CN104597194B - High performance liquid chromatography-fluorescence detection method for 3-chloro-1,2-propanediol - Google Patents

Abstract

The invention discloses a high-performance liquid chromatography-fluorescence detection method for 3-MCPD. The method comprises the following steps: 1) treating the 3-MCPD aqueous solution with a periodate solution to crack the 3-MCPD into chloroacetaldehyde ; 2) remove excess periodate, eliminate side reactions; 3) fluorescence derivatization reaction: react chloroacetaldehyde with fluorescence derivatization reagent to generate a target substance with fluorescent effect; 4) HPLC-FLD measurement: carry out the target substance The 3-MCPD is separated and quantified according to its chromatographic peak area; the fluorescent derivatization reagent is an o-amino N-heterotwo-membered cyclic compound. In the present invention, a novel fluorescent derivatization reagent that is convenient and easy to obtain, good in selectivity, high in fluorescence efficiency, and strong in hydrophobicity is used in high-performance liquid chromatography-fluorescence detection 3-MCPD, so that the target product can be detected in reversed-phase liquid chromatography. It has a good retention behavior, which is conducive to improving the accuracy and sensitivity of the test method.

Description

High performance liquid chromatography-fluorescence detection method for 3-chloro-1,2-propanediol

technical field

The invention relates to a high performance liquid chromatography-fluorescence detection method for 3-chloro-1,2-propanediol, belonging to the field of analytical chemistry.

Background technique

3-Chloro-1,2-propanediol is also known as 3-chloropropanediol or chloroglycerin, its English name is 3-monochloro-1,2-propanediol, abbreviated as 3-MCPD, its chemical structure formula is: CH ₂ Cl-CHOH-CH ₂ OH. 3-MCPD is a kind of monochlorodihydric alcohol, which is a colorless liquid with a sweet taste, freezing point -40°C, boiling point 213°C (decomposition), relative density 1.3218 (20°C), refractive index 1.4809 (20°C), Soluble in water, ethanol, ether and acetone, slightly soluble in toluene, insoluble in benzene, petroleum ether and carbon tetrachloride; unstable, gradually turns rice yellow after standing, easy to absorb moisture.

Toxicity studies in recent years have shown that 3-MCPD has strong hazards to human health and has become an internationally recognized food chemical pollutant. The oral median lethal dose LD ₅₀ is 150mg/kg.bw. When male rats are continuously orally exposed to a dose of 3-MCPD above 1mg/kg/day, sperm motility and fecundity can be reduced, and the dose is greater than or equal to 10-20mg/kg/day, and the sperm of the rats has a significant morphology ②Genotoxicity: At present, most research institutions and scholars believe that 3-MCPD is a non-genotoxic carcinogen, and the tentative maximum tolerated daily dose (TDI) is 2μg/kg.bw; ③Carcinogenicity: The British Committee on Carcinogenicity (Committeoncarcinoginicity) believes that 3-MCPD can cause cancer in animal experiments; and the British Mutagenicity Committee (CommitteonMutagenicity) believes that 3-MCPD is a non-genotoxic carcinogen in vivo experiments; ④Neurotoxicity: Mice and rats are equally sensitive to the neurotoxic effects of 3-MCPD, especially to symmetrical damage to the brainstem. When the dose of 3-MCPD was greater than 25mg/kg.bw/day, the central nervous system damage of experimental animals showed a significant dose-effect relationship.

The contamination of chloropropanols in food was first found in condiments (such as chicken essence and soy sauce) made of acid hydrolyzed vegetable protein (HVP), and later in malt extract, modified starch, meat extract, Detected in biscuits, bread, sausages, cereals, etc. In 2006, ZelinkováZ. et al. of the Czech Republic first found that chloropropanol fatty acid esters existed in edible oils. In 2007, SeefelderW. et al. found that the content of chloropropanol fatty acid esters in refined oils ranged from 0.2 to 20 mg/kg, and the refined vegetable oils were divided into rapeseed oil, soybean oil, sunflower oil, safflower oil, and walnut. Oil and palm oil are in increasing numerical order.

In the environmental water body, the discharge of industrial wastewater will lead to a certain degree of 3-MCPD pollution, such as the discharge of acid hydrolyzed vegetable protein production wastewater, and the use of wet strength agent-polyamide polyamine epichlorohydrin resin in the pulp production process. An important source of 3-MCPD pollution, if it is not treated, it will lead to pollution of nearby water bodies; in addition, the use of polyamine flocculants in the water treatment process will also produce a certain degree of 3-MCPD pollution.

Because 3-MCPD is seriously harmful to the human body, many countries have established limit standards to control this pollutant in food and drinking water. For example, the European Union formulated limit standards in 2001, stipulating that it should not exceed 20μg/kg.bw per day intake; my country has also established a national standard for the detection of 3-MCPD in soy sauce. The British Drinking Water Inspectorate (DrinkingWaterInspectorate) stipulates that the maximum content of 3-MCPD in drinking water shall not exceed 0.1mg/L. Since the safety limit of 3-MCPD in food and drinking water reaches the ppb level, it is necessary to adopt a highly sensitive detection method to meet the monitoring requirements. At present, the commonly used methods are mainly gas chromatography (GC) and gas chromatography-mass spectrometry (GC- MS). Due to the high polarity and boiling point of 3-MCPD, 3-MCPD needs to be enriched and extracted from the matrix during the determination process, and then derivatized. Commonly used extraction methods include liquid-liquid extraction (LLE), matrix solid-phase dispersion extraction (MSPD), solid-phase microextraction (SPME), etc.; while derivatization methods mainly include ketone derivatives and boric acid derivatives (phenylboronic acid, Butylboronic acid), N,O-bistrimethyl-trifluoroacetamide (BSTFA), anhydride derivatives (HFBA), heptafluorobutyryl imidazole (HFBI), etc. However, due to the good water solubility and high boiling point of 3-MCPD, the analytical method based on gas chromatography for the determination of 3-MCPD has the disadvantages of complex and cumbersome pretreatment process, harsh derivatization conditions, and high instrument maintenance costs. In view of this, it is of great significance to develop a low-cost, easy-to-operate high-sensitivity detection method based on high-performance liquid chromatography. Patent CN102565270 first reported a combination of periodic acid oxidative cleavage, base or nucleoside (nucleotide ) derivatized reversed-phase liquid chromatography-fluorescence detection method, the method has low test cost, simple operation and high sensitivity, and can be applied to the determination of 3-MCPD in foods such as oils and soy sauce. However, due to the use of fluorescence derivatization The reagent is too hydrophilic, and the retention of the target product in reversed-phase liquid chromatography is weak. When measuring complex food samples, it may be easily interfered by the matrix effect, which affects the accuracy and reproducibility of the measurement. Therefore, the development of new fluorescent properties Good, more hydrophobic fluorescent derivatization reagents will be more conducive to the strengths and advantages of this method.

Contents of the invention

The purpose of the present invention is to provide a kind of high-performance liquid chromatography-fluorescence detection method of 3-MCPD, this method is by using a kind of novel fluorescent derivatization reagent that is convenient and easy to get, good selectivity, high fluorescence efficiency, strong hydrophobicity It is used in high-performance liquid chromatography-fluorescence detection 3-MCPD, so that the target product has better retention behavior in reversed-phase liquid chromatography, which is conducive to improving the accuracy and sensitivity of the test method.

In order to achieve the above object, the technical scheme adopted in the present invention is:

After the test sample 3-MCPD aqueous solution is treated with periodate solution to make the 3-MCPD therein cracked into chloroacetaldehyde, the following steps are carried out:

1) remove unreacted excess periodate, eliminate side reactions;

2) Fluorescent derivatization reaction: react chloroacetaldehyde with a fluorescent derivatization reagent to generate a target substance with a fluorescent effect;

3) HPLC-FLD determination: the target substance is separated, and 3-MCPD is quantified according to its chromatographic peak area;

The fluorescent derivatization reagent is an o-aminobenzo N-heterotwo-membered ring compound having the following general structural formula:

Wherein, the dotted ring structure represents a nitrogen-containing heterocycle or a substituted nitrogen heterocycle structure.

Further, in said step 1), the method for removing excessive periodate is to add sulfite solution or lead salt solution, and the addition amount is 1 by the ratio of sulfite or lead ion to periodate: (1- 2), the added volume is 10-80% of the volume of the 3-MCPD aqueous solution, the reaction temperature is 5-30°C, and the reaction time is 10-120min.

Further, in the step 2), the fluorescent derivatization reagent is 2-aminoquinoline, 1-aminoisoquinoline, 3-aminoisoquinoline, 2-aminoquinoxaline, 4-aminoquinazoline or 2 -Amino-4-hydroxyquinoline.

Further, in the step 2), the concentration of the fluorescent derivatization reagent is 0.002-0.02mol/L, the reaction temperature is 37-100°C, and the reaction time is 10-240min.

Further, in the step 3), the HPLC adopts reverse phase chromatography mode, the stationary phase includes but not limited to alkali-resistant silica gel ODS, zirconium matrix ODS or polymer matrix ODS packing, and the mobile phase includes but not limited to methanol-buffer Or acetonitrile-buffer solution, the ratio is (5%:95%)-(100%:0%); the FLD excitation wavelength is 260-320nm, and the detection wavelength is 350-470nm; the pH of the buffer solution is 7-13.

The 3-MCPD aqueous solution can be various environmental water samples such as filtered and centrifuged river water, river water, lake water, groundwater, factory waste water, tap water, drinking water (such as pure water, mineral water).

Described 3-MCPD aqueous solution also can be to make through following steps processing animal and vegetable oil product:

a) Weigh the oil sample;

b) adding hexane and an acetic acid solution containing NaCl for extraction, and discarding the upper organic phase;

c) Repeat the extraction once with hexane, and discard the upper organic phase.

The 3-MCPD aqueous solution can also be processed through the following steps to obtain solid oil-containing products, and the solid oil-containing products include but are not limited to fried instant noodles, potato chips, deep-fried dough sticks, oil cakes:

a) Weigh the sample mass of the oil-containing product and pulverize it;

b) add hexane to extract more than 2 times, and combine the extracts;

c) adding water to the combined extracts for extraction, and discarding the upper organic phase.

The 3-MCPD aqueous solution can also be obtained by solid-phase extraction of non-fat products, including but not limited to soy sauce and hydrolyzed vegetable protein.

By using the method of the invention, the content of the 3-chloro-1,2-propanediol fatty acid ester in the aqueous solution can also be detected by hydrolyzing the 3-chloro-1,2-propanediol fatty acid ester into 3-MCPD.

The beneficial effects of the present invention are:

1) The fluorescence derivatization reagent adopted in the present invention is convenient and easy to obtain, has good selectivity and high fluorescence efficiency, so that the detection sensitivity of the method is very high;

2) The fluorescent derivatization reagent adopted in the present invention has good hydrophobicity, and the target product is relatively retained in the reversed-phase chromatographic separation, which is easy to eliminate matrix effect interference and improves the measurement accuracy;

3) Using ordinary liquid chromatography-fluorescence method for determination, the equipment investment is greatly reduced compared with GC-MS, and the requirements for operators are lower, so it is easy to popularize and apply.

Description of drawings

Figure 1 is a chromatogram of adenine as a derivatization reagent for the determination of tap water spiked (15.0ng/mL).

Fig. 2 is 2-aminoquinoline is the chromatogram of the derivatization reagent determination water sample near the outlet of paper mill.

Figure 3 is a chromatogram of commercially available rice bran oil measured with 2-aminoquinoxaline as a derivatization reagent.

detailed description

The present invention will be further described below in conjunction with specific embodiments.

Embodiment 1: Take the downstream water sample of the paper mill and centrifuge it at high speed, filter the supernatant with a 0.45 μm filter membrane, measure 1 mL of the treated water sample, add 0.1 mL of sodium periodate solution (0.1 mol/L) to carry out the oxidation reaction , the reaction temperature was 30°C, and the reaction time was 20min; after the reaction, 0.2mL Na ₂ SO ₃ solution (0.1mol/L) was added, mixed and reacted at 25°C for 20min to remove excess unreacted sodium periodate. Then add 1mL of 0.002mol/L 2-aminoquinoline for derivatization reaction, and react at 90°C for 180min; after the reaction is completed, set the volume to 5mL, and use HPLC-FLD method for detection: the stationary phase is silica gel matrix reversed-phase ODS Packing, mobile phase is methanol-pH7.0PBS buffer solution (45:55, v/v); excitation wavelength is 260nm, fluorescence detection wavelength is 383nm; external standard method for quantification. The measurement was repeated 10 times, and the content of 3-MCPD in the water sample was 11.2±0.7 μg/kg. The results of the standard addition test showed that the recovery rate was between 97-103%.

Embodiment two: get the Yangtze River water sample and high-speed centrifugation, the supernatant is filtered with a 0.45 μm filter membrane, measure the water sample after 1mL of treatment, add 0.1mL sodium periodate solution (0.1mol/L) to carry out oxidation reaction, react The temperature was 30°C, and the reaction time was 20min; after the reaction, 0.15mL Na ₂ SO ₃ solution (0.1mol/L) was added, mixed well and reacted at 15°C for 120min to remove excess unreacted sodium periodate. Then add 1mL of 0.02mol/L 1-aminoisoquinoline for derivatization reaction, react at 37°C for 240min; after the reaction is completed, set the volume to 5mL, and use HPLC-FLD method for detection: the stationary phase is zirconium-based ODS packing , the mobile phase is acetonitrile-pH8.0PBS buffer solution (5:95, v/v); the excitation wavelength is 260nm, and the fluorescence detection wavelength is 470nm; the external standard method is used for quantification. The measurement was repeated 10 times, and the content of 3-MCPD in the water sample was 5.2±0.5 μg/kg. The results of the standard addition test showed that the recovery rate was between 96-101%.

Embodiment three: get tap water sample and high-speed centrifugation, supernatant is filtered with 0.45 μm filter membrane, measure the water sample after 1mL treatment, add 0.1mL sodium periodate solution (0.1mol/L) and carry out oxidation reaction, reaction temperature The temperature was 30°C, and the reaction time was 20min; after the reaction, 0.15mL Na ₂ SO ₃ solution (0.12mol/L) was added, mixed well, and reacted at 5°C for 10min to remove excess unreacted sodium periodate. Then add 1mL of 0.008mol/L 2-aminoquinoxaline for derivatization reaction, and react at 50°C for 100min; after the reaction is completed, set the volume to 5mL, and use HPLC-FLD method for detection: the stationary phase is polymer matrix ODS Packing, the mobile phase is methanol-pH9.0PBS buffer solution (25:75, v/v); the excitation wavelength is 320nm, and the fluorescence detection wavelength is 470nm; the external standard method is used for quantification. The measurement was repeated 10 times, and the content of 3-MCPD in the water sample was 4.5±0.6 μg/kg. The results of the standard addition test showed that the recovery rate was between 98-105%.

Example 4: Disperse 10 mL of first-grade soybean oil in 20 mL of n-hexane, stir evenly, add 6 mL of 0.01 g/mL NaCl solution, stir magnetically for 20 min, take 1 mL of the lower aqueous phase, and add 0.1 mL of periodic acid Sodium solution (0.1mol/L) was oxidized, the reaction temperature was 30°C, and the reaction time was 20min; after the reaction was completed, 0.1mL Pb(NO ₃ ) ₂ solution (0.2mol/L) was added, and after mixing, the reaction was carried out at 25°C for 120min To remove excess unreacted sodium periodate. Then add 1mL of 0.002mol/L 2-amino-4-hydroxyquinoline for derivatization reaction, and react at 60°C for 240min; Matrix reversed-phase ODS filler, mobile phase is acetonitrile-pH10.0PBS buffer (35:65, v/v); excitation wavelength is 300nm, fluorescence detection wavelength is 350nm; external standard method for quantification. The measurement was repeated 10 times, and the content of 3-MCPD in the oil sample was 19.6±0.2 μg/kg. The results of the standard addition test showed that the recovery rate was between 95-102%.

Example 5: Disperse 10 mL of first-grade rapeseed oil in 20 mL of n-heptane, stir evenly, add 6 mL of 0.05 g/mL NaCl solution, stir magnetically for 20 min, take 1 mL of the lower aqueous phase, and add 0.1 mL of high Sodium iodate solution (0.1mol/ _L ) was oxidized, the reaction temperature was 30°C, and the reaction time was 20min _; React for 10 min to remove excess unreacted sodium periodate. Then add 1mL of 0.002mol/L 3-aminoisoquinoline for derivatization reaction, and react at 100°C for 10min; after the reaction is completed, set the volume to 5mL, and use HPLC-FLD method for detection: the stationary phase is zirconium matrix reversed phase ODS filler, the mobile phase is methanol-pH11.0 calcium hydroxide buffer solution (55:45, v/v); the excitation wavelength is 320nm, and the fluorescence detection wavelength is 350nm; the external standard method is used for quantification. The measurement was repeated 10 times, and the content of 3-MCPD in the water sample was 31.2±0.4 μg/kg. The results of the standard addition test showed that the recovery rate was between 95-103%.

Example 6: Weigh 0.1g of first-grade soybean oil, uniformly disperse and dissolve in 0.5mL tert-butyl methyl ether/ethyl acetate (v/v8:2), add 0.5mLH ₂ SO ₄ /n-propanol (v/v, 0.5%) solution, vortexed and mixed, ultrasonicated in a water bath at 45°C for 15 min, then added 1 mL of 0.5 mol/L sodium methoxide solution for rapid hydrolysis for 3 min; after hydrolysis was completed, quickly added 3 mL of n-heptane and 3 mL of 3.3% glacial 0.01g/mL NaCl) to terminate the reaction; remove the upper organic phase after full extraction, extract twice with 3mL n-heptane to remove the remaining non-polar components in the solution, and then adjust the pH value of the lower aqueous phase to neutral. Take 1 mL of the lower aqueous phase, add 0.1 mL of sodium periodate solution (0.1 mol/L) for oxidation reaction, the reaction temperature is 30 ° C, and the reaction time is 20 min; after the reaction, add 0.15 mL of Na ₂ SO ₃ solution (0.1 mol/L ), after mixing, react at 20°C for 30 minutes to remove excess unreacted sodium periodate. Then add 1mL of 0.005mol/L 2-aminoquinoxaline for derivatization reaction, react at 37°C for 240min; Phase ODS filler, mobile phase is acetonitrile-pH12.0 calcium hydroxide buffer solution (65:35, v/v); excitation wavelength is 300nm, fluorescence detection wavelength is 383nm; external standard method for quantification. The measurement was repeated 10 times, and the total content of free and bound 3-MCPD in the oil was 635±15 μg/kg. When measuring the content of free 3-MCPD, the 3-MCPD extraction process was carried out first, and the other operations were the same as above, and the measurement result was 20.3±0.4 μg/kg, and the standard addition test results showed that the recovery rate was between 94-102%.

Example 7: Weigh 0.1g lard, uniformly disperse and dissolve in 0.5mL tert-butyl methyl ether/ethyl acetate (v/v8:2), add 0.5mLH ₂ SO ₄ /n-propanol (v/v, 0.5% ) solution, vortexed and mixed in a water bath at 45°C for 15 min, then added 1 mL of 0.5 mol/L sodium methoxide solution for rapid hydrolysis for 3 min; g/mLNaCl) to terminate the reaction; remove the upper organic phase after full extraction, extract twice with 3mL n-heptane to remove the remaining non-polar components in the solution, and then adjust the pH value of the lower aqueous phase to neutral. Take 1 mL of the lower aqueous phase, add 0.1 mL of sodium periodate solution (0.1 mol/L) for oxidation reaction, the reaction temperature is 30 ° C, and the reaction time is 20 min; after the reaction, add 0.1 mL of Pb(NO ₃ ) ₂ solution (0.2 mol/L) /L), after mixing, react at 25°C for 20 minutes to remove excess unreacted sodium periodate. Then add 1mL of 0.02mol/L 4-aminoquinazoline for derivatization reaction, react at 70°C for 100min; after the reaction is completed, set the volume to 5mL, and use HPLC-FLD method for detection: the stationary phase is zirconium matrix reversed phase ODS filler, the mobile phase is methanol-pH13.0 calcium hydroxide buffer solution (75:25, v/v); the excitation wavelength is 260nm, and the fluorescence detection wavelength is 400nm; the external standard method is used for quantification. The measurement was repeated 10 times, and the total content of free and bound 3-MCPD in the oil was 197±6 μg/kg. When measuring the content of free 3-MCPD, the 3-MCPD extraction process was carried out first, and other operations were the same as above, and the measurement result was 12.7±0.3 μg/kg, and the standard addition test results showed that the recovery rate was between 99-104%.

Embodiment 8: Weigh 8g fried instant noodles, pulverize and extract 3 times with hexane, 50mL each time, combine the extracts and remove hexane; evenly disperse and dissolve in 0.5mL tert-butyl methyl ether/ethyl acetate (v/v8 :2), add 0.5mL H ₂ SO ₄ /n-propanol (v/v, 0.5%) solution, vortex and mix well, then sonicate in a water bath at 45°C for 15min, then add 1mL0.5mol/L sodium methoxide solution for rapid hydrolysis for 3min Add 3mL of n-heptane and 3mL of 3.3% glacial acetic acid (dissolved in 0.01g/mLNaCl) to terminate the reaction quickly after the completion of hydrolysis; remove the upper organic phase after sufficient extraction, extract twice with 3mL of n-heptane, remove the solution Residual non-polar ingredients, then adjust the pH value of the lower aqueous phase to neutral. Take 1 mL of the lower aqueous phase, add 0.1 mL of sodium periodate solution (0.1 mol/L) for oxidation reaction, the reaction temperature is 30 ° C, and the reaction time is 20 min; after the reaction, add 0.15 mL of Na ₂ SO ₃ solution (0.1 mol/L ), after mixing, react at 25°C for 120min to remove excess unreacted sodium periodate. Then add 1mL of 0.01mol/L 4-aminoquinazoline for derivatization reaction, and react at 100°C for 10min; after the reaction is completed, set the volume to 5mL, and use HPLC-FLD method for detection: the stationary phase is zirconium matrix reversed phase ODS filler, mobile phase is acetonitrile-pH10.0PBS buffer solution (85:15, v/v); excitation wavelength is 280nm, fluorescence detection wavelength is 350nm; external standard method is used for quantification. The measurement was repeated 10 times, and the total content of free and bound 3-MCPD in the oil was 215±8 μg/kg. When measuring the content of free 3-MCPD, the 3-MCPD extraction process was carried out first, and other operations were the same as above, and the measurement result was 16.1±0.2 μg/kg, and the standard addition test results showed that the recovery rate was between 98-104%.

Embodiment 9: Weigh 8g fried potato chips, crush them and extract them with hexane for 3 times, 50mL each time, combine the extracts and remove the hexane; evenly disperse and dissolve in 0.5mL tert-butyl methyl ether/ethyl acetate (v/ v8:2), add 0.5mL H ₂ SO ₄ /n-propanol (v/v, 0.5%) solution, vortex and mix well, then sonicate in a water bath at 45°C for 15min, then add 1mL 0.5mol/L sodium methoxide solution for rapid hydrolysis 3min; after the completion of hydrolysis, quickly add 3mL of n-heptane and 3mL of 3.3% glacial acetic acid (dissolved in 0.01g/mLNaCl) to terminate the reaction; after sufficient extraction, remove the upper organic phase, extract twice with 3mL of n-heptane, and remove the solution The remaining non-polar components in the solution, and then adjust the pH value of the lower aqueous phase to neutral. Take 1 mL of the lower aqueous phase, add 0.1 mL of sodium periodate solution (0.1 mol/L) for oxidation reaction, the reaction temperature is 30 ° C, and the reaction time is 20 min; after the reaction, add 0.2 mL of Na ₂ SO ₃ solution (0.1 mol/L ), after mixing, react at 20°C for 10 minutes to remove excess unreacted sodium periodate. Then add 1mL of 0.02mol/L 2-amino-4-hydroxyquinoline for derivatization reaction, react at 80°C for 30min; Reversed-phase ODS filler with material matrix, the mobile phase is methanol-pH10.0PBS buffer solution (95:5, v/v); the excitation wavelength is 310nm, and the fluorescence detection wavelength is 460nm; the external standard method is used for quantification. The measurement was repeated 10 times, and the total content of free and bound 3-MCPD in the oil was 310±5 μg/kg. When measuring the content of free 3-MCPD, the 3-MCPD extraction process was carried out first, and other operations were the same as above, and the measurement result was 27.3±0.8 μg/kg, and the standard addition test results showed that the recovery rate was between 96-101%.

Example 10: Weigh 8g of prepared soy sauce, mix it with 12mL of 5mol/L NaCl solution, load it on a solid phase extraction column, first elute with 80mL of hexane-ether (90:10, v/v), and then use 250mL of ether Elution, collect the ether eluate and remove the solvent, add 1mL of water and shake evenly, then add 0.1mL sodium periodate solution (0.1mol/L) to carry out oxidation reaction, the reaction temperature is 30 °C, the reaction time is 20min; the reaction is completed Then add 0.1mL Pb(NO ₃ ) ₂ solution (0.15mol/L), mix well and react at 30°C for 10min to remove excess unreacted sodium periodate. Then add 1mL of 0.005mol/L 1-aminoisoquinoline for derivatization reaction, react at 70°C for 200min; after the reaction is completed, set the volume to 5mL, and use HPLC-FLD method for detection: the stationary phase is silica gel reversed-phase ODS Packing, mobile phase is acetonitrile; excitation wavelength is 310nm, fluorescence detection wavelength is 450nm; external standard method for quantification. The measurement was repeated 10 times, and the content of 3-MCPD in the oil sample was 23.6±0.3 μg/kg, and the result of the standard addition test showed that the recovery rate was between 98-103%.

Embodiment 11: Weigh 8g of hydrolyzed vegetable protein, mix it with 12mL of 5mol/LNaCl solution, load it into a solid phase extraction column, first elute with 80mL of hexane-ether (90:10, v/v), and then use Elute with 250mL of ether, collect the ether eluate and remove the solvent, add 1mL of water and oscillate evenly, then add 0.1mL of sodium periodate solution (0.1mol/L) for oxidation reaction, the reaction temperature is 30°C, and the reaction time is 20min; After the reaction, add 0.2mL Na ₂ SO ₃ solution (0.1mol/L), mix well and react at 20°C for 100min to remove excess unreacted sodium periodate. Then add 1mL of 0.015mol/L 1-aminoisoquinoline for derivatization reaction, react at 40°C for 200min; after the reaction is completed, set the volume to 5mL, and use HPLC-FLD method for detection: the stationary phase is zirconium matrix reversed phase ODS filler, the mobile phase is methanol-pH12.0 calcium hydroxide buffer solution (45:55, v/v); the excitation wavelength is 290nm, and the fluorescence detection wavelength is 390nm; the external standard method is used for quantification. The measurement was repeated 10 times, and the content of 3-MCPD in the oil sample was 25.6±0.3 μg/kg, and the result of the standard addition test showed that the recovery rate was between 97-104%.

Table 1 is a comparison of the conditions and effects of adenine, 2-aminoquinoline, and 2-aminoquinoxaline as derivatization reagents for determining the content of 3-MCPD.

Table 1 Comparison of the assay effects of three different derivatization reagents

It can be seen from Figures 1-3 and Table 1 that after derivatization with new fluorescent derivatization reagents 2-aminoquinoline and 2-aminoquinoxaline, the hydrophobicity of the target product is significantly stronger than that of adenine-derived products, and the concentration of methanol in the mobile phase After increasing multiple times, the retention time is still longer than that of adenine-derived products, which improves the column efficiency and the anti-impurity interference ability, the measurement accuracy and reproducibility are also better, and the detection limit of the method is also lower.

Claims (8)

1. a kind of 3-chloro-1, the high-performance liquid chromatography-fluorescence detection method of 2-propanediol, comprise that test sample 3-MCPD aqueous solution is processed and 3-MCPD wherein is cracked into chloroacetaldehyde through periodate solution Step, it is characterized in that: the method also includes the following steps: 1) remove unreacted excess periodate, eliminate side reactions; 2) Fluorescent derivatization reaction: react chloroacetaldehyde with a fluorescent derivatization reagent to generate a target substance with a fluorescent effect; the fluorescent derivatization reagent is 2-aminoquinoline, 1-aminoisoquinoline, 3-aminoisoquinoline line, 2-aminoquinoxaline, 4-aminoquinazoline or 2-amino-4-hydroxyquinoline; 3) HPLC-FLD determination: the target substance is separated, and 3-MCPD is quantified according to its chromatographic peak area; the HPLC adopts reverse phase chromatography mode, and the stationary phase is alkali-resistant silica gel ODS, zirconium matrix ODS or polymeric Material matrix ODS filler, mobile phase is methanol-buffer or acetonitrile-buffer, the ratio is (5%:95%)-(100%:0%); The pH of described buffer is 7-13; The fluorescent derivatization reagent is an o-aminobenzo N-heterotwo-membered ring compound having the following general structural formula: Wherein, the dotted ring structure represents a nitrogen-containing heterocycle or a substituted nitrogen heterocycle structure. 2. according to the described 3-chloro-1 of claim 1, the high performance liquid chromatography-fluorescence detection method of 2-propanediol, it is characterized in that: In described step 1), the method for removing excessive periodate is to add sulfite solution or lead salt solution, and the addition amount is 1: (1-2) by the ratio of sulfite or lead ion and periodate, The added volume is 10-80% of the volume of the 3-MCPD aqueous solution, the reaction temperature is 5-30°C, and the reaction time is 10-120min. 3. according to claim 1 and 2 described 3-chloro-1, the high performance liquid chromatography-fluorescence detection method of 2-propanediol, it is characterized in that: In the step 2), the concentration of the fluorescent derivatization reagent is 0.002-0.02mol/L, the reaction temperature is 37-100°C, and the reaction time is 10-240min. 4. according to claim 1 and 2 described 3-chloro-1, the high performance liquid chromatography-fluorescence detection method of 2-propanediol, it is characterized in that: In the step 3), the excitation wavelength of FLD is 260-320nm, and the detection wavelength is 350-470nm. 5. according to claim 1 and 2 described 3-chloro-1, the high performance liquid chromatography-fluorescence detection method of 2-propanediol, it is characterized in that: The 3-MCPD aqueous solution is filtered and centrifuged river water, river water, lake water, ground water, factory waste water, tap water or drinking water. 6. according to claim 1 and 2 described 3-chloro-1, the high performance liquid chromatography-fluorescence detection method of 2-propanediol, it is characterized in that: The 3-MCPD aqueous solution is obtained through the following steps of processing animal and vegetable oil products: a) Weigh the oil sample; b) adding hexane and an acetic acid solution containing NaCl for extraction, and discarding the upper organic phase; c) Repeat the extraction once with hexane, and discard the upper organic phase. 7. according to claim 1 and 2 described 3-chloro-1, the high performance liquid chromatography-fluorescence detection method of 2-propanediol, it is characterized in that: The 3-MCPD aqueous solution is obtained through the following steps of processing solid oil-containing products, and the solid oil-containing products include fried instant noodles, potato chips, deep-fried dough sticks or oil cakes: a) Weigh the sample mass of the oil-containing product and pulverize it; b) add hexane to extract more than 2 times, and combine the extracts; c) adding water to the combined extracts for extraction, and discarding the upper organic phase. 8. according to claim 1 and 2 described 3-chloro-1, the high performance liquid chromatography-fluorescence detection method of 2-propanediol, it is characterized in that: The 3-MCPD aqueous solution is obtained through solid-phase extraction of non-fat products, and the non-fat products are soy sauce or hydrolyzed vegetable protein.