CN108333276B - Method for identifying high fructose syrup adulterated honey - Google Patents
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Abstract
The invention provides a method for identifying adulterated honey with high fructose syrup, which is characterized in that α -dicarbonyl compounds are used as target substances, and a honey sample containing the target substances is detected to be the adulterated honey with high fructose syrup, wherein α -dicarbonyl compounds with indicating effect comprise one or more of 1, 2-diketone-pentose, 2, 3-diketone-pentose, 3-deoxy-1, 2-diketone-pentose and 1-deoxy-2, 3-diketone-pentose.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for detecting honey doped with high fructose syrup.
Background
The honey is natural sweet substance obtained by collecting nectar, secretion or honeydew of plants by bees, combining with secretion, and brewing. Since honey has important nutritive value and health care function, it is popular with people for a long time. Honey is a food which is easy to adulterate. Driven by benefits, in recent years, some individual households and manufacturers mix sugar, invert sugar, fructose, glucose high fructose syrup, fructose and the like into honey to manufacture adulterated honey, some manufacturers even use the glucose, the glucose high fructose syrup, the honey essence and the like to manufacture the honey to be put on the market in the form of natural honey, and cheat consumers to obtain violence, so that the product reputation of the natural honey in China is damaged. Particularly, a novel adulteration method appears in recent years, namely adulteration of rice high fructose syrup and beet high fructose syrup, because the adulteration method is close to the C isotope of honey, and some polysaccharides are removed by combining a relatively advanced manufacturing process, so that the true and false of the honey can not be distinguished by the conventional detection methods, such as a high fructose starch high fructose syrup determination method in the honey, thin-layer chromatography and ion chromatography, a carbon-4 plant sugar content determination method, a stable carbon isotopologue method and the like. Meanwhile, due to the existence of multiple methods, the raw materials are detected by enterprises, and the multiple methods are often used for simultaneous detection, so that the enterprise cost is greatly increased, and the healthy development of the industry is not facilitated. Therefore, an identification method capable of simultaneously determining various high fructose syrup adulterated honey is urgently needed. In recent years, a plurality of high fructose syrup identification methods are simultaneously tried by successive articles and some laboratory methods, but the method has poor scientificity and high misjudgment rate because no clear target or standard substance is used as support.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for simultaneously identifying various types of high fructose syrup adulterated honey.
The technical scheme for realizing the above purpose of the invention is as follows:
a method for identifying high fructose syrup adulterated honey takes α -dicarbonyl compound as a target substance, and a honey sample containing the target substance is detected to be the high fructose syrup adulterated honey;
α -dicarbonyl compounds having an indicating function include one or more of 1, 2-diketo-pentose, 2, 3-diketo-pentose, 3-deoxy-1, 2-diketo-pentose, and 1-deoxy-2, 3-diketo-pentose.
Further, the detection method is one or two of liquid chromatography fluorescence detection method, high performance liquid chromatography time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry.
Preferably, one of o-phenylenediamine, 5, 6-diamino-2, 4-dihydroxypyrimidine, 2,4, 5-triamino-6-hydroxypyrimidine is used as the derivatizing agent.
Wherein the derivatization condition is that 1-20 g of honey sample is taken, excessive derivatization agent is added under the condition of pH value of 3.0-9.0, the reaction temperature is from 10 ℃ to 130 ℃, and the reaction lasts for 30 min-24 h.
The preparation method of the α -dicarbonyl compound quantitative standard substance comprises the steps of taking ribose, glucose and a derivative with a molar ratio of 1: 1: 2 (0.5-2), reacting in a buffer solution with a pH value of 3.0-9.0, preferably 4.0-5.0, at 100-150 ℃ for 50-80 min, and cooling in ice water after reaction.
In the method, a positive threshold value is set in such a manner that when any one or more of these 4 α -dicarbonyl compounds are detected in a sample and the content of any one of the 4 compounds exceeds a predetermined level, i.e., the content of 1, 2-diketo-pentose, 2, 3-diketo-pentose is not less than 50. mu.g/kg, the content of 3-deoxy-1, 2-diketo-pentose, and the content of 1-deoxy-2, 3-diketo-pentose is not less than 20. mu.g/kg, the sample can be judged to be a high fructose syrup adulteration (addition of more than 20%) in combination with the detection limit of the method.
One preferable technical scheme of the invention is that the method comprises the following steps:
1) derivatization: putting the honey sample in a buffer solution with the pH value of 3.0-9.0, wherein the preferable pH value is 4-5; adding a derivatization agent solution with the mass concentration of 0.1-5%, and reacting for 10-20 h under the conditions of normal temperature and light protection, wherein the proportion of the honey sample to the derivatization agent solution is 1-3 g: 1 mL;
2) and (3) purification: purifying the reaction solution by a reverse phase column, washing with water, eluting with a mobile phase, diluting to constant volume with water, filtering with a filter membrane,
3) and (3) chromatographic detection: detection using liquid chromatography fluorescence or mass spectrometry.
Wherein, the reversed phase chromatographic column in the step 2) is one of C18, C8 and phenyl packed column; the mobile phase is acetonitrile water solution or methanol water solution, and the flow rate is 0.2-1.0 mL/min.
In the liquid chromatography fluorescence detection in the step 3), the excitation wavelength of the fluorescence detector is 270-370 nm, and the emission wavelength is 420-520 nm.
The mass spectrometry method in the step 3) is an electrospray mass spectrometry method, the temperature of atomizing gas is 250-350 ℃, the flow rate of atomizing gas is 6-12L/min, the pressure of atomizing gas is 30-50 psi, the temperature of sheath gas is 260-350 ℃, the flow rate of sheath gas is 6-12L/min, the ionization voltage is 4000kv, the fragment voltage is 60-175, the scanning mode is a positive ion mode, and the full scanning and multi-reaction monitoring modes are adopted. The monitoring ion is the derivatization reaction product of the three different derivatizing agents described above. In the full-scanning mode, the quasi-molecular ions of the compounds and the fragments with the characteristics thereof are monitored, and one molecule or two molecules H are lost2O, loss of CH2O、CH2CO and CHOH ═ CHOH. When multiple reaction monitoring mode is used, the excimer ions M + H are used toAnd M + H-18, M + H-36 and M + H-30 are used as monitoring ion pairs for quantitative detection.
The technical scheme of the invention has the following advantages:
according to the invention, through research on a processing technology of adding honey into high fructose syrup, the types and contents of α -dicarbonyl compounds are different due to different raw materials and processing modes, so that a method capable of identifying the adulterated honey of various types of high fructose syrups is determined by taking α -dicarbonyl compounds as target substances.
The method provided by the invention is simple and effective, can be used for one-time analysis and can cover the main high fructose syrup adulterated honey at present.
Aiming at the problems that α -dicarbonyl compound has small molecular weight, is easy to dissolve in water and can not directly prepare the pure product of the compound, α -dicarbonyl compound is generated in a Maillard reaction system of pentose and cysteine, and the compound can easily react with o-phenylenediamine, 5, 6-diamino-2, 4-dihydroxypyrimidine and 2,4, 5-triamino-6-hydroxypyrimidine to generate stable quinoxaline and pteridine derivatives.
Drawings
FIG. 1 Mass Spectrometry TIC plots of 1, 2-diketo-pentose, 2, 3-diketo-pentose in high fructose syrup and honey.
FIG. 2 is a graph of mass spectra TIC of 3-deoxy-1, 2-dione-pentose and 1-deoxy-2, 3-dione-pentose in high fructose syrup and honey.
FIG. 3 is a chromatogram of 4 kinds of characteristics of high fructose syrup and honey by fluorescence chromatography.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the examples, unless otherwise specified, the methods used are conventional in the art.
Example 1
1. Common high fructose syrup samples of experimental samples 11 rice high fructose syrup samples, No. DM1-DM 11; 23 corn high fructose syrup samples with numbers of YM1-YM 23; 9 beet high fructose syrup samples with the numbers of TC1-TC 9; 14 sucrose high fructose syrups with numbers of ZT1-ZT 14; 6 cassava high fructose syrup.
2. Real honey samples for experiments: 32 rape honeys with the number YC1-YC 32; 33 acacia honey with the number of YH1-YH 33; 15 jujube honey with the number ZH1-ZH 15; 12 linden honey with the number of DS1-DS 12; 30 vitex negundo honey with the number of JT1-JT 30; 5 litchi honey with the serial number of LZ1-LZ 5; 3 longan honey with the serial number of LY1-LY 3; 4 buckwheat and honey are numbered as QM1-QM 4; 5 sunflower honey with the number of XRK1-XRK 5; 7 manuka honey with the number of MNK1-MNK 7; clover honey 4, number SYC1-SYC 3; 32 mixed honeys of various flowers are numbered DHZ1-DHZ 32; 13 medium honey with the number of ZF1-ZF 13.
3. Sample extraction and derivatization (in the three derivatization agent tests of examples 1, 3 and 4, the derivatization agent concentration is mass concentration): weighing 2g of high fructose syrup or honey sample, dissolving the high fructose syrup or honey sample in 2mL of phosphate buffer solution with the pH value of 5.0, adding 1mL of 1% o-phenylenediamine aqueous solution, and reacting for 24 hours at normal temperature in a dark place;
4. sample purification- ① derivatized sample was directly measured after filtration through a microporous membrane (this is an experiment where differences were found at the beginning). ② derivatized sample was purified in batches through an OASIS HLB (6mL, 200mg, activated with 5mL methanol and 10mL water before use) column, and after the sample was eluted with 3mL water, eluted with 3mL acetonitrile, and then added with water to a constant volume of 10mL, and measured after filtration through a membrane.
Typical information on derivatives of o-phenylenediamine is as follows:
o-phenylenediamine derivatives of 1, 2-diketo-pentoses:
o-phenylenediamine derivative of 2, 3-diketo-pentose:
o-phenylenediamine derivative of 3-deoxy-1, 2-dione-pentose:
o-phenylenediamine derivative of 1-deoxy-2, 3-dione-pentose:
5. the α -dicarbonyl compound derivatives are analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF/MS), and compared with the difference between α -dicarbonyl compounds of fructose syrup and natural honey, the α -dicarbonyl compound derivatives are used for screening a characteristic α -dicarbonyl compound indicator, and the specific conditions of the liquid chromatography tandem mass spectrometry are as follows:
liquid chromatography, namely gilent 6545UHPLC-Q-TOF/MS, chromatographic column Agilent AQ C18, 2.1 × 100, 3.5 mu m, column temperature of 30 ℃, mobile phase, namely methanol A + 0.2% formic acid water solution B, gradient elution is carried out for 0-3min, A + B is 10+90, 3-15min, A + B is 60+40, 15-20min, A + B is 10+90, 20-21min, A + B is 90+10, 21-30min, A + B is 90+10, flow rate is 0.2mL/min, ultraviolet detection wavelength is 280nm, 320nm, needle insertion volume is 10 mu L;
mass spectrometry ESI +, temperature of atomizing gas: 320 ℃, flow rate of atomizing gas: 6L/min, pressure of atomizing gas: 40psi, temperature of sheath gas: 320 ℃, flow rate of sheath gas: 16L/min, ionization voltage: 4000kv, fragmentation voltage 115, scanning mode: full scan, scanning range 60-500, secondary fragment, Target MS/MS mode, linear collision 0-25eV, three derivatives mass spectrometry fragments are identified, mass spectrometry TIC charts of α -dicarbonyl compound derivative (o-phenylenediamine) in high fructose syrup and natural honey are shown in FIGS. 1 and 2, FIG. 1 is a comparison of difference between 1, 2-dione-pentose and 2, 3-dione-pentose in high fructose syrup and honey (extraction 221.0921 (retention time 1.908, 2.089), no related compound is extracted at this position in honey chart, it can be seen that content in high fructose syrup is significantly higher than in normal honey), FIG. 2 is a comparison of difference between 3-deoxy-1, 2-dione-pentose and 1-dione-2 in honey (trace amount of related compound in high fructose syrup is seen in honey is shown in graph 112.2).
As can be seen from FIG. 1, the high fructose syrup and honey have significant difference in α -dicarbonyl compounds, the α -dicarbonyl compounds 1, 2-diketo-pentose, 2, 3-diketo-pentose shared by natural honey and high fructose syrup, but the content is significantly different (comparison of chromatographic peak heights), the 3-deoxy-1, 2-diketo-pentose, 1-deoxy-2, 3-diketo-pentose, 1, 4-dideoxy-2, 3-diketo-pentose, etc. α -dicarbonyl compounds in high fructose syrup have significantly higher content than natural honey, and can be used as indicators for identifying various types of high fructose syrup faked honey, the typical mass spectrum information (mass number error + -5 ppm) of o-phenylenediamine, 5, 6-diamino-2, 4-dihydroxypyrimidine, 1, 2-diketo-pentose, 2, 3-deoxy-1, 2-diketo-pentose, 1-deoxy-2, 3-dione-derivatives,
wherein the 1, 2-diketone-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 221.092020]＝203.0181,[M+H-2H20]＝185.0707,[M+H-H20-CH2O]＝173.0075，[M+H-2H20-CO]＝157.0757；
2, 3-diketo-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 221.092120]＝203.0182,[M+H-2H20]＝185.0709,[M+H-H20-CH2CO]＝173.0074；[M+H-H20-CH2CO]＝161.0075；
3-deoxy-1, 2-dione-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 205.097120]＝187.0864,[M+H-2H20]＝169.0758,[M+H-H20-CH2O]＝157.0757；
1-deoxy-2, 3-dione-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 205.097020]＝187.0865,[M+H-2H20]＝169.0757,[M+H-CH2O]＝175.0865。
6. Preparation of main characteristic substance derivatives in order to accurately quantify 4 kinds of α -dicarbonyl compound content and distribution in high fructose syrup and honey, need to prepare 4 kinds of compound standard substance, because α -dicarbonyl compound molecular weight is small, easily soluble in water, it is impossible to prepare the compound directly to obtain pure product, solution in Maillard reaction system of pentose and cysteine can generate α -dicarbonyl compound, at the same time, the compound can easily react with o-phenylenediamine, 5, 6-diamino-2, 4-dihydroxypyrimidine and 2,4, 5-triamino-6-hydroxypyrimidine to generate stable quinoxaline and pyridine derivatives, because o-phenylenediamine is cheap and easy to purchase, this example selects o-phenylenediamine as derivatization reagent, and prepares quinoxaline derivatives of various kinds of α -dicarbonyl compounds according to the reaction, and uses as standard substance, accurate quantification of α -dicarbonyl compound in high fructose syrup and honey, filter membrane α -dicarbonyl compound reaction liquid, 0.1M ribose and 0.1M glucose, 0.2.82-cysteine and 0.1M phosphate buffer solution are put in a chromatographic box after 0.1M of pteridine, 0.115 is added in a liquid phase chromatographic analysis, the pH is 0.2.2 min, and is added in a liquid phase after being dried, and the reaction is 60 mL, and is used for analysis, and is used for liquid chromatography.
The liquid chromatographic conditions of the standard substance are that a chromatographic column Zorbax SB-C18, 21.2 × mm, a mobile phase is water and methanol, a gradient elution procedure is that the chromatographic column Zorbax SB-C18, 21.2 × mm, the mobile phase is water and methanol, the gradient elution procedure is that the chromatographic column is 0-8 min, 30% B, 8-20 min, 30% B, 20-30 min, 60% B, 30% B, the flow rate is 18ml/min, the sample amount is 2ml, the detection wavelength is 320nm, eluent of α -dicarbonyl compound quinoxaline derivatives corresponding to each peak is taken according to the peak emergence time, wherein 8-10min is 1, 2-diketone-pentose, 10.5-12.5min is 2, 3-diketone-pentose, 15-17min is 3-deoxy-1, 2-diketone-pentose, 17.7-19.5min is 1-deoxy-2, 3-diketone-pentose, the frozen liquid is dried to obtain a mass spectrum, the mass spectrum is used for determining the purity of 320nm and 254nm substances by comparing the liquid chromatographic column, and the purity of the freeze-carbonyl compound is more than 96 percent of honey derivatives required by high resolution analysis and high purity 36539 analysis.
7. Liquid chromatography fluorescence detection analysis of α -dicarbonyl compounds in high fructose syrup and honey 1) sample treatment according to step 3, weighing 2g of high fructose syrup or honey sample, dissolving the high fructose syrup or honey sample in 2mL of phosphate buffer solution with pH value of 5.0, adding 1% o-phenylenediamine, reacting at normal temperature in a dark place for 24h, directly measuring the derivatized sample after being filtered by a microporous filter membrane, 2) purifying the derivatized sample liquid by OASIS HLB (6mL and 200mg, activating the sample liquid by 5mL of methanol and 10mL of water before use) in batches, washing the sample liquid by 3mL of water, eluting the sample liquid by 3mL of acetonitrile, adding water to fix the volume to 10mL, filtering the sample liquid by the filter membrane, and measuring by a liquid chromatography fluorescence detector (WATERS UPLC-FLD), wherein the chromatographic column is ACQUITY BEH C18,1.7 mu m,2.1 × 100mm, the column is 30 ℃, the flow rate is 0.4mL/min, the mobile phase is applied, the mobile phase is eluted by a mobile phase A, the mobile phase is 1min, the mobile phase B is 10min, the mobile phase is eluted by the wavelength of 2.7-80% methanol, the mobile phase B, the mobile phase is set by the wavelength of 2.1.7 min, the wavelength of 3-10 nm after the fluorescence detector, and the wavelength of the fluorescence emission is set from the wavelength of the.
Typical chromatograms of high fructose syrup and honey samples are shown in figure 3. FIG. 3 is a chromatogram of 4 kinds of characteristic substances in high fructose syrup and honey by fluorescence chromatography (from the chart, it can be seen that the content of four compounds in syrup is obviously higher than that of four compounds in honey).
The detection limits LOD of the 4 compounds are respectively 4.5 mu g/kg of 1, 2-diketone-pentose; 4.6 mug/kg of 2, 3-diketo-pentose; 3.3. mu.g/kg of 3-deoxy-1, 2-dione-pentose; the 1-deoxy-2, 3-dione-pentose was 3.2. mu.g/kg.
All high fructose syrup samples contained α -dicarbonyl compounds, particularly 2, 3-diketo-pentose, 3-deoxy-1, 2-diketo-pentose and 1, 4-dideoxy-2, 3-diketo-pentose, in trace amounts in ordinary honey, and remained high in the syrup, and considering the detection limit of the binding method, a positive threshold was set such that when any one or more of the 4 α -dicarbonyl compounds were arbitrarily detected in the sample, and as long as the content of any one of the 4 compounds exceeded the specification, that is, the content of 1, 2-diketo-pentose, 2, 3-diketo-pentose was not less than 50 μ g/kg, the content of 3-deoxy-1, 2-diketo-pentose, and the content of 1-deoxy-2, 3-diketo-pentose was not less than 20 μ g/kg, the sample was judged to be high fructose syrup adulterated (addition of more than 20%).
By adding different high fructose syrups into natural honey, respectively adding 10%, 20%, 30% and 50%, analyzing the added samples according to the processing and detecting methods, and analyzing 4 α -dicarbonyl compounds, various high fructose syrups with 20% of added can be accurately identified.
The method is simple and effective, and can cover the main high fructose syrup adulterated honey at present through one-time analysis, and the prepared standard product can accurately quantify 4 α -dicarbonyl compounds in high fructose syrup and honey samples, thereby providing an effective scientific basis for scientifically identifying the high fructose syrup adulterated honey.
Example 2
Sample source: 20 suspected high fructose syrup adulteration samples are sent and detected by a general station of bee industry in Shandong province, and 15 samples are determined by a stable carbon isotope ratio method through a carbon-4 plant sugar content determination method in GB/T18932.1-2002 honey, so that the corn high fructose syrup adulteration is confirmed. The other 2 samples were considered to be sugar beet high fructose syrup adulterated samples, 2 were rice high fructose syrup adulterated samples, and 1 was malt high fructose syrup adulterated samples.
Sample treatment: weighing 2g of sample, adding 2mL of phosphate buffer solution with pH of 6.0 to dissolve the sample, adding 1mL of 1% o-phenylenediamine solution, mixing for 1min, reacting overnight in a dark place for 12h, filtering by 0.22 mu m, and measuring by using ultra performance liquid chromatography tandem mass spectrometry.
The conditions of the liquid chromatography tandem mass spectrometry are that an Agilent 6495 tandem mass spectrometer and a chromatographic column Agilent ZorbaxSB-Aq are 2.1 × 100mm and 3.5um, the column temperature is 30 ℃, the mobile phase comprises A0.1 percent formic acid aqueous solution and B methanol, the gradient elution is carried out for 0 to 2min and 10 percent B, the gradient elution is carried out for 2 to 10min and 60 percent B, the gradient elution is carried out for 10 to 15min and 80 percent B, the gradient elution is carried out for 15 to 16min and 10 percent B, the gradient elution is carried out for 16 to 25min and 10 percent B, the flow rate is 0.2ml/min, and the needle insertion volume is 10 mu L;
mass spectrometry analysis: an ionization mode: electrospray positive ion mode (ESI +), equipped with an ion funnel device; temperature of atomized gas: 320 ℃, atomizing gas flow rate: 6L/min; atomizing gas pressure: 45 psi; temperature of sheath gas: 320 ℃; flow rate of sheath gas: 12L/min; ionization voltage: 4 kv; the scanning mode is as follows: multiple reaction detection (MRM), specific detection ions are shown in Table 1 below
TABLE 14 conditions for monitoring ion pairs and data acquisition of α -dicarbonyl quinoxaline derivatives
a is a quantitative ion.
And (4) analyzing results:
2, 3-diketone-pentose (the content range is 76-190 mug/kg), 3-deoxy-1, 2-diketone-pentose (the content range is 35-290 mug/kg) and the content of the 2, 3-diketone-pentose is obviously larger than a set threshold value are detected from 20 samples and 15 corn high fructose syrup samples, the high fructose syrup is judged to be adulterated, and the result is consistent with the national standard isotope method;
2 suspected beet adulteration samples, detecting 2, 3-diketone-pentose (the content is 97 mug/kg and 136 mug/kg respectively) and 3-deoxy-1, 2-diketone-pentose (77 mug/kg and 139 mug/kg) and judging the samples as positive samples;
2 suspected rice high fructose syrup adulteration samples, detecting 2, 3-diketone-pentose (the content is 67 mug/kg and 103 mug/kg respectively) and 3-deoxy-1, 2-diketone-pentose (the content is 79 mug/kg and 67 mug/kg respectively) and judging the samples as positive samples;
3-deoxy-1, 2-dione-pentose (the content is 309 mu g/kg) is detected from 1 malt high fructose syrup sample, the maltose content of the sample is analyzed by liquid chromatography, the maltose content of the sample is more than 50 percent, and the sample is obviously an adulteration sample (the maltose content of a natural honey sample is less than or equal to 5 percent), and the result is consistent with the result identified by the method.
The method is compared with the common honey adulteration detection method shown in the following table 2.
Table 2 test results of honey samples as described in example 2
As can be seen from Table 2, compared with the existing method, the method can realize one-time analysis and judge various types of high fructose syrup adulterated honey.
Example 3
The honey samples were as in example 1. The step 3 is: weighing 2g of high fructose syrup or honey sample, dissolving the high fructose syrup or honey sample in 2mL of phosphate buffer solution with the pH value of 5.0, adding 1mL of 1% 5, 6-diamino-2, 4-dihydroxypyrimidine aqueous solution, reacting the mixture at 100 ℃ in a dark place for 1h, and cooling the mixture by ice cubes.
The other operations were the same as in example 1.
The derivatives obtained when diffracted with different derivatizing agents have the following structures:
the 5, 6-diamino-2, 4-dihydroxypyrimidine derivatives of 1, 2-diketo-pentoses are:
the 5, 6-diamino-2, 4-dihydroxypyrimidine derivatives of 2, 3-diketo-pentoses are:
the 5, 6-diamino-2, 4-dihydroxypyrimidine derivatives of 3-deoxy-1, 2-dione-pentoses are:
the 5, 6-diamino-2, 4-dihydroxypyrimidine derivatives of 1-deoxy-2, 3-dione-pentoses are:
the derivatizing agent 5, 6-diamino-2, 4-dihydroxypyrimidine, typical mass spectrometric information (mass number error ± 5ppm) is:
wherein the 1, 2-diketone-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 254.088220]＝236.0779,[M+H-2H20]＝218.0761,[M+H-H20-CH2O]＝206.0036，[M+H-2H20-CO]＝190.7531；
2, 3-diketo-pentose excimer ion [ M + H]254.0884, major debris is separatedThe seed is [ M + H-H20]＝236.0776,[M+H-2H20]＝218.0763,[M+H-H20-CH2O]＝206.0034，[M+H-H20-CH2CO]＝194.0037；
3-deoxy-1, 2-dione-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 238.093220]＝220.0827,[M+H-2H20]＝202.0720,[M+H-H20-CH2O]＝190.0719；
1-deoxy-2, 3-dione-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 238.093020]＝220.0824,[M+H-2H20]＝202.0718,[M+H-CH2O]＝207.7555；
1, 4-dideoxy-2, 3-dione-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 222.097120]＝204.0878,[M+H-CH2CHOH]＝178.0711；
3-deoxyglucosenone excimer ion [ M + H ] ═ 268.1043; [ M + H-H20] ═ 250.0934, [ M + H-2H20] ═ 232.0828, [ M + H-H20-CH2O ] ═ 220.0824, [ M + H-2H20-CO ] ═ 204.0883
Buspirone excimer [ M + H ] ═ 192.0879.
Example 4
Weighing 2g of high fructose syrup or honey sample, dissolving the high fructose syrup or honey sample by using 2mL of phosphate buffer solution with the pH value of 9.0, adding 1mL of 1% 2,4, 5-triamino-6-hydroxypyrimidine aqueous solution, reacting the mixture at 60 ℃ in a dark place for 1 hour, and cooling the mixture by ice cubes.
The other operations were the same as in example 1. The corresponding information for a typical 2,4, 5-triamino-6-hydroxypyrimidine derivative is as follows:
2,4, 5-triamino-6-hydroxypyrimidine derivatives of 1, 2-diketo-pentose
2,4, 5-triamino-6-hydroxypyrimidine derivatives of 2, 3-diketo-pentose:
2,4, 5-triamino-6-hydroxypyrimidine derivatives of 3-deoxy-1, 2-dione-pentose
2,4, 5-triamino-6-hydroxypyrimidine derivatives of 1-deoxy-2, 3-dione-pentose sugars:
typical mass spectrometric information (mass number error. + -. 5ppm) is 2,4, 5-triamino-6-hydroxypyrimidine
Wherein the 1, 2-diketone-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 255.072320]＝237.0618,[M+H-2H20]＝219.0601,[M+H-H20-CH2O]＝206.9876，[M+H-2H20-CO]＝191.7371；
2, 3-diketo-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 255.072420]＝237.0616,[M+H-2H20]＝219.0603,[M+H-H20-CH2O]＝206.9877，[M+H-H20-CH2CO]＝194.9876；
3-deoxy-1, 2-dione-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 239.077320]＝221.0667,[M+H-2H20]＝203.0560,[M+H-H20-CH2O]＝191.0559；
1-deoxy-2, 3-dione-pentose excimer ion [ M + H]The predominant fragment ion is [ M + H-H ] 239.077020]＝221.0665,[M+H-2H20]＝203.0558,[M+H-CH2O]＝208.7394；
3-deoxyglucuronone excimer ion [ M + H]＝255.0723；[M+H-H20]＝237.0614，[M+H-2H20]＝219.0511，[M+H-H20-CH2O]＝207.0514，[M+H-2H20-CO]＝191.0563
Buspirone excimer [ M + H ] ═ 193.0722.
Although the present invention has been described in the foregoing by way of examples, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (9)
1. A method for identifying high fructose syrup adulterated honey is characterized in that α -dicarbonyl compound is used as a target substance, and a honey sample containing the target substance is detected to be the high fructose syrup adulterated honey;
α -dicarbonyl compounds having an indicating function include one or more of 1, 2-diketo-pentose, 2, 3-diketo-pentose, 3-deoxy-1, 2-diketo-pentose, 1-deoxy-2, 3-diketo-pentose;
wherein, one of o-phenylenediamine, 5, 6-diamino-2, 4-dihydroxypyrimidine and 2,4, 5-triamino-6-hydroxypyrimidine is used as a derivatization agent, the derivatization condition is that 1-20 g of honey sample is taken, excessive derivatization agent is added under the condition of pH value of 3.0-9.0, the reaction temperature is from 10 ℃ to 130 ℃, and the reaction is carried out for 30 min-24 h;
the positive threshold value is set so that a sample is judged to be a high fructose syrup adulterated when any one or more of the 4 α -dicarbonyl compounds are detected in the sample and the content of any one of the 4 compounds exceeds the following specification, i.e., the content of 1, 2-diketo-pentose, 2, 3-diketo-pentose is not less than 50. mu.g/kg, the content of 3-deoxy-1, 2-diketo-pentose, and the content of 1-deoxy-2, 3-diketo-pentose is not less than 20. mu.g/kg.
2. The method of claim 1, wherein the detection is performed by one or both of liquid chromatography fluorescence detection, high performance liquid chromatography time-of-flight mass spectrometry, and liquid chromatography tandem mass spectrometry.
3. The method according to claim 1, wherein the standard substance for quantifying α -dicarbonyl compound is prepared by reacting ribose, glucose and a derivatizing agent at a molar ratio of 1: 1: 2 (0.5-2) in a buffer solution having a pH of 3.0-9.0 at 100-150 ℃ for 50-80 min, and cooling the reaction product in ice water.
4. The method of claim 3, wherein the pH is in the range of 4.0 to 5.0.
5. A method according to any one of claims 1 to 4, comprising the steps of:
1) derivatization: adding a derivative solution with the mass concentration of 0.1-5% into a buffer solution with the pH = 3.0-9.0, reacting for 10-20 h at normal temperature under the condition of keeping out of the sun, wherein the proportion of the honey sample to the derivative solution is 1-3 g: 1 mL;
2) and (3) purification: purifying the reaction solution by a reverse phase column, washing with water, eluting with a mobile phase, diluting to constant volume with water, filtering with a filter membrane,
3) and (3) chromatographic detection: detection using liquid chromatography fluorescence or mass spectrometry.
6. The method of claim 5, wherein the pH is in the range of 4.0 to 5.0.
7. The method of claim 5, wherein the reverse phase chromatography column of step 2) is one of C18, C8, phenyl packed column; the mobile phase is acetonitrile water solution or methanol water solution, and the flow rate is 0.2-1.0 mL/min.
8. The method according to claim 5, wherein in the liquid chromatography fluorescence detection in step 3), the excitation wavelength of the fluorescence detector is 270-370 nm, and the emission wavelength is 420-520 nm.
9. The method according to claim 5, wherein the mass spectrometry of step 3) is electrospray mass spectrometry, the temperature of the atomizing gas is 250-350 ℃, the flow rate of the atomizing gas is 6-12L/min, the pressure of the atomizing gas is 30-50 psi, the temperature of the sheath gas is 260-350 ℃, the flow rate of the sheath gas is 6-12L/min, the ionization voltage is 4000kv, the fragment voltage is 60-175, the scanning mode is positive ion mode, full scanning mode and multiple reaction monitoring mode.
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