CN104597194B - The high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol - Google Patents
The high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol Download PDFInfo
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Abstract
The invention discloses a kind of high performance liquid chromatography-fluorescence method of 3-MCPD, the method comprises the steps: 1) make 3-MCPD therein be cracked into 2-Chloro-1-ethanal through periodate solution process 3-MCPD aqueous solution；2) remove excess of periodate, eliminate side reaction；3) fluorescence derivation reaction: 2-Chloro-1-ethanal and fluorescence derivatization are reacted and generates the target substance with fluorescent effect；4) HPLC-FLD measures: target substance is easily separated, and according to its chromatographic peak area, 3-MCPD is carried out quantitatively；Described fluorescence derivatization is adjacent assorted two membered cyclic compound of amino N.The novel fluorescence derivatization reagent that the present invention passes through a class to be conveniently easy to get, selectivity is good, fluorescence efficiency is high, hydrophobicity is stronger is used for high performance liquid chromatography-fluorescence 3-MCPD, make target product have good retention behavior in reversed-phase liquid chromatography, be conducive to improving accuracy and the sensitivity of method of testing.
Description
Technical field
The present invention relates to a kind of high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol, belong to analytical chemistry field.
Background technology
3-chlorine-1,2-propylene glycol is also known as 3-propylene glycol of chlorine or glycerin chlorohydrin, and its English is called 3-monochloro-1,2-propanediol, writes a Chinese character in simplified form 3-MCPD, and its chemical structural formula is: CH2Cl-CHOH-CH2OH.3-MCPD is an a kind of chloro dihydroxylic alcohols, for colourless pleasantly sweet liquid, freezing point-40 DEG C, boiling point 213 DEG C (decomposition), relative density 1.3218 (20 DEG C), refractive index 1.4809 (20 DEG C), it is dissolved in water, ethanol, ether and acetone, it is slightly soluble in toluene, insoluble in benzene, petroleum ether and carbon tetrachloride；Instability, fades to rice yellow, the easy moisture absorption after placement.
Toxicity research in recent years shows, health is had stronger harm by 3-MCPD, has become as a kind of internationally recognized Chemical Contaminants, and its toxicity main manifestations is: the 1. rat oral median lethal dose(LD 50) LD of acute and chronic toxicity: 3-MCPD50For 150mg/kg.bw.During male rat continuous per os contamination more than 3-MCPD1mg/kg/day dosage, it is possible to occurring that sperm motility weakens and reproductivity reduces, dosage is more than or equal to 10-20mg/kg/day, it is seen that the sperm of rat has significant morphological change and injury of epididymis；2. genetoxic: current most of research institutions and scholar think that 3-MCPD belongs to non-genotoxic carcinogen, tentative maximum tolerated dose every day (TDI) is 2 μ g/kg.bw；3. carcinogenecity: carcinogenic committee of Britain (Committeeoncarcinoginicity) is thought, 3-MCPD can cause carcinogenic in zoopery；And Britain causes variation committee (CommitteeonMutagenicity) and thinks, during 3-MCPD tests in vivo, it is the carcinogen of a nongenotoxic；4. neurotoxicity: mice is identical to the neurotoxic effect sensitivity of 3-MCPD with rat, especially the symmetry of brain stem is damaged.When 3-MCPD poisoning dosage is more than 25mg/kg.bw/day, laboratory animal central nervous system injury presents significant dose-effect relationship.
In food, first the pollution of chloropropyl alcohol is find in the flavoring agent (such as chicken essence and soy sauce etc.) being raw material with acid hydrolyzed vegetable protein (HVP), later detection in Fructus Hordei Germinatus extract, modified starch, meat extract, cookies, bread, sausage, cereals etc. successively again.First Zelinkov á Z. of Czech in 2006 et al. finds there is chloropropyl alcohol fatty acid ester in edible oil.SeefelderW. in 2007 et al. research finds that the content range of chloropropyl alcohol fatty acid ester is between 0.2～20mg/kg in polished fat, and wherein refining vegetable oil is incremented by according to Oleum Brassicae campestris, soybean oil, Oleum Helianthi, safflower oil, walnut oil and palmitic number sequence.
In environment water, owing to the discharge of industrial wastewater can cause that a degree of 3-MCPD pollutes, it is all produce the 3-MCPD important sources polluted as acid hydrolyzed vegetable protein produces the use of wet strength agent-Polyamide Polyamine Epichlorohydrin Resin in the discharge of waste water, pulp production, if not treated meeting causes the pollution of neighbouring water body；It addition, the use of polyamines flocculant also can produce a degree of 3-MCPD pollution in water treatment procedure.
Just owing to 3-MCPD is serious to harm, many countries have all formulated limit standard these pollutant to control in food and drinking water, as European Union has worked out limit standard in calendar year 2001, it is stipulated that must not exceed the intake of μ g/kg.bw every day 20；China also establishes the national standard of 3-MCPD in detection soy sauce.In Britain's drinking water inspection body (DrinkingWaterInspectorate) regulation drinking water, the maximum level of 3-MCPD must not exceed 0.1mg/L.Owing in food and drinking water, 3-MCPD limits safely and all reaches ppb level, therefore highly sensitive detection method must be adopted to can be only achieved monitoring requirement, means conventional at present are mainly gas chromatogram (GC), gas chromatography-mass spectrography (GC-MS).Owing to the polarity of 3-MCPD is higher with boiling point, mensuration process all needs 3-MCPD is enriched with from substrate, is extracted, then perform the derivatization.Conventional extracting process has liquid-liquid extraction (LLE), matrix solid phase dispersion to extract (MSPD), solid-phase microextraction (SPME) etc.；Derivatization method then mainly has ketone to derive, boric acid class derivative (phenylboric acid, butyl boron dihydroxide), the double; two trimethyl-trifluoroacetamide (BSTFA) of N, O-, anhydrides derive (HFBA), heptafluoro-butyryl imidazole (HFBI) etc..But, due to 3-MCPD good water solubility, boiling point is higher, therefore it is complicated, loaded down with trivial details to have pretreatment process based on the analysis method of gas Chromatographic Determination 3-MCPD, and derivatising condition is harsh, instrument working service high in cost of production shortcoming.nullGiven this，Develop low based on the cost of high performance liquid chromatography、Simple and easy to do high-sensitivity detecting method tool is of great significance，Patent CN102565270 reported first one cracks in conjunction with periodate oxidation、Reversed-phase liquid chromatography-the fluorescence detection of base or nucleoside (nucleotide) derivatization，The method testing cost is low、Simple to operate、Highly sensitive，It is applicable to oils and fats、The mensuration of 3-MCPD in the food such as soy sauce，But，Fluorescence derivatization hydrophilic owing to using is too strong，Target product retains more weak in reversed-phase liquid chromatography，It is likely to be vulnerable to the interference of matrix effect when measuring foodstuff complex sample，The accuracy of impact mensuration and repeatability，Therefore，Development of new fluorescence property is good、The higher fluorescence derivatization of hydrophobicity would be more advantageous in the strong point and the advantage that play the method.
Summary of the invention
It is an object of the invention to provide a kind of high performance liquid chromatography-fluorescence method of 3-MCPD, the novel fluorescence derivatization reagent that the method is passed through a class to be conveniently easy to get, selectivity is good, fluorescence efficiency is high, hydrophobicity is stronger is used for high performance liquid chromatography-fluorescence 3-MCPD, make target product have good retention behavior in reversed-phase liquid chromatography, be conducive to improving accuracy and the sensitivity of method of testing.
To achieve these goals, the technical solution used in the present invention is:
By test sample 3-MCPD aqueous solution after periodate solution process makes 3-MCPD therein be cracked into 2-Chloro-1-ethanal, carry out following steps:
1) remove unreacted excess of periodate, eliminate side reaction；
2) fluorescence derivation reaction: 2-Chloro-1-ethanal and fluorescence derivatization are reacted and generates the target substance with fluorescent effect；
3) HPLC-FLD measures: target substance is easily separated, and according to its chromatographic peak area, 3-MCPD is carried out quantitatively；
Described fluorescence derivatization is adjacent assorted two membered ring compounds of amino benzo N being structured with formula:
Wherein, dotted line circulus represents nitrogen heterocyclic ring or substituted nitrogen heterocyclic ring structure.
Further, described step 1) in, remove the method for excess of periodate for adding sulfite solution or lead salt solution, addition is 1:(1-2 in the ratio of inferior sulfate radical or lead ion and periodate), add the 10-80% that volume is described 3-MCPD aqueous solution volume, reaction temperature is 5-30 DEG C, and the response time is 10-120min.
Further, described step 2) in, fluorescence derivatization is 2-quinolin-2-ylamine, 1-aminoisoquinoline, 3-aminoisoquinoline, 2-aminoquinoxaline, 4-amido quinazoline or 2-amino-4-hydroxy quinoline.
Further, described step 2) in, the concentration of fluorescence derivatization is 0.002-0.02mol/L, and reaction temperature is 37-100 DEG C, and the response time is 10-240min.
Further, described step 3) in, HPLC adopts reversed phase chromatography pattern, fix and include but not limited to the silica gel ODS of alkali resistance, zirconio matter ODS or polymeric matrix ODS filler mutually, mobile phase includes but not limited to methanol-buffer or acetonitrile-buffer, and ratio is (5%:95%)-(100%:0%)；FLD excitation wavelength is 260-320nm, and detection wavelength is 350-470nm；The pH of described buffer is 7-13.
Described 3-MCPD aqueous solution can be through multiple environmental water samples such as filtration, the river of centrifugal treating, river, lake water, subsoil water, plant chimney stalk, tap water, drinking waters (such as pure water, mineral water).
Described 3-MCPD aqueous solution can also be process vegetable and animals oils fat prod through following steps to prepare:
A) oil sample is weighed；
B) add hexane and the acetic acid solution containing NaCl extracts, discard upper organic phase；
C) repeat to extract once with hexane, discard upper organic phase.
Described 3-MCPD aqueous solution can also be process solid-state through following steps to prepare containing oil and fat product, and described solid-state includes but not limited to fried instant noodle containing oil and fat product, potato chips, fritters of twisted dough, oil cake:
A) weigh the sample quality containing oil and fat product and pulverize；
B) add hexane to carry out extracting more than 2 times, combining extraction liquid；
C) add water in merge extract in extract, discard upper organic phase.
Described 3-MCPD aqueous solution can also is that and prepares through Solid-Phase Extraction non-oil lipid product, and described non-oil lipid product includes but not limited to soy sauce, hydrolyzed vegetable protein.
The inventive method is utilized to be hydrolyzed to 3-MCPD also by by 3-chlorine-1,2-propylene glycol aliphatic ester, thus detecting the content of 3-chlorine-1,2-propylene glycol aliphatic ester in aqueous solution.
The beneficial effects of the present invention is:
1) fluorescence derivatization of the present invention is conveniently easy to get, selectivity is good, fluorescence efficiency is high so that the method detection sensitivity is very high；
2) fluorescence derivatization hydrophobicity of the present invention is good, and target product retains stronger in reversed phase chromatography separation, it is easy to gets rid of matrix effect interference, improves accuracy of measurement；
3) using common liquid chromatograph fluorescence method to be measured, equipment investment relatively GC-MS is greatly lowered, and operator is required relatively low, it is easy to popularization and application.
Accompanying drawing explanation
Fig. 1 is adenine be derivatization reagent measure tap water mark-on (15.0ng/mL) chromatogram.
Fig. 2 is 2-quinolin-2-ylamine is that derivatization reagent measures the chromatogram of water sample near the discharge outlet of paper mill.
Fig. 3 is 2-aminoquinoxaline be derivatization reagent measure commercially available Testa oryzae oil chromatogram.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described.
Embodiment one: take downstream, paper mill water sample high speed centrifugation, supernatant, with 0.45 μm of membrane filtration, measures the water sample after 1mL processes, and adds 0.1mL sodium periodate solution (0.1mol/L) and carries out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.2mLNa is added after having reacted2SO3Solution (0.1mol/L), mixes and reacts 20min at latter 25 DEG C to remove excessive unreacted sodium metaperiodate.The 2-quinolin-2-ylamine of the 0.002mol/L then adding 1mL performs the derivatization reaction, reacts 180min at 90 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of silica matrix mutually, and mobile phase is methanol-pH7.0PBS buffer (45:55, v/v)；Excitation wavelength is 260nm, and fluoroscopic examination wavelength is 383nm；Quantified by external standard method.Replication 10 times, obtaining 3-MCPD content in water sample is 11.2 ± 0.7 μ g/kg, and mark-on result of the test shows that the response rate is between 97-103%.
Embodiment two: take the Changjiang river water sample high speed centrifugation, supernatant, with 0.45 μm of membrane filtration, measures the water sample after 1mL processes, and adds 0.1mL sodium periodate solution (0.1mol/L) and carries out oxidation reaction, and reaction temperature is 30 DEG C, and the response time is 20min；0.15mLNa is added after having reacted2SO3Solution (0.1mol/L), mixes and reacts 120min at latter 15 DEG C to remove excessive unreacted sodium metaperiodate.The 1-aminoisoquinoline of the 0.02mol/L then adding 1mL performs the derivatization reaction, reacts 240min at 37 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is zirconio matter ODS filler mutually, and mobile phase is acetonitrile-pH8.0PBS buffer (5:95, v/v)；Excitation wavelength is 260nm, and fluoroscopic examination wavelength is 470nm；Quantified by external standard method.Replication 10 times, obtaining 3-MCPD content in water sample is 5.2 ± 0.5 μ g/kg, and mark-on result of the test shows that the response rate is between 96-101%.
Embodiment three: take from water sample high speed centrifugation, supernatant, with 0.45 μm of membrane filtration, measures the water sample after 1mL processes, and adds 0.1mL sodium periodate solution (0.1mol/L) and carries out oxidation reaction, and reaction temperature is 30 DEG C, and the response time is 20min；0.15mLNa is added after having reacted2SO3Solution (0.12mol/L), mixes and reacts 10min at latter 5 DEG C to remove excessive unreacted sodium metaperiodate.The 2-aminoquinoxaline of the 0.008mol/L then adding 1mL performs the derivatization reaction, reacts 100min at 50 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is polymeric matrix ODS filler mutually, and mobile phase is methanol-pH9.0PBS buffer (25:75, v/v)；Excitation wavelength is 320nm, and fluoroscopic examination wavelength is 470nm；Quantified by external standard method.Replication 10 times, obtaining 3-MCPD content in water sample is 4.5 ± 0.6 μ g/kg, and mark-on result of the test shows that the response rate is between 98-105%.
Embodiment four: 10mL one-level soybean oil is scattered in 20mL normal hexane, after stirring, add the NaCl solution of 6mL0.01g/mL, magnetic agitation 20min, standing measures lower floor aqueous phase 1mL, adding 0.1mL sodium periodate solution (0.1mol/L) and carry out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.1mLPb (NO is added after having reacted3)2Solution (0.2mol/L), mixes and reacts 120min at latter 25 DEG C to remove excessive unreacted sodium metaperiodate.The 2-amino-4-hydroxy quinoline of the 0.002mol/L then adding 1mL performs the derivatization reaction, reacts 240min at 60 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of zirconio matter mutually, and mobile phase is acetonitrile-pH10.0PBS buffer (35:65, v/v)；Excitation wavelength is 300nm, and fluoroscopic examination wavelength is 350nm；Quantified by external standard method.Replication 10 times, obtaining 3-MCPD content in oil sample is 19.6 ± 0.2 μ g/kg, and mark-on result of the test shows that the response rate is between 95-102%.
Embodiment five: 10mL one-level Oleum Brassicae campestris is scattered in 20mL normal heptane, after stirring, add the NaCl solution of 6mL0.05g/mL, magnetic agitation 20min, standing measures lower floor aqueous phase 1mL, adding 0.1mL sodium periodate solution (0.1mol/L) and carry out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.1mLPb (NO is added after having reacted3)2Solution (0.1mol/L), mixes and reacts 10min at latter 25 DEG C to remove excessive unreacted sodium metaperiodate.The 3-aminoisoquinoline of the 0.002mol/L then adding 1mL performs the derivatization reaction, reacts 10min at 100 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of zirconio matter mutually, and mobile phase is methanol-pH11.0 calcium hydroxide buffer solution (55:45, v/v)；Excitation wavelength is 320nm, and fluoroscopic examination wavelength is 350nm；Quantified by external standard method.Replication 10 times, obtaining 3-MCPD content in water sample is 31.2 ± 0.4 μ g/kg, and mark-on result of the test shows that the response rate is between 95-103%.
Embodiment six: weigh 0.1g one-level soybean oil, dispersed is dissolved in 0.5mL t-butyl methyl ether/ethyl acetate (v/v8:2), adds 0.5mLH2SO4/ normal propyl alcohol (v/v, 0.5%) solution, whirlpool mixing after under 45 DEG C of water-baths ultrasonic 15min, be subsequently added 1mL0.5mol/L sodium methoxide solution fast hydrolyzing 3min；The glacial acetic acid (being dissolved in 0.01g/mLNaCl) being rapidly added 3mL normal heptane and 3mL3.3% after being hydrolyzed terminates reaction；Remove upper organic phase after fully extracting, by 3mL normal heptane extracting twice, remove the nonpolarity element of residual in solution, then lower floor's aqueous pH values is adjusted to neutrality.Taking off a layer aqueous phase 1mL, add 0.1mL sodium periodate solution (0.1mol/L) and carry out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.15mLNa is added after having reacted2SO3Solution (0.1mol/L), mixes and reacts the 30min excessive unreacted sodium metaperiodate of removing at latter 20 DEG C.The 2-aminoquinoxaline of the 0.005mol/L then adding 1mL performs the derivatization reaction, reacts 240min at 37 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of polymeric matrix mutually, and mobile phase is acetonitrile-pH12.0 calcium hydroxide buffer solution (65:35, v/v)；Excitation wavelength is 300nm, and fluoroscopic examination wavelength is 383nm；Quantified by external standard method.Replication 10 times, obtaining the total content of free state and combined state 3-MCPD in oil is 635 ± 15 μ g/kg.When measuring the content of free state 3-MCPD, first carrying out 3-MCPD extraction process, other operation is same with upper, and obtaining measurement result is 20.3 ± 0.4 μ g/kg, and mark-on result of the test shows that the response rate is between 94-102%.
Embodiment seven: weigh 0.1g Adeps Sus domestica, dispersed is dissolved in 0.5mL t-butyl methyl ether/ethyl acetate (v/v8:2), adds 0.5mLH2SO4/ normal propyl alcohol (v/v, 0.5%) solution, whirlpool mixing after under 45 DEG C of water-baths ultrasonic 15min, be subsequently added 1mL0.5mol/L sodium methoxide solution fast hydrolyzing 3min；The glacial acetic acid (being dissolved in 0.01g/mLNaCl) being rapidly added 3mL normal heptane and 3mL3.3% after being hydrolyzed terminates reaction；Remove upper organic phase after fully extracting, by 3mL normal heptane extracting twice, remove the nonpolarity element of residual in solution, then lower floor's aqueous pH values is adjusted to neutrality.Taking off a layer aqueous phase 1mL, add 0.1mL sodium periodate solution (0.1mol/L) and carry out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.1mLPb (NO is added after having reacted3)2Solution (0.2mol/L), mixes and reacts the 20min excessive unreacted sodium metaperiodate of removing at latter 25 DEG C.The 4-amido quinazoline of the 0.02mol/L then adding 1mL performs the derivatization reaction, reacts 100min at 70 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of zirconio matter mutually, and mobile phase is methanol-pH13.0 calcium hydroxide buffer solution (75:25, v/v)；Excitation wavelength is 260nm, and fluoroscopic examination wavelength is 400nm；Quantified by external standard method.Replication 10 times, obtaining the total content of free state and combined state 3-MCPD in oil is 197 ± 6 μ g/kg.When measuring the content of free state 3-MCPD, first carrying out 3-MCPD extraction process, other operation is same with upper, and obtaining measurement result is 12.7 ± 0.3 μ g/kg, and mark-on result of the test shows that the response rate is between 99-104%.
Embodiment eight: weigh 8g fried instant noodle, extracts 3 times with hexane after pulverizing, and each 50mL, combining extraction liquid also removes hexane；Dispersed it is dissolved in 0.5mL t-butyl methyl ether/ethyl acetate (v/v8:2), adds 0.5mLH2SO4/ normal propyl alcohol (v/v, 0.5%) solution, whirlpool mixing after under 45 DEG C of water-baths ultrasonic 15min, be subsequently added 1mL0.5mol/L sodium methoxide solution fast hydrolyzing 3min；The glacial acetic acid (being dissolved in 0.01g/mLNaCl) being rapidly added 3mL normal heptane and 3mL3.3% after being hydrolyzed terminates reaction；Remove upper organic phase after fully extracting, by 3mL normal heptane extracting twice, remove the nonpolarity element of residual in solution, then lower floor's aqueous pH values is adjusted to neutrality.Taking off a layer aqueous phase 1mL, add 0.1mL sodium periodate solution (0.1mol/L) and carry out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.15mLNa is added after having reacted2SO3Solution (0.1mol/L), mixes and reacts the 120min excessive unreacted sodium metaperiodate of removing at latter 25 DEG C.The 4-amido quinazoline of the 0.01mol/L then adding 1mL performs the derivatization reaction, reacts 10min at 100 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of zirconio matter mutually, and mobile phase is acetonitrile-pH10.0PBS buffer (85:15, v/v)；Excitation wavelength is 280nm, and fluoroscopic examination wavelength is 350nm；Quantified by external standard method.Replication 10 times, obtaining the total content of free state and combined state 3-MCPD in oil is 215 ± 8 μ g/kg.When measuring the content of free state 3-MCPD, first carrying out 3-MCPD extraction process, other operation is same with upper, and obtaining measurement result is 16.1 ± 0.2 μ g/kg, and mark-on result of the test shows that the response rate is between 98-104%.
Embodiment nine: weigh 8g potato chips, extracts 3 times with hexane after pulverizing, and each 50mL, combining extraction liquid also removes hexane；Dispersed it is dissolved in 0.5mL t-butyl methyl ether/ethyl acetate (v/v8:2), adds 0.5mLH2SO4/ normal propyl alcohol (v/v, 0.5%) solution, whirlpool mixing after under 45 DEG C of water-baths ultrasonic 15min, be subsequently added 1mL0.5mol/L sodium methoxide solution fast hydrolyzing 3min；The glacial acetic acid (being dissolved in 0.01g/mLNaCl) being rapidly added 3mL normal heptane and 3mL3.3% after being hydrolyzed terminates reaction；Remove upper organic phase after fully extracting, by 3mL normal heptane extracting twice, remove the nonpolarity element of residual in solution, then lower floor's aqueous pH values is adjusted to neutrality.Taking off a layer aqueous phase 1mL, add 0.1mL sodium periodate solution (0.1mol/L) and carry out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.2mLNa is added after having reacted2SO3Solution (0.1mol/L), mixes and reacts the 10min excessive unreacted sodium metaperiodate of removing at latter 20 DEG C.The 2-amino-4-hydroxy quinoline of the 0.02mol/L then adding 1mL performs the derivatization reaction, reacts 30min at 80 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of polymeric matrix mutually, and mobile phase is methanol-pH10.0PBS buffer (95:5, v/v)；Excitation wavelength is 310nm, and fluoroscopic examination wavelength is 460nm；Quantified by external standard method.Replication 10 times, obtaining the total content of free state and combined state 3-MCPD in oil is 310 ± 5 μ g/kg.When measuring the content of free state 3-MCPD, first carrying out 3-MCPD extraction process, other operation is same with upper, and obtaining measurement result is 27.3 ± 0.8 μ g/kg, and mark-on result of the test shows that the response rate is between 96-101%.
Embodiment ten: weigh 8g blended soy sauce, mix with the 5mol/LNaCl solution of 12mL, loading solid-phase extraction column, first with 80mL hexane-ether (90:10, v/v) eluting, then 250mL ether eluting is used instead, collecting diethyl ether eluant desolvation, add water 1mL shaken well, is subsequently adding 0.1mL sodium periodate solution (0.1mol/L) and carries out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.1mLPb (NO is added after having reacted3)2Solution (0.15mol/L), mixes and reacts 10min at latter 30 DEG C to remove excessive unreacted sodium metaperiodate.The 1-aminoisoquinoline of the 0.005mol/L then adding 1mL performs the derivatization reaction, reacts 200min at 70 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of silica gel mutually, and mobile phase is acetonitrile；Excitation wavelength is 310nm, and fluoroscopic examination wavelength is 450nm；Quantified by external standard method.Replication 10 times, obtaining 3-MCPD content in oil sample is 23.6 ± 0.3 μ g/kg, and mark-on result of the test shows that the response rate is between 98-103%.
Embodiment 11: weigh 8g hydrolyzed vegetable protein, mix with the 5mol/LNaCl solution of 12mL, loading solid-phase extraction column, first with 80mL hexane-ether (90:10, v/v) eluting, then 250mL ether eluting is used instead, collecting diethyl ether eluant desolvation, add water 1mL shaken well, is subsequently adding 0.1mL sodium periodate solution (0.1mol/L) and carries out oxidation reaction, reaction temperature is 30 DEG C, and the response time is 20min；0.2mLNa is added after having reacted2SO3Solution (0.1mol/L), mixes and reacts 100min at latter 20 DEG C to remove excessive unreacted sodium metaperiodate.The 1-aminoisoquinoline of the 0.015mol/L then adding 1mL performs the derivatization reaction, reacts 200min at 40 DEG C；It is settled to 5mL after having reacted, adopts the detection of HPLC-FLD method: fixing is the anti-phase ODS filler of zirconio matter mutually, and mobile phase is methanol-pH12.0 calcium hydroxide buffer solution (45:55, v/v)；Excitation wavelength is 290nm, and fluoroscopic examination wavelength is 390nm；Quantified by external standard method.Replication 10 times, obtaining 3-MCPD content in oil sample is 25.6 ± 0.3 μ g/kg, and mark-on result of the test shows that the response rate is between 97-104%.
Table 1 is adenine, 2-quinolin-2-ylamine, 2-aminoquinoxaline is condition and the effectiveness comparison that derivatization reagent measures 3-MCPD content.
1 three kinds of different derivatization reagents of table measure effectiveness comparison
From Fig. 1-3 and table 1, after adopting novel fluorescence derivatization reagent 2-quinolin-2-ylamine, 2-aminoquinoxaline derivatization, the hydrophobicity of target product is remarkably reinforced than adenine derivative products, after in mobile phase, methanol concentration increases many times, retention time is still long than adenine derivative products, this is higher with anti-impurity interference performance for the raising of post effect, and accuracy of measurement and repeatability are also better, and the detection limit of method is also lower.
Claims (8)
1. a 3-chloro-1, the high performance liquid chromatography-fluorescence method of 2-propylene glycol, the 3-MCPD therein step being cracked into 2-Chloro-1-ethanal is made including being processed through periodate solution by test sample 3-MCPD aqueous solution, it is characterised in that: the method is further comprising the steps of:
1) remove unreacted excess of periodate, eliminate side reaction；
2) fluorescence derivation reaction: 2-Chloro-1-ethanal and fluorescence derivatization are reacted and generates the target substance with fluorescent effect；Described fluorescence derivatization is 2-quinolin-2-ylamine, 1-aminoisoquinoline, 3-aminoisoquinoline, 2-aminoquinoxaline, 4-amido quinazoline or 2-amino-4-hydroxy quinoline；
3) HPLC-FLD measures: target substance is easily separated, and according to its chromatographic peak area, 3-MCPD is carried out quantitatively；Described HPLC adopts reversed phase chromatography pattern, and fixing is the silica gel ODS of alkali resistance, zirconio matter ODS or polymeric matrix ODS filler mutually, and mobile phase is methanol-buffer or acetonitrile-buffer, and ratio is (5%:95%)-(100%:0%)；The pH of described buffer is 7-13；
Described fluorescence derivatization is adjacent assorted two membered ring compounds of amino benzo N being structured with formula:
Wherein, dotted line circulus represents nitrogen heterocyclic ring or substituted nitrogen heterocyclic ring structure.
2. the high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol according to claim 1, it is characterised in that:
Described step 1) in, remove the method for excess of periodate for adding sulfite solution or lead salt solution, addition is 1:(1-2 in the ratio of inferior sulfate radical or lead ion and periodate), add the 10-80% that volume is described 3-MCPD aqueous solution volume, reaction temperature is 5-30 DEG C, and the response time is 10-120min.
3. the high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol according to claim 1 or claim 2, it is characterised in that:
Described step 2) in, the concentration of fluorescence derivatization is 0.002-0.02mol/L, and reaction temperature is 37-100 DEG C, and the response time is 10-240min.
4. the high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol according to claim 1 or claim 2, it is characterised in that:
Described step 3) in, FLD excitation wavelength is 260-320nm, and detection wavelength is 350-470nm.
5. the high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol according to claim 1 or claim 2, it is characterised in that:
Described 3-MCPD aqueous solution is through filtration, the river of centrifugal treating, river, lake water, subsoil water, plant chimney stalk, tap water or drinking water.
6. the high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol according to claim 1 or claim 2, it is characterised in that:
Described 3-MCPD aqueous solution is to process vegetable and animals oils fat prod through following steps to prepare:
A) oil sample is weighed；
B) add hexane and the acetic acid solution containing NaCl extracts, discard upper organic phase；
C) repeat to extract once with hexane, discard upper organic phase.
7. the high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol according to claim 1 or claim 2, it is characterised in that:
Described 3-MCPD aqueous solution is to process solid-state through following steps to prepare containing oil and fat product, and described solid-state includes fried instant noodle, potato chips, fritters of twisted dough or oil cake containing oil and fat product:
A) weigh the sample quality containing oil and fat product and pulverize；
B) add hexane to carry out extracting more than 2 times, combining extraction liquid；
C) add water in merge extract in extract, discard upper organic phase.
8. the high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol according to claim 1 or claim 2, it is characterised in that:
Described 3-MCPD aqueous solution is to prepare through Solid-Phase Extraction non-oil lipid product, and described non-oil lipid product is soy sauce or hydrolyzed vegetable protein.
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| CN110208406B (en) * | 2019-05-31 | 2020-11-20 | 浙江大学 | Detection method and application of mercapturic acid adduct for evaluating short-term exposure of 3-chloro-1,2-propanediol and ester thereof |
| CN112505006B (en) * | 2020-11-06 | 2022-06-24 | 宁波工程学院 | Device and method for identifying olive oil quality based on three-dimensional fluorescence characteristic peak |
| CN112946157B (en) * | 2021-02-08 | 2023-10-27 | 武汉轻工大学 | Application of fluorescent thiourea derivatization reagent in detection of 3-chloro-1, 2-propanediol |
| CN115326774B (en) * | 2022-09-15 | 2024-04-16 | 武汉轻工大学 | 3-MCPD detection method based on fluorescence analysis and catechol |
| CN115753722A (en) * | 2022-12-10 | 2023-03-07 | 武汉轻工大学 | 3-MCPD detection method based on dopamine fluorescence quenching effect |
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