Rapid detection of α-dicarbonyl compounds in honey based on direct mass spectrometry

Abstract

The present invention relates to the field of detection technology, and in particular to rapid detection of α-dicarbonyl compounds in honey based on direct mass spectrometry. The present invention uses nanoliter electrospray ion source mass spectrometry based on the principle of droplet reaction acceleration to achieve rapid derivatization and detection of α-dicarbonyl compounds in honey. Compared with the traditional chromatography-mass spectrometry method, the present invention does not require complex sample pretreatment, shortens the derivatization time from several hours to milliseconds, thereby achieving rapid analysis, and the entire mass spectrometry data acquisition process can be completed within 5 minutes, which has good application prospects.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to rapid detection of alpha-dicarbonyl compounds in honey based on direct mass spectrometry technology. Background Art

[0002] Honey is a natural sweet substance that is brewed by bees after collecting nectar or secretions from plants through their own enzymes and special substances. "Compendium of Materia Medica": Honey has five functions as a medicine: clearing away heat and detoxifying, tonifying the middle, removing toxins, moistening dryness, and relieving pain. "Ben Jing": It is used to treat evil spirits in the heart and abdomen, various convulsions, epilepsy, and deficiencies in the five internal organs, replenishing qi and tonifying the middle, relieving pain and detoxifying, and harmonizing all medicines. "Bie Lu": It nourishes the spleen, eliminates irritability, inability to eat or drink, stops diarrhea, muscle pain, mouth sores, and brightens the eyes and ears. Honey is not only a nutritious natural nourishing food, but also widely used in traditional Chinese medicine clinics, Chinese medicine processing, and Chinese medicine preparations as a commonly used Chinese medicine. It has many functions such as regulating the spleen and stomach, relieving pain, moistening the lungs and relieving coughs, moistening the intestines and laxatives, moisturizing the skin and regenerating muscles, and detoxification.

[0003] The conversion of nectar into honey must be completed entirely by bees, and no human intervention is allowed during the maturation process. Thermal processing promotes the accumulation of Maillard reaction products (MRPs), reduces enzyme activity, and loses heat-sensitive components, which seriously affects the quality and safety of honey. Honey is a good matrix for the Maillard reaction. It is rich in monosaccharides (glucose and fructose account for about 75%) and contains a variety of free amino acids. α-Dicarbonyl compounds are a class of highly reactive small molecules generated in the intermediate stage of the Maillard reaction, which can be used as an indicator to distinguish natural mature honey from artificial concentrated honey. Today, the method used to detect concentrated honey is mainly based on chromatography-mass spectrometry. Since α-dicarbonyl compounds cannot be directly detected, a derivatization reaction before analysis is essential, and this process often takes several hours, and is followed by complicated pretreatment processes such as purification and enrichment. Droplet reactions allow reactions to be accelerated under mild conditions, even for reactions under certain harsh conditions; therefore, droplet-accelerated reactions show great potential in organic synthesis, reaction mechanism elucidation, and reaction monitoring. Due to its high sensitivity and specificity, mass spectrometry is often used to monitor droplet reactions. The present invention uses nanoESI-MS to quickly derivatize α-dicarbonyl compounds in honey, overcoming the background interference generated during sample analysis and realizing its application in more complex food matrices. Summary of the invention

[0004] In view of the problems mentioned in the background technology, the present invention provides a method for rapid detection of α-dicarbonyl compounds in honey based on nano-electrospray ion source mass spectrometry (nanoESI-MS), which specifically adopts the following technical scheme:

[0005] The present invention provides a method for rapidly detecting α-dicarbonyl compounds in honey based on nanoESI-MS, comprising the following steps:

[0006] Step 1): Prepare the honey to be tested into a honey sample solution for later use;

[0007] Step 2): α-dicarbonyl compounds in the honey sample solution were detected by nanoESI-MS, the substances were qualitatively identified based on the secondary and tertiary fragment information of the standard, and relatively quantitatively identified based on the response intensity of the secondary fragment ions with higher responses.

[0008] As a further preferred embodiment of the above scheme, the preparation method of the honey sample solution in step 1) is: stir the honey to be tested at room temperature, add water to make up the volume, and centrifuge to obtain the supernatant to obtain the honey sample solution.

[0009] As a further preferred embodiment of the above scheme, in step 2), nanoESI-MS detection is performed using a LTQ-XL linear ion trap (LTQXL, Thermo Scientific, San Jose, CA).

[0010] As a further preferred embodiment of the above scheme, in the detection: the LTQ capillary temperature is 300° C., the tube lens voltage is 100 V; the spectrum is acquired in positive ion mode, and the secondary mass spectrum is acquired by collision induced dissociation (CID).

[0011] As a further preferred embodiment of the above scheme, the mass spectrometry scanning range is m / z 50-500, and the mass spectrometry data are analyzed on the software ThermoXcalibur Qual Browser 4.0.

[0012] As a further preferred embodiment of the above scheme, the detection adopts a pointed θ-shaped borosilicate glass capillary with an od of 1.5 mm.

[0013] As a further preferred embodiment of the above scheme, the tip θ-shaped borosilicate glass capillary is divided into two independent channels separated by a layer of glass in the middle; the honey sample solution is added to one of the channels, and the derivatization agent is added to the other channel to achieve rapid derivatization of α-dicarbonyl compounds.

[0014] As a further preferred embodiment of the above scheme, the derivatizing agent is o-phenylenediamine; the o-phenylenediamine is a 10 mg / mL aqueous solution.

[0015] As a further preferred embodiment of the above scheme, the MS of each substance obtained by collision induced dissociation is 2 and MS 3 Mass spectra are compared with references and standards to identify or predict metabolites.

[0016] As a further preferred embodiment of the above scheme, in the MS of m / z 235 2 In the mass spectrum, m / z 217 with the highest peak in the secondary mass spectrum was used as the relative quantitative ion.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The method of the present invention can achieve rapid derivatization, shortening the derivatization time of α-dicarbonyl compounds from several hours to milliseconds.

[0019] 2. The detection method of the present invention can realize rapid analysis. High-sugar honey samples do not need to undergo complex sample pretreatment, and the entire mass spectrometry data acquisition process can be completed within 5 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the nanoESI-MS device.

[0021] Figure 2 This is the primary mass spectrum of the honey sample collected by nanoESI-MS in positive ion mode.

[0022] Figure 3 These are the secondary and tertiary mass spectra of α-dicarbonyl compounds in honey samples.

[0023] Figure 4 Changes in the content of α-dicarbonyl compounds m / z 235 in honeys from different plant sources under thermal processing conditions at 55°C. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0026] Example

[0027] 1. Sample collection and processing

[0028] Immature honeycombs (numbered IH 1-IH 4) were placed in new honeycombs in the morning and collected in the evening of the same day; artificial concentrated honey (AH) was prepared in the laboratory using a rotary evaporator (immature honey IH 4 was concentrated by heating at 85°C for 2 h; rotary evaporator, parameter settings: speed 90 rpm, air pressure 100 mbar); all samples were stored at -20°C. One hour before analysis, the honey samples were placed at room temperature (25°C) and manually stirred to ensure homogeneity; 1.0 g of honey sample was diluted to 5.0 mL, centrifuged at 8000 rpm for 10 min, and the supernatant was stored at -80°C.

[0029] 2. Fabrication of Nanopipette

[0030] The nanopipette is a pipette with a conical inner diameter in the nanometer range, which is used to load samples and form an emission nozzle for sample electrospray as an ion source. The borosilicate glass capillary is melt-cut and drawn into a nanopipette using a P-97 drawing instrument.

[0031] 3. NanoESI-MS sample detection

[0032] Mass spectrometry (MS) experiments were mainly performed on a linear ion trap (LTQ XL, Thermo Scientific, San Jose, CA). The LTQ capillary temperature was 300 °C, the tube lens voltage was 100 V, the spectra were acquired in positive ion mode, and the secondary mass spectra were acquired by collision-induced dissociation (CID). The mass spectrometry scan range was m / z 50-500, and the mass spectrometry data were analyzed on the software ThermoXcalibur Qual Browser 4.0. NanoESI tip θ-type borosilicate glass capillary: od = 1.5 mm; a 2.25 kV direct current (DC) was connected to the back of the capillary, and the device was as follows Figure 1 shown.

[0033] 4. Qualitative and quantitative analysis of substances

[0034] Collision induced dissociation (CID) MS of each substance 2 and MS 3Mass Spectrometry and References (Marceau, E., & Yaylayan, VAProfiling of alpha-Dicarbonyl Content of Commercial Honeys fromDifferent Botanical Origins: Identification of 3,4-Dideoxyglucoson-3-ene(3,4-DGE) and Related Compounds.Journal of Agricultural and Food Chemistry,2009,57(22),10837-10844;Mittelmaier,S.,Fuenfrocken,M.,Fenn,D.,&Pischetsrieder,M.3-Deoxygalactosone,a new glucose degradation product in peritoneal dialysisfluids:Identification,quantification by HPLC / DAD / MSMS and its pathway offormation.Analytical and Bioanalytical Chemistry, 2011, 399 (4), 1689-1697; Gensberger, S., Mittelmaier, S., Glomb, MA, & Pischetsrieder, M. Identification and quantification of six major alpha-dicarbonyl process contaminants in high-fructose corn syrup. Analytical and Bioanalytical Chemistry, 2012, 403 (10), 2923-2931), standard (3-deoxyglucosone (3-DG), 2-keto-D-Glucose (GS); purchased from Toronto Research Chemicals (Toronto, Canada)) for comparison and identification or prediction of metabolites. MS at m / z 235 2 In mass spectrometry, m / z 217 was used as a relative quantitative ion to detect changes in its content.

[0035] 5. α-Dicarbonyl compounds in honey samples

[0036] The present invention uses a widely used derivatizing agent o-phenylenediamine (OPD) to derivatize α-dicarbonyl compounds in the sample to form a stable quinoxaline form. The droplets formed by ESI can be used as a microreactor to achieve reaction acceleration, and the derivatization can be completed on a millisecond scale. The acceleration of the reaction in the microdroplets is due to the larger surface area to volume ratio, drastic pH changes and increased reactant concentrations. OPD solution (10 mg / mL) is added to one channel of the θ-type borosilicate glass capillary, and the honey sample solution (i.e., the supernatant obtained in step 1) is added to the other channel. The derivatization reaction is almost completed simultaneously with the formation of the spray. The primary mass spectrum of the derivatized honey sample is shown in FIG. Figure 2 As shown, α-dicarbonyl compounds are displayed as [M+H] in positive ion mode. + Peak. Four α-dicarbonyl compounds were identified in the honey samples. Figure 3 The secondary and tertiary mass spectra of α-dicarbonyl compounds in honey samples are shown, respectively, m / z 235 (3-deoxyglucosone quinoxaline, 3-DG qx ,3-deoxyglucosone;1-deoxyglucosone quinoxaline,1-DG qx ,1-deoxyglucosone;3-deoxygalactosone quinoxaline,3-DGal qx , 3-deoxygalactosidone), m / z 251 (glucosonequinoxaline, GS qx , glucosone), m / z 217 (3,4-dideoxyglucoson-3-ene quinoxaline, 3,4-DGE qx , 3,4-dideoxyglucosone-3-ene), m / z 219 (1,4-dideoxyglucosone quinoxaline, 1,4-DDG qx , 1,4-dideoxyglucosone). High content of reducing sugars and free amino acids promotes the formation of α-dicarbonyl compounds in honey under heating conditions. The content of α-dicarbonyl compounds m / z 235 in honey from different plant sources under 55℃ heat processing conditions (using a rotary evaporator, parameter settings: speed 90rpm, air pressure 100mbar; heating and concentrating at 55℃ for 2h) is shown in the figure. Figure 4 As shown (groups 1, 2, 3, and 4 in the figure correspond to immature water honey IH 1-IH 4 and artificial concentrated honey AH 1-AH 4 under 55°C heating conditions (the same as the above 55°C heating step parameters); * indicates p≤0.05, ** indicates p≤0.01, and *** indicates p≤0.001), from Figure 4It can be seen that the content of m / z 235 in honeys of different botanical and geographical origins was significantly upregulated under heat treatment at 55°C. In summary, nanoESI-MS technology can quickly detect α-dicarbonyl compounds in honey, and m / z 235 can be used as a potential marker for detecting thermally processed honey.

[0037] The embodiments described above only express several preferred embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but are not intended to limit the present invention. It should be noted that for those skilled in the art, the present invention may also have various changes and modifications, and any modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for rapid detection of α-dicarbonyl compounds in honey based on nanoliter electrospray ion source mass spectrometry, characterized in that: The following steps are involved: Step 1): Prepare the honey to be tested into a honey sample solution for later use; Step 2): α-dicarbonyl compounds in the honey sample solution were detected by nano-electrospray ion source mass spectrometry, the substances were qualitatively identified based on the secondary and tertiary fragment information of the standard, and relative quantification was performed based on the response intensity of the secondary fragment ions with higher responses; Nano-electrospray ion source mass spectrometry detection uses an LTQ-XL linear ion trap, and the detection uses a tip θ-type borosilicate glass capillary, which is divided into two independent channels separated by a layer of glass in the middle; a honey sample solution is added to one of the channels, and a derivatizing agent is added to the other channel to achieve rapid derivatization of α-dicarbonyl compounds.

2. The method for rapid detection of α-dicarbonyl compounds in honey based on nanoliter electrospray ion source mass spectrometry according to claim 1, characterized in that: The preparation method of the honey sample solution in step 1) is: stir the honey to be tested at room temperature, add water to make up the volume, and centrifuge to obtain the supernatant to obtain the honey sample solution.

3. The method for rapid detection of α-dicarbonyl compounds in honey based on nanoliter electrospray ion source mass spectrometry according to claim 1, characterized in that: In the detection, the LTQ capillary temperature was 300° C., the tube lens voltage was 100 V, the spectrum was acquired in positive ion mode, and the secondary mass spectrum was acquired by collision induced dissociation.

4. The method for rapid detection of α-dicarbonyl compounds in honey based on nanoliter electrospray ion source mass spectrometry according to claim 1, characterized in that: The tip of the θ-type borosilicate glass capillary has an OD = 1.5 mm.

5. The method for rapid detection of α-dicarbonyl compounds in honey based on nanoliter electrospray ion source mass spectrometry according to claim 1, characterized in that: The derivatizing agent is o-phenylenediamine.

6. The method for rapid detection of α-dicarbonyl compounds in honey based on nanoliter electrospray ion source mass spectrometry according to claim 3, characterized in that: The MS of each substance obtained by collision induced dissociation 2 and MS 3 Mass spectra are compared with references and standards to identify or predict metabolites.

7. The method for rapid detection of α-dicarbonyl compounds in honey based on nanoliter electrospray ion source mass spectrometry according to claim 1, characterized in that: MS at m / z 235 2 In the mass spectrum, m / z 217 with the highest peak in the secondary mass spectrum was used as the relative quantitative ion.

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