutral alumina powder, fully shaking and then centrifuging to obtain supernatant C and precipitate C;
(5) combining the methanol solution in the step (2) and the supernatant C in the step (4), carrying out nitrogen-blown concentration, fixing the volume of methanol to 2.0mL, and filtering with a filter membrane to obtain a test solution;
(B) high performance liquid chromatography-high resolution mass spectrometry detection
And detecting the test solution by using a high performance liquid chromatography-high resolution mass spectrometer, and quantifying by adopting an external standard method to obtain the content of the 7 chloropropanol esters in the milk powder.
In the step (1), the fat sample is extracted from the milk powder sample according to the steps 17.1.2, 17.2 and 17.3 in the third method of GB 5009.6-2016 (determination of fat in food).
Further, in the step (1), the volume consumption of the methanol is 8-20 mL/g, preferably 10mL/g, based on the mass of the fat sample.
In the step (1), the extraction is generally performed by shaking for 5-10 min and ultrasonic extraction for 20-30 min.
In the step (1), centrifugation is generally carried out at a rotating speed of 12000-15000 r/min.
In the step (2), the C18 solid phase extraction column is activated by methanol and water in sequence before use.
In step (2), the organic phase A is usually equilibrated for 5min after loading.
In said step (2), the volume of the eluent is generally 2 column volumes.
In the step (2), the volume consumption of the methanol solution for re-dissolution is 1-3 mL/g, preferably 2mL/g, based on the mass of the fat sample.
In the step (3), the volume usage amount of the mixed solvent of dichloromethane and methanol in a volume ratio of 8:2 is 8-20 mL/g, preferably 10mL/g, based on the mass of the fat sample.
In the step (3), the secondary extraction is generally performed by oscillating for 5-10 min and ultrasonic extracting for 20-30 min.
In the step (3), centrifugation is generally carried out at a rotating speed of 12000-15000 r/min.
In the step (4), the volume of the methanol solution for re-dissolution is 1-3 mL/g, preferably 2mL/g, based on the mass of the fat sample.
In the step (4), the mass ratio of the dosage of the neutral alumina powder to the fat sample is 0.5-1: 1, preferably 0.5: 1.
The neutral alumina powder particle size is preferably 100-200 mesh.
In the step (5), the filter membrane is a 0.22 μm filter membrane.
Further, the sample pretreatment in the step (1) is preferably performed by the following steps:
(1) extracting a fat sample from 5.0g of milk powder sample, adding 10mL of methanol solution into the obtained fat sample, shaking for 5min, ultrasonically extracting for 20min, and centrifuging at a high speed of 12000r/min to obtain an organic phase A and residue A; (2) purifying the upper organic phase A by using a C18 solid phase extraction column, balancing for 5min, leaching with methanol, eluting with 12mL of mixed solvent of dichloromethane and methanol at a volume ratio of 8:2, concentrating the obtained eluent by nitrogen blow to near dryness, and adding 2.0mL of methanol solution for redissolving; (3) adding 10mL of mixed solvent of dichloromethane and methanol with the volume ratio of 8:2 into the residue A for secondary extraction, shaking for 5min, ultrasonically extracting for 20min, and centrifuging at high speed of 12000r/min to obtain an organic phase B and a residue B; (4) taking the upper organic phase B, carrying out nitrogen blowing concentration to be nearly dry, adding 2.0mL of methanol solution for redissolving, adding 0.5g of neutral alumina powder for full oscillation, and carrying out high-speed centrifugation at 12000r/min to obtain supernatant C and precipitate C; (5) and (3) combining the 2.0mL methanol solution in the step (2) and the supernatant C in the step (4), carrying out nitrogen blowing concentration, fixing the volume of the methanol to 2.0mL, and filtering through a 0.22 mu m filter membrane to obtain a sample solution to be analyzed.
Further, a high-resolution mass spectrometer is adopted in the step (B) through a high performance liquid chromatography-quadrupole electrostatic field orbitrap.
Further, in the step (B), the conditions of the high performance liquid chromatography are as follows:
c18 column, mobile phase a: 0.02mM sodium acetate in methanol-water solution, wherein the volume ratio of methanol to water is 9: 1; mobile phase B: 0.02mM sodium acetate in isopropanol-methanol, wherein the volume ratio of isopropanol to methanol is 1: 1; gradient elution.
Further, the flow rate of the liquid chromatography was 0.3mL/min, and the amount of the sample was 5.0. mu.L.
Further, the gradient elution procedure of the liquid chromatography is shown in Table 1
TABLE 1 gradient elution procedure
In the step (B), the conditions of the high-resolution mass spectrum are as follows:
mass spectrum conditions: full scan measurements were performed in positive/negative ion switching mode, mass range: m/z 100-1000, negative ion mode 2800V, positive ion mode 3500V, ion transmission tube temperature 320 ℃, sheath gas (N) 2 )35L/h, auxiliary gas (N) 2 )15L/h, and the temperature of a gasification chamber is 380 ℃; the In-source cracking voltage (In-source CID) is 40eV, and the maximum injection time is 250 ms; two stage adoption auto-trigger mode (dd-MS) 2 ) The relative collision energy range is 10-40%.
In the step (B), the operation steps of external standard method quantification are as follows: detecting the test solution by using a liquid chromatography-high resolution mass spectrometer to obtain an extracted ion current chromatogram of the test solution, comparing the peak area of each component to be detected of the test solution with the standard curve of the corresponding standard substance, calculating to obtain the concentration of each component to be detected in the test solution, correspondingly converting to obtain the content of each component to be detected in the fat sample, and then converting to obtain the content of each component to be detected in the milk powder sample.
The standard curve of the standard substance is obtained according to the following method: preparing standard working solutions with different concentrations from chloropropanol ester, such as glycidol-palmitate, glycidol-stearate, 3-chloropropanol-2-stearate monoester, 3-chloropropanol-1-palmitate monoester, 3-chloropropanol-1, 2-palmitate diester, 2-chloropropanol-1, 3-stearate diester and 3-chloropropanol-1-palmitic acid-2-stearate diester, detecting by using a liquid chromatography-high resolution mass spectrometer under the same condition according to the step (B), and drawing a standard curve according to the concentration of the standard and the peak area of an extraction ion chromatogram of the corresponding standard.
The pretreatment of the invention is two-stage extraction and purification, the first stage adopts methanol oscillation extraction and C18 solid phase extraction column purification, the extraction and purification work of glycidyl ester and partial chloropropanol monoester can be completed, the second stage adopts methanol extraction, dichloromethane auxiliary extraction, neutral alumina powder purification, the extraction and purification of chloropropanol diester can be completed while grease interference is removed, by combining the first and second extraction solutions, the extraction rate of polar and medium polarity glycidyl ester (glycidyl-palmitate, glycidyl-stearate) and chloropropanol monoester (3-chloropropanol-2-stearate and 3-chloropropanol-1-palmitate) and low polarity chloropropanol diester can be ensured simultaneously, and the excessive extraction of other fat components can be avoided at the same time.
According to the method, the secondary extracting solution is qualitatively and quantitatively determined by a high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS) technology, the high-throughput screening of the chloropropanol ester in the milk powder can be realized, the detection limit of the method is 0.02mg/kg, the quantitative limit is 0.05mg/kg, the recovery rate is more than or equal to 80.6%, the standard deviation is lower than 10%, and the method has strong applicability to the milk powder matrix with high protein and high fat. The invention provides a high-flux screening method for chloropropanol ester in milk powder for the first time, and the method is convenient for monitoring the content of chloropropanol ester in milk powder.
Drawings
FIG. 17 shows a structural formula of a chloropropanol ester standard substance, wherein in FIG. 1, A is a structural formula of glycidyl ester; b is chloropropanol monoester structural formula; c is structural formula of chloropropanol diester.
Fig. 27 is an extracted ion chromatogram of chloropropanol ester standard and 3 isotope internal standards thereof, wherein in fig. 2, a: glycidol-palmitate, b: glycidyl-stearate, c: 3-chloropropanol-1-palmitic acid monoester, d: 3-chloropropanol-2-stearic acid monoester, e: 3-chloropropanol-1, 2-palmitic acid diester, f: -chloropropanol-1-palmitic acid-2-stearic acid diester, g: 2-chloropropanol-1, 3-stearic acid diester, h: d5-glycidol-palmitate, i: d5-3-chloropropanol-2-stearic acid monoester, j: d5-3-chloropropanol-1, 2-palmitic acid diester.
FIG. 3 shows the normalized recovery of 7 representative chloropropanol esters from fat samples of two milk powders at different ratios of dichloromethane, wherein in FIG. 3, graph A is an infant formula and graph B is a formula for special medical applications.
FIG. 47 elution profiles of representative chloropropanol esters and palmitic acid triglycerides at different elution volumes.
Fig. 5 shows the oil removal rate of neutral alumina powder to 5.0g of two milk powders and the recovery rate curve (n is 3%, percent) of 7 representative chloropropanol esters after being purified by neutral alumina powder, in fig. 5, curve a is the oil removal rate of infant formula milk powder, and curve B is the oil removal rate of formula milk powder for special medical use.
Detailed Description
The technical solution of the present invention is further described with specific examples, but the scope of the present invention is not limited thereto.
Example 1
Instruments and reagents
Q-active quadrupole electrostatic field orbitrap high resolution mass spectrometer (Semmer Feishel science ThermoFisher Scientific Co.) was equipped with a source of H-ESI II. The liquid chromatography system is an UltiMate3000 high pressure liquid chromatography band autosampler. Milli-Q high purity water generator (Millipore, USA), refrigerated centrifuge (SIGMA, Germany), vortex shaker (Heldolph, Germany), filter membrane (DIKMA, PTFE 0.22 μm), C18 solid phase extraction column (WATERS, USA, 150mg/6 cc).
The chloropropanol ester standard is purchased from Toronto Research Chemicals, Canada and Tsukashima Foods industries, Japan, and has a purity of 95% or more. The method comprises the following steps: (A) glycidyl ester: 1 glycidyl-palmitate (glycol palmitate), 2 glycidyl-stearate (glycol stearate); (B) chloropropanol monoester: 33-chloropropanol-2-stearic acid monoester (2-stearate-3-chloropropanediol) and 43-chloropropanol-1-palmitic acid monoester (1-palmitat-3-chloropropanediol); (C) chloropropanol diester: 53-chloropropanol-1, 2-palmitic acid diester (1, 2-bis-palmitat-3-chloropropionic), 62-chloropropanol-1, 3-stearic acid diester (1, 3-bis-stearate-2-chloropropionic) and 73-chloropropanol-1-palmitic acid-2-stearic acid diester (1-palmitat-2-stearate-3-chloropropionic), and corresponding isotopic internal standards D5-3-chloropropanol-1, 2-palmitate (D5-1, 2-bis-palmitate-3-chloropropionate), D5-3-chloropropanol-2-stearate monoester (D5-2-stearate-3-chloropropionate), and D5-Glycidol-palmitate (D5-glycol palmitate). The standard substance has a structural formula shown in figure 1.
Weighing the above appropriate amount of standard substances, dissolving with dichloromethane, diluting with methanol (containing 0.02mM sodium acetate) to constant volume, and making into 1.0mg/mL standard stock solution, and storing at-20 deg.C in dark place. Sodium acetate (chromatographically pure, Sigma-Aldrich, USA), methanol, dichloromethane and acetonitrile, etc., all were chromatographically pure, purchased from Merck, Germany, and the experimental water was Milli-Q ultrapure water (18.2. omega. M.cm).
Chromatographic and mass spectral conditions
Liquid phase conditions: syncronis C18(2.1mm x 150mm,1.7 μm) was the stationary phase, mobile phase a: methanol-water (9:1, V: V, containing 0.02mM sodium acetate), mobile phase B: isopropanol-methanol (1:1, V: V, containing 0.02mM sodium acetate) solution at a flow rate of 0.3mL/min, sample size of 5.0. mu.L. The gradient is shown in table 1.
TABLE 1 ESI + /ESI - Liquid chromatography elution gradient in mode
Mass spectrum conditions: the mass spectrum is subjected to full-scan measurement in a positive/negative ion conversion mode, and the mass range is as follows: m/z 100-1000 (cycle duration 256ms), resolution 70,000(m/z 200), and Automatic Gain Control (AGC) target value 5e 5 (ii) a 2800V in negative ion mode, 3500V in positive ion mode, 320 ℃ in ion transmission tube temperature, and sheath gas (N) 2 )35L/h, auxiliary gas (N) 2 )15L/h, and the temperature of a gasification chamber is 380 ℃; MS collection time is 0.0-20.0min, source cracking voltage (In-source CID) is 40eV, maximum injection time is 250MS, and positive and negative ion correction is respectively carried out on the instrument before a sample runs. Two stage adoption auto-trigger mode (dd-MS) 2 ) Resolution 35,000FWHM (m/z 200), Automatic Gain Control (AGC) target 5.0e 5 Relative collision energy ranges from 10-40%, retention time windows are based on target RT + -1.0 min. The extracted ion chromatogram of 7 mixed chloropropanol esters and 3 internal isotope standards thereof is shown in figure 2. In fig. 2, a: glycidol-palmitate, b: glycidyl-stearate, c: 3-chloropropanol-1-palmitic acid monoester, d: 3-chloropropanol-2-stearic acid monoester, e: 3-chloropropanol-1, 2-palmitic acid diester, f: -chloropropanol-1-palmitic acid-2-stearic acid diester, g: 2-chloropropanol-1, 3-stearic acid diester, h: d5-glycidol-palmitate, i: d5-3-chloropropanol-2-stearic acid monoester, j: d5-3-chloropropanol-1, 2-palmitic acid diester.
Sample pretreatment process
The milk powder substrate comprises: infant formula milk powder, modified milk powder and formula milk powder for special medical purposes comprise a full-nutrition formula, an amino acid formula, a deep hydrolysis formula and the like, and the brands cover common milk powder production companies at home and abroad. Referring to step 17.1.2, step 17.2 and step 17.3 in method III of GB 5009.6-2016 fat determination in food, 5.0g of milk powder sample is taken, about 1g of extracted fat is accurately weighed, then an internal standard is added into the extracted fat sample, and pretreatment is carried out.
The pretreatment steps are as follows: (1) adding 10mL of methanol solution into a fat sample extracted from 5.0g of milk powder sample, shaking for 5min, ultrasonically extracting for 20min, and centrifuging at a high speed of 12000r/min to obtain an organic phase A and a residue A; (2) purifying the upper organic phase A with C18 purification column, balancing for 5min, eluting with methanol, eluting with 12mL 80% dichloromethane-methanol (V: V) as eluent, concentrating the eluate by nitrogen blowing, and dissolving with 2.0mL methanol solution; (3) adding 10mL of 80% dichloromethane-methanol solution (V: V) into residue A, extracting for the second time, shaking for 5min, ultrasonically extracting for 20min, and centrifuging at high speed of 12000r/min to obtain organic phase B and residue B; (4) taking the upper organic phase B, carrying out nitrogen blowing concentration to be nearly dry, adding 2.0mL of methanol solution for redissolving, adding 0.5g of neutral alumina powder (100-200 meshes) for full shaking, and carrying out high-speed centrifugation at 12000r/min to obtain supernatant C and precipitate C; (5) and (3) combining the 2.0mL methanol solution in the step (2) and the supernatant C in the step (4), carrying out nitrogen blowing concentration, fixing the volume of the methanol to 2.0mL, and filtering through a 0.22 mu m filter membrane to obtain a sample solution to be analyzed. All experiments were processed in 3 replicates and the experimental data are presented as mean ± SD.
Qualitative and quantitative analysis
And (3) qualitative analysis: exactfinder using high-throughput screening software TM (Thermo Scientific) qualitative analysis of chloropropanoate, requiring accurate mass errors of less than 5 x 10 -6 Meanwhile, the retention time, the isotope distribution, the main secondary fragments and the secondary mass spectrogram identification degree are compared, and comprehensive judgment is carried out to obtain an accurate qualitative result and avoid the occurrence of false positive results.
Quantitative analysis: preparing a mixed standard solution (1.0 mu g/mL) of 7 chloropropanol ester standard substances, and diluting the working solution step by step, wherein the concentration ranges of the working solution are 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 200 ng/mL. And (4) drawing a standard working curve by taking the concentration as an abscissa and the peak area of the standard product as an ordinate, and taking the standard working curve as a basis for quantifying the target object.
Detecting the test solution by using a liquid chromatography-high resolution mass spectrometer under the same condition to obtain an extracted ion current chromatogram of the test solution, comparing the peak area of each component to be detected of the test solution with the standard curve of the corresponding standard substance, calculating to obtain the concentration of each component to be detected in the test solution, correspondingly converting to obtain the content of each component to be detected in the fat sample to be detected, and converting to obtain the content of each component to be detected in the milk powder sample.
Results and discussion
Selection of extraction solvent
Currently, regarding the extraction of chloropropanol ester compounds in edible oil, a single organic reagent of n-hexane, diethyl ether, dichloromethane, acetonitrile and methanol or a binary and ternary mixed solution is mostly adopted as an extraction solvent. In the experiment, acetonitrile, methanol, normal hexane, ether and dichloromethane are respectively selected as extraction solvents to extract a fat sample extracted from a milk powder sample, 5.0g of the fat sample extracted from the milk powder sample is extracted, the dosage of the extraction solvent is 10mL, and the extraction is carried out once. The fat sample was obtained as follows: through preliminary experiments, negative infant formula milk powder and formula milk powder with special medical application are selected as blank matrixes, and different representative chloropropanol ester-glycidyl ester (glycidol-palmitate, glycidol-stearate) are added into extracted fat by referring to a method of GB 5009.6-2016 (determination of fat in food); chloropropanol monoesters (3-chloropropanol-2-stearic acid monoester and 3-chloropropanol-1-palmitic acid monoester); and chloropropanol diesters (3-chloropropanol-1, 2-palmitic acid diester, 2-chloropropanol-1, 3-stearic acid diester, and 3-chloropropanol-1-palmitic acid-2-stearic acid diester) as internal standards, and the recovery rates were calculated by examining peak area values, as methodological verification was performed for the extraction solvent.
The recovery rates of 7 representative chloropropanol ester compounds in infant formula milk powder and formula milk powder for special medical application under different extraction solvents are shown in Table 2, and the results show that acetonitrile and methanol are adopted as the extraction solvents, the peak shape is good, the interference is less, the extraction effect on partial and medium polarity glycidyl ester (glycidyl-palmitate, glycidyl-stearate) and chloropropanol monoester (3-chloropropanol-2-stearate monoester and 3-chloropropanol-1-palmitate monoester) is good, but the extraction effect on low polarity chloropropanol diester (3-chloropropanol-1, 2-palmitate diester, 2-chloropropanol-1, 3-stearate diester and 3-chloropropanol-1-palmitate-2-stearate diester) is not ideal. The n-hexane, the ether and the dichloromethane are used as extraction solvents, and the extraction effect of the low-polarity chloropropanol diester is improved, and the extraction effect of other compounds is not the same as that of acetonitrile and methanol extract. In addition, by measuring the residual oil values in the n-hexane, ether and dichloromethane extract, it can be known that the interference of n-hexane and ether is serious compared with dichloromethane oil. Therefore, in order to simultaneously meet the extraction requirements of the common 3 types of chloropropanol ester compounds with different physicochemical properties, the invention adopts a graded extraction and purification system. Firstly, a methanol solution with good solubility and stronger permeability is selected as a first-stage extraction solvent, so that the extraction efficiency of partial polarity and medium polarity glycidyl ester and chloropropanol monoester is ensured, and the effect of reducing interference of precipitated protein is achieved; then, considering that dichloromethane is used as an extraction solvent, oil interference is small, and mutual solubility with methanol is strong, dichloromethane is used as an auxiliary extraction solvent in the second stage, and a proper amount of methanol is added, so that the extraction rate of low-polarity chloropropanol diester is ensured, and excessive extraction of oil components such as triglyceride and diglyceride caused by direct extraction of dichloromethane can be avoided. Therefore, it was finally confirmed that the extraction solvent was a methanol solution and a methanol-dichloromethane solution in this order. The two groups of solvents have wide polar coverage range, and completely meet the extraction requirements of three types of chloropropanol ester compounds.
Table 2 recovery of 7 representative chloropropanol ester compounds in infant formula and formula for special medical use under different extraction solutions (n ═ 6%)
P is palmitic acid, S is stearic acid, MCPD is chloropropanol;
a is infant formula milk powder, and B is formula milk powder for special medical purposes.
Methylene chloride ratio optimization
The dichloromethane is used as a secondary extraction solvent, and the proportion of the dichloromethane has great influence on the extraction efficiency of the chloropropanol ester compounds, especially the low-polarity chloropropanol diester compounds. In the experiment, the conditions of the standard recovery rates of the 7 representative chloropropanol esters when the volume of dichloromethane in a mixed solvent of dichloromethane and methanol accounts for 30%, 50%, 80% and 100% during the secondary extraction are respectively considered. The standard recovery of 7 representative chloropropanol esters at different dichloromethane ratios is shown in fig. 3, where a is a formula for infants and B is a formula for special medical applications, (n ═ 6,%).
The results in fig. 3 show that polar glycidyl ester (glycidyl-palmitate, glycidyl-stearate) and chloropropanol monoester with medium polarity (3-chloropropanol-2-stearate monoester and 3-chloropropanol-1-palmitate monoester) mainly exist in the methanol extract, are not greatly influenced by the ratio of dichloromethane, and the recovery rate of the compound is stably maintained above 70%. However, the recovery rate of the low-polarity chloropropanol diester (3-chloropropanol-1, 2-palmitic acid diester, 2-chloropropanol-1, 3-stearic acid diester and 3-chloropropanol-1-palmitic acid-2-stearic acid diester) is greatly influenced by the ratio of dichloromethane, and when the dichloromethane accounts for 30% and 50%, the extraction efficiency is poor, and the recovery rate is less than 50%; when the dichloromethane accounts for 80% and 100%, the extraction rate gradually tends to be stable, and the recovery rate values all reach more than 70%. Considering the influence of oil components such as triglyceride in the fat matrix, the ratio of dichloromethane in the secondary extract is finally determined to be 80%. As can be seen from FIG. 3, the recovery rates of 7 representative chloropropanol esters in both the infant formula (FIG. 3A) and the special medical formula (FIG. 3B) were maintained smoothly at above 70%.
Selection of purification conditions
The fat sample is firstly extracted by a first-stage extracting solution-methanol, and extraction target compounds mainly comprise partial-polarity and medium-polarity glycidyl ester (glycidyl-palmitate, glycidyl-stearate) and chloropropanol monoester (3-chloropropanol-2-stearic acid monoester and 3-chloropropanol-1-palmitic acid monoester) and the like, and a small amount of low-polarity chloropropanol diester (3-chloropropanol-1, 2-palmitic acid diester, 2-chloropropanol-1, 3-stearic acid diester and 3-chloropropanol-1-palmitic acid-2-stearic acid diester) and the like. In addition, certain impurity interference including fat-soluble vitamins, mycotoxins and the like exists in the inevitable methanol extraction solution, and the small molecular compounds can be effectively removed by using a C18 solid phase extraction small column. The leaching and elution conditions of the C18 purifying column are optimized in detail in the experiment, and the experimental process is as follows: taking 10 mul (C1.0 mg/mL) of the mixed standard solution of the 7 representative chloropropanol ester compounds, fixing the volume of methanol to 10mL, using a C18 solid phase extraction column (WATERS company, USA, the filling amount is 150mg, the column volume is 6cc, each C18 purification column is activated by methanol and water in sequence before use, balancing for 5min after sample loading, respectively using 12mL of dichloromethane-methanol (V: V) solution with 0%, 10%, 20%, 30%, 50%, 80% and 100%, collecting eluent, blowing nitrogen for concentration, fixing the volume of methanol to 2.0mL, filtering by a filter membrane PTFE, measuring by LC-MS, and determining the recovery rate of the 7 representative chloropropanol ester compounds by the C18 purification column under different elution conditions as shown in Table 3 (n 6%)
The results show that: in 0% dichloromethane-methanol (V: V) eluate, no target compound was detected; in 10 percent, 20 percent and 30 percent of dichloromethane-methanol (V: V) eluent, the glycidyl ester and the chloropropanol monoester are gradually eluted, and the chloropropanol diester with strong hydrophobicity cannot be eluted; the 80% and 100% dichloromethane-methanol (V: V) eluents achieved complete elution of all target compounds, and as shown in table 3, 80% dichloromethane-methanol (V: V) was determined as the eluent from the viewpoint of minimizing interference of the grease compounds. Therefore, according to the above experimental results, the C18 purge conditions were finally determined as: and (3) balancing the methanol extract for 5min after sample loading, leaching by using pure methanol, effectively removing the interference of micromolecular impurities in the milk powder, and finally eluting by using 80% dichloromethane-methanol (V: V) as an eluent.
"-" indicates no detection
The eluent with enough volume can well ensure the recovery rate of the chloropropanol ester compound, 80 percent dichloromethane-methanol (V: V) is used as the eluent, some of the moderately polar glycidyl esters (glycidyl-palmitate, glycidyl-stearate) and chloropropanol monoesters (3-chloropropanol-2-stearate and 3-chloropropanol-1-palmitate) are first eluted from the column by addition of dichloromethane, then, with the stepwise permeation of dichloromethane, a part of chloropropanol diester (3-chloropropanol-1, 2-palmitic acid diester, 2-chloropropanol-1, 3-stearic acid diester and 3-chloropropanol-1-palmitic acid-2-stearic acid diester) with low polarity is finally eluted. Meanwhile, the main oil and fat compound contained in the milk powder, palmitic acid triglyceride (TAG for short) ([ M + Na)] + m/z 805.7) will also co-elute with the target analyte, resulting in a strong ionization suppression effect in subsequent mass spectrometry analysis. Based on this, in the experiment, the effects (in terms of peak area) of the eluent volumes of 0mL, 3.0mL, 6.0mL, 9mL, 12mL, 15mL, 18mL and 21mL on the representative 7 chloropropanol ester compounds (C ═ 1.0ug/mL) and elution of palmitic acid triglyceride (C ═ 1.0ug/mL, in terms of peak area) were examined. The elution profiles of 7 representative chloropropanol esters and palmitic acid triglycerides at different elution volumes are shown in figure 4. The results show that: as can be seen from the elution curves of the 7 representative chloropropanol diesters, compared with triglyceride, the elution of glycidyl ester and chloropropanol monoester is completed firstly, the elution of chloropropanol diester is slightly earlier than that of palmitic acid triglyceride, when the volume of the eluent is 15mL, the elution of the 7 representative chloropropanol esters can be completed, but the elution volume of the C18 purification column under the condition of 80% dichloromethane-methanol (V: V) as the eluent is finally determined to be 12mL from the viewpoints of maximally reducing the interference of the palmitic acid triglyceride as the lipid compound in milk powder and saving nitrogen-blowing concentration time and cost.
The second-stage extraction adopts 80% dichloromethane-methanol (V: V) as the extracting solution, and inevitably leads to the extraction of oil components and impurities while ensuring the extraction rate of low-polarity chloropropanol diesterThe existence of the chloropropanol compound can generate strong ionization inhibition effect and raise the base line level in the subsequent mass spectrometry, thereby greatly reducing or even not detecting the content value of the chloropropanol compound with low content. Studies have shown that neutral alumina powder can adsorb grease well by hydrogen bonding forces, but excessive use can cause loss of target compounds. Based on the method, the addition amount of the neutral alumina powder is optimized in detail in the experiment, and the experimental process is as follows: taking 10 mu L (C is 1.0mg/mL) of the mixed standard solution of the 7 representative chloropropanol ester compounds, after nitrogen is blown to be dry, redissolving a secondary extracting solution (80% dichloromethane-methanol) organic phase B obtained in the step (3) by adopting fat samples of infant formula milk powder and formula milk powder for special medical purposes, and purifying the obtained mixed solution by using neutral alumina powder according to the step (4), wherein the specific steps are as follows: blowing and concentrating the mixed liquid nitrogen to be nearly dry, adding 2.0mL of methanol solution, respectively adding 0.1g, 0.2g, 0.5g, 1.0g, 2.0g and 5.0g of neutral alumina powder, fully shaking, and centrifuging at high speed of 12000r/min to obtain supernatant C and precipitate C; measuring the accurate mass value of the residual oil after the mixed liquid and the supernatant C are fully blown with nitrogen, and calculating the oil removal rate, wherein the oil removal rate (M) is equal to the oil removal rate (%) (M) 0 -M X )/M 0 X 100, wherein M X Refers to the residual oil lipid content, M, after the addition of neutral alumina powder 0 Refers to the residual grease quality value without any treatment. The oil removal rates of the infant formula (a) and the special medical formula (B) and the recovery rates of the 7 representative chloropropanol esters (n is 3%,%) were compared by adding 0.1g, 0.2g, 0.5g, 1.0g, 2.0g and 5.0g of neutral alumina powder, as shown in fig. 5, where the curve a is the oil removal rate of the infant formula and the curve B is the oil removal rate of the special medical formula. When the neutral alumina powder is added by more than or equal to 1.0g, the grease is fully adsorbed, the recovery rate conditions of 7 representative chloropropanol esters are comprehensively considered, and when the addition amount of the neutral alumina powder is 0.5g, the recovery rate of the chloropropanol ester reaches the optimal value (the recovery rate is more than or equal to 90%).
The effect of the two-stage extraction and purification process described above on the recovery of each spiked sample was examined by adding the 7 representative chloropropanol esters described above (0.05mg/kg) to the extracted fat sample. The recovery (n ═ 6,%) of 7 representative chloropropanol esters (0.05mg/kg) in the fat samples of the two milk powders after the two-stage extraction, purification process is shown in table 4, and the results show that: the recovery rate of 7 representative chloropropanol esters is stably maintained to be more than 80 percent (between 82.9 and 92.6 percent), wherein polar glycidyl ester (glycidyl-palmitate, glycidyl-stearate) and chloropropanol monoester with medium polarity (3-chloropropanol-2-stearate and 3-chloropropanol-1-palmitate) mainly exist in a primary extracting solution, and after the primary extracting solution is purified by a C18 small column, the recovery rate is stably maintained to be between 70.6 and 77.7 percent; chloropropanol diester (3-chloropropanol-1, 2-palmitic acid diester, 2-chloropropanol-1, 3-stearic acid diester and 3-chloropropanol-1-palmitic acid-2-stearic acid diester) mainly exist in the secondary extracting solution, and after oil is absorbed by neutral alumina powder, the recovery rate is stably maintained between 71.9% and 73.3%, thereby fully verifying the feasibility of the purification method.
Table 47 recovery rates of representative chloropropanol esters (0.05mg/kg) after two-stage extraction and purification processes (n ═ 6,%)
P is palmitic acid, S is stearic acid, MCPD is chloropropanol;
analysis of spiked recovery results
According to the optimized optimal fractional extraction system, multistage purification extraction and liquid chromatography-mass spectrometry combined technology analysis are carried out on chloropropanol ester in the two milk powders, and the obtained results are shown in table 5. The result shows that the linear relation of the chloropropanol ester is good (r is r) within the linear range of 0.01-0.2 mu g/mL 2 Not less than 0.999), the detection limit is 0.02mg/kg, the quantification limit is 0.05mg/kg, the standard recovery rate is 80.6-94.2%, and the standard deviation is lower than 10%.
TABLE 57 Experimental data on the addition level, linear range and recovery of representative chloropropanol esters in infant and special medical formula
P is palmitic acid, S is stearic acid, MCPD is chloropropanol;
a is infant formula milk powder, and B is formula milk powder for special medical purposes.
The application comprises the following steps according to the polarity range of 3 large chloropropanol ester compounds: glycidyl ester, chloropropanol monoester and chloropropanol diester, and high-protein and high-fat attribute characteristics in a milk powder substrate sample, and a method for extracting and purifying chloropropanol ester compounds in milk powder in a grading manner is established. Firstly, methanol and dichloromethane/methanol (8:2, V: V) are used as extraction solvents, the polar coverage of the two solvents is wide, and the extraction requirement of 3 types of chloropropanol ester compounds is completely met. The first stage adopts methanol oscillation extraction and C18 small column purification, so that effective extraction and separation of glycidyl ester and chloropropanol monoester can be realized; the second stage adopts methanol extraction, dichloromethane auxiliary extraction and neutral alumina powder purification, and can realize the extraction and separation of the dichloropropanol diester while removing grease. Secondly, the high-flux detection of the chloropropanol ester compounds in the milk powder can be realized by combining the first and second extracting solutions. The method has strong compatibility with different milk powder matrixes and compounds with different physicochemical properties, and lays an experimental foundation for enriching the types of chloropropanol esters in milk powder and exploring the toxicology of chloropropanol esters in infant milk powder in the later period.
Claims (10)
1. The method for rapidly determining the content of chloropropanol ester in milk powder by liquid chromatography-high resolution mass spectrometry comprises the following steps of (1) quickly determining the content of chloropropanol ester in milk powder, wherein the chloropropanol ester is one or more of glycidol-palmitate, glycidol-stearate, 3-chloropropanol-2-stearate monoester, 3-chloropropanol-1-palmitate monoester, 3-chloropropanol-1, 2-palmitate diester, 2-chloropropanol-1, 3-stearate diester and 3-chloropropanol-1-palmitate-2-stearate diester; characterized in that the method comprises the following steps:
(A) sample pretreatment
(1) Extracting a fat sample from a milk powder sample, extracting the fat sample with methanol, and centrifuging to obtain an organic phase A and residue A;
(2) purifying the organic phase A obtained in the step (1) by using a C18 solid phase extraction column, eluting by using methanol, eluting by using a mixed solvent of dichloromethane and methanol with the volume ratio of 8:2 as an eluent, blowing nitrogen to concentrate the obtained eluent to be nearly dry, and adding a methanol solution for redissolving;
(3) adding the residue A obtained in the step (1) into a mixed solvent of dichloromethane and methanol in a volume ratio of 8:2 for secondary extraction, and centrifuging to obtain an organic phase B and a residue B;
(4) taking the organic phase B in the step (3), carrying out nitrogen blowing concentration to be nearly dry, adding a methanol solution for redissolving, adding neutral alumina powder, fully shaking and then centrifuging to obtain supernatant C and precipitate C;
(5) combining the methanol solution in the step (2) and the supernatant C in the step (4), carrying out nitrogen-blown concentration, fixing the volume of methanol to 2.0mL, and filtering with a filter membrane to obtain a test solution;
(B) high performance liquid chromatography-high resolution mass spectrometry detection
And detecting the test solution by using a high performance liquid chromatography-high resolution mass spectrometer, and quantifying by adopting an external standard method to obtain the content of the 7 chloropropanol esters in the milk powder.
2. The method of claim 1, wherein in step (2), the volume of eluent is 2 column volumes of the C18 solid phase extraction column.
3. The method according to claim 1, wherein in the step (4), the mass ratio of the amount of the neutral alumina powder to the fat sample is 0.5-1: 1.
4. the method as set forth in claim 1, wherein in the step (4), the neutral alumina powder has a particle size of 100-200 mesh.
5. The method according to claim 1, wherein in the step (B), the conditions of the high performance liquid chromatography are as follows:
c18 column, mobile phase a: 0.02mM sodium acetate in methanol-water solution, wherein the volume ratio of methanol to water is 9: 1; mobile phase B: 0.02mM sodium acetate in isopropanol-methanol, wherein the volume ratio of isopropanol to methanol is 1: 1; and (4) gradient elution.
6. The method of claim 5, wherein in step (B), the gradient elution procedure for liquid chromatography is as follows
Gradient elution procedure
7. The method of claim 1, wherein in step (B), the conditions for high resolution mass spectrometry are:
mass spectrum conditions: full scan measurements were performed in positive/negative ion switching mode, mass range: m/z 100-1000, negative ion mode 2800V, positive ion mode 3500V, ion transmission tube temperature 320 ℃, sheath gas N 2 Flow rate 35L/h, auxiliary gas N 2 The flow is 15L/h, and the temperature of the gasification chamber is 380 ℃; the source cracking voltage is 40eV, and the maximum injection time length is 250 ms; two stage adoption auto-trigger mode (dd-MS) 2 ) The relative collision energy range is 10-40%.
8. The method of claim 1, wherein in step (B), the external standard quantification step is: detecting the test solution by using a liquid chromatography-high resolution mass spectrometer to obtain an extracted ion current chromatogram of the test solution, comparing the peak area of each component to be detected of the test solution with the standard curve of the corresponding standard substance, calculating to obtain the concentration of each component to be detected in the test solution, correspondingly converting to obtain the content of each component to be detected in the fat sample, and then converting to obtain the content of each component to be detected in the milk powder sample.
9. The method according to claim 1, wherein in the step (1), the volume dosage of the methanol is 8-20 mL/g based on the mass of the fat sample; in the step (3), the volume usage amount of the mixed solvent of dichloromethane and methanol in a volume ratio of 8:2 is 8-20 mL/g based on the mass of the fat sample.
10. The method of claim 1, wherein the pre-treating of the sample in step (a) is performed by:
(1) extracting a fat sample from 5.0g of milk powder sample, adding 10mL of methanol solution into the obtained fat sample, shaking for 5min, ultrasonically extracting for 20min, and centrifuging at a high speed of 12000r/min to obtain an organic phase A and residue A; (2) purifying the upper organic phase A by using a C18 solid phase extraction column, balancing for 5min, leaching with methanol, eluting with 12mL of mixed solvent of dichloromethane and methanol at a volume ratio of 8:2, concentrating the obtained eluent by nitrogen blow to near dryness, and adding 2.0mL of methanol solution for redissolving; (3) adding 10mL of mixed solvent of dichloromethane and methanol with the volume ratio of 8:2 into the residue A for secondary extraction, shaking for 5min, ultrasonically extracting for 20min, and centrifuging at high speed of 12000r/min to obtain an organic phase B and a residue B; (4) taking the upper organic phase B, carrying out nitrogen blowing concentration to be nearly dry, adding 2.0mL of methanol solution for redissolving, adding 0.5g of neutral alumina powder for full oscillation, and carrying out high-speed centrifugation at 12000r/min to obtain supernatant C and precipitate C; (5) and (3) combining the 2.0mL methanol solution in the step (2) and the supernatant C in the step (4), carrying out nitrogen blowing concentration, fixing the volume of the methanol to 2.0mL, and filtering through a 0.22 mu m filter membrane to obtain a sample solution to be analyzed.
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