阅读说明：本技术 一种检测蜂蜜中33种生物碱的方法 (Method for detecting 33 alkaloids in honey ) 是由 徐敦明 张志刚 林建忠 杜凤君 袁文萱 赖国银 林立毅 吴敏 于 2019-11-08 设计创作，主要内容包括：本发明涉及一种检测蜂蜜中33种生物碱的方法,使用乙腈作为提取溶剂,采用PSA粉末对试样进行净化,选择0.1％甲酸水-甲醇作为流动相进行梯度洗脱,经Agilent ZORBAX Eclipse Plus C18反相液相色谱柱分离,在电喷雾正离子多反应监测模式下,以保留时间和特征离子对信息进行定性分析和定量分析。该方法在0.5～50μg/L浓度范围内,33种生物碱均具有良好的线性关系,其操作简单,灵敏度高,可以快速地进行定性和定量分析,从而为蜂蜜中有毒生物碱的安全评价提供依据。(The invention relates to a method for detecting 33 alkaloids in honey, which uses acetonitrile as an extraction solvent, adopts PSA powder to purify a sample, selects 0.1% formic acid water-methanol as a mobile phase to carry out gradient elution, separates the sample by an Agilent ZORBAX Eclipse Plus C18 reversed phase liquid chromatographic column, and carries out qualitative analysis and quantitative analysis on information by retention time and characteristic ions under an electrospray positive ion multiple reaction monitoring mode. The method has the advantages that within the concentration range of 0.5-50 mug/L, the 33 alkaloids have good linear relation, the operation is simple, the sensitivity is high, qualitative and quantitative analysis can be rapidly carried out, and therefore a basis is provided for safety evaluation of toxic alkaloids in honey.)

1. A method for detecting 33 alkaloids in honey is characterized in that: the method comprises the following steps:

sample pretreatment: accurately weighing 4-6g of honey into a 50mL centrifuge tube, adding 3-7mL of secondary water, 200 +/-20 mu L of ammonia water, adding 10 +/-2 mL of acetonitrile, and uniformly mixing; then adding sodium chloride to saturation, whirling for 1-2min, centrifuging, and taking the upper layer; adding 10mL of +/-2 acetonitrile into the lower-layer water phase, performing vortex for 1-3min, centrifuging, and combining the upper-layer organic phases; the combined upper organic phases were nitrogen blown dry and washed with 0.05M HCl: diluting methanol to 2mL (V/V) at a constant volume of 8:2 to obtain an extracting solution; adding 0.1-0.3g N-propyl ethylenediamine powder into the extracting solution, uniformly mixing for 1-2min by vortex, centrifuging for 2-5min at 10000-;

high performance liquid chromatography-tandem mass spectrometry detection: the pretreated sample is sent into a high performance liquid chromatography and a mass spectrum in sequence for analysis,

the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: agilent Eclipse Plus C18, mobile phase a: 0.1 plus or minus 0.05 percent of formic acid aqueous solution; mobile phase B: methanol; mobile phase elution procedure: 0.0-1.0 min, 40% B; 1.0-3.0 min, 40-60% B; 3.0-5.0 min, 60-70% B; 5.0-5.1 min, 70% -90% B; 5.10-10 min, 90% -100% B; 10.0-12.0 min 100% B; column temperature: 35-45 ℃; sample introduction amount: 10.0 +/-1.0 mu L; flow rate: 0.1-0.3 mL/min;

the conditions of the mass spectrum were as follows:

an ion source: electrospray ion source, positive ion scanning mode; parameters were adjusted to optimize the alkaloid target compound assay conditions: the temperature of atomizing gas is 250-;

and qualitatively judging the existence of 33 alkaloids in the honey according to the analysis result, or quantitatively calculating the contents of the 33 alkaloids in the honey:

the qualitative judgment method comprises the following steps of qualitatively judging 33 alkaloids in honey to be mesaconine, hypaconitine, brucine, nuciferine, podophylline, aconitine, arecoline, pilocarpine, gelsemine, physostigmine, solanine, D-tetrahydroxyjatrorrhizine, dehydropeganine, matrine, protopine, pilocarpine hydrochloride, laburnine, Senecine, scopolamine, tetrahydropalmatine, berberine hydrochloride, benzoylprotopine, evodiamine, colchicine, strychnine, pseudoephedrine hydrochloride, camptothecin, atropine, ephedrine hydrochloride, monocrotaline, sophoridine, chelerythrine and barbital, and qualitatively judging by adopting 4 identification points;

the 28 alkaloids in the honey are calculated quantitatively and are selected from mesaconine, hypaconitine, brucine, nuciferine, podophylline, aconitine, arecoline, pilocarpine, gelsemine, physostigmine, solanine, D-tetrahydroxyjatrorrhizine, harmine, matrine, protopine, pilocarpine hydrochloride, laburnine, Senecine, scopolamine, aconitine, berberine hydrochloride, benzoylprotopine, evodiamine, colchicine, strychnine, pseudoephedrine hydrochloride, camptothecin and atropine; the quantitative calculation method comprises the following steps: preparing a series of standard solutions, carrying out sample injection with the same volume according to the conditions of the high performance liquid chromatography, establishing a standard curve by taking the sample injection concentration as a horizontal coordinate and the peak area of a quantitative ion pair as a vertical coordinate, and carrying out quantification by an external standard curve method.

2. A method of detecting 33 alkaloids in honey as claimed in claim 1, wherein: the sample pretreatment comprises the following steps: accurately weighing 5g of honey into a 50mL centrifuge tube, adding 5mL of secondary water, 200 mu L of ammonia water and 10mL of acetonitrile, and uniformly mixing; then adding sodium chloride to saturation, whirling for 2min, centrifuging, and taking the upper layer; adding 10mL of acetonitrile into the lower-layer water phase, vortexing for 2min, centrifuging, and combining the upper-layer organic phases; the combined upper organic phases were nitrogen blown dry and washed with 0.05M HCl: diluting methanol to 2mL (V/V) at a constant volume of 8:2 to obtain an extracting solution; adding 0.2g N-propyl ethylenediamine powder into the extractive solution, mixing uniformly for 1min, centrifuging at 15000r/min for 3min, collecting supernatant, filtering with 0.22 μm microporous membrane, and testing.

3. A method of detecting 33 alkaloids in honey as claimed in claim 1, wherein: the conditions of the high performance liquid chromatography are as follows: a chromatographic column: agilent Eclipse Plus C18(3.0X 50mm, 1.8 μm), mobile phase A: 0.1% aqueous formic acid; mobile phase B: methanol; mobile phase elution procedure: 0.0-1.0 min, 40% B; 1.0-3.0 min, 40-60% B; 3.0-5.0 min, 60-70% B; 5.0-5.1 min, 70% -90% B; 5.10-10 min, 90% -100% B; 10.0-12.0 min 100% B; column temperature: 40 ℃; sample introduction amount: 10.0 μ L; flow rate: 0.2 mL/min.

4. A method of detecting 33 alkaloids in honey as claimed in claim 1, wherein: the conditions of the mass spectrum are as follows: an ion source: electrospray ion source (ESI), positive ion scanning mode; parameters were adjusted to optimize the alkaloid target compound assay conditions: the temperature of atomizing gas is 300 ℃, the flow rate of drying gas is 8.0L/min, the pressure of the atomizer is 35psi, the temperature of sheath flow gas is 400 ℃, the flow rate of sheath flow gas is 12.0L/min, the voltage of a capillary tube is 3500V, and the voltage of a nozzle is 500V.

5. A method of detecting 33 alkaloids in honey as claimed in claim 1, wherein: the method for detecting the 33 alkaloids in the honey has good linear relation to the 33 alkaloids within the concentration range of 0.5-50 mug/L.

6. A method of detecting 33 alkaloids in honey as claimed in claim 1, wherein: the method for detecting 33 alkaloids in honey has detection limit (signal-to-noise ratio S/N is 3) and quantification limit (signal-to-noise ratio S/N is 10), wherein the detection limit of 33 alkaloids is 0.33-6.7 mug/kg, and the quantification limit is 1.0-20 mug/kg.

7. A method of detecting 33 alkaloids in honey as claimed in claim 1, wherein: the method for detecting 33 alkaloids in honey comprises the steps of carrying out a labeling recovery test on honey matrix at the level of 2, 4 and 20 mu g/kg, and carrying out matrix matching curve correction on 28 alkaloids, wherein the recovery rate ranges from 64.5% to 100.2% and the relative standard deviation ranges from 4.7% to 14.6% by 28 alkaloids, such as neoaconitine, hypaconitine, brucine, nuciferine, podophylline, aconitine, arecoline, pilocarpine, gelseminine, physostigmine, solanine, D-tetrahydroxyjatrorrhizine, harmine, matrine, protopine, pilocarpine hydrochloride, labelline, muricine, scopolamine, tetrahydropalmatine fumarate, coptisine hydrochloride, benzoylaconitine, evodiamine, colchicine, strychnine hydrochloride, pseudoephedrine hydrochloride and atropine.

Technical Field

The invention relates to an alkaloid detection and analysis technology, in particular to a method for detecting 33 kinds of alkaloids in honey.

Background

The alkaloid is an alkaline substance with a nitrogen-containing heterocyclic ring structure, and has high-efficiency pharmacological action and physiological activity. Clinically, the opioid alkaloid with therapeutic dose has the functions of sedation, analgesia and intestinal peristalsis inhibition; semen Strychni alkaloid has effects of treating numbness of limbs, carbuncle, sore, toxic swelling, and sore throat; the nuciferine alkaloid has effects of clearing away heat and toxic materials, reducing blood fat, reducing weight, stopping bleeding, and removing blood stasis. However, many alkaloids are extremely toxic, and intake of milligram levels can cause harm to human bodies, and have fatal toxicity. Food poisoning or drug addiction can be caused by excessive food intake, misuse of foods containing toxic alkaloids and drugs. For example, long-term consumption of foods containing opioid alkaloids can result in addiction, and in severe cases, damage to the nervous system and digestive system; excessive administration of strychnine can cause excitation and hyperreflexia of the central nervous system, leading to fatigue and paralysis of the central nervous system and death, which seriously harms human health.

The honey is a natural sweet substance and is brewed by collecting plant nectar from bees, wherein the variety of nectar determines the quality of honey. Honey is obtained by collecting nectar from bees, and in nature, some plant pollens and nectars contain alkaloids with natural toxins, such as pyrrolizidine alkaloid and isobuinine alkaloid. In recent years, alkaloid poisoning events occur in various foods including bee products, more and more researchers attract attention, and residual analysis research is carried out on the alkaloid poisoning events, but at present, the research on the alkaloid is not much in China, and the methods for measuring the alkaloid mainly comprise a chemical analysis method, a spectrophotometry method, a thin-layer chromatography, a high-performance liquid chromatography tandem mass spectrometry method and the like. Chemical analysis method and spectrophotometry method are simple and convenient to operate, low in cost and suitable for the method for measuring the total alkaloid content. Thin-layer chromatography and high performance liquid chromatography can be used for measuring the content of the monomer alkaloid by virtue of the separation advantages of chromatography. The high performance liquid chromatography tandem mass spectrometry combines the high performance on-line separation capability of liquid chromatography and the high selectivity and high sensitivity of mass spectrometry, has the capability of simultaneously detecting various target substances with different properties, and shows wider and wider application prospects in the analysis of multiple drug residues. Pyrrolizidine alkaloids such as senecine and Kreilin can be enriched and purified by strong cation solid phase extraction column, and then qualitative and quantitative analysis can be carried out by high performance liquid chromatography tandem mass spectrometry. Most of the existing liquid chromatogram-tandem mass spectrometry methods are suitable for detecting one type of alkaloid with similar chemical properties, and an effective detection method for simultaneously detecting multiple types of alkaloid with huge chemical property differences is not established.

Patent application CN110161143A discloses a chromatography-mass spectrometry combined method for simultaneously detecting 20 alkaloids in food, belonging to the technical field of food detection. Extracting an alkaloid target object in a sample by using acetonitrile and Na2 EDTA-Mclvaine buffer solution to obtain a solution to be detected; and then, sequentially carrying out chromatographic analysis and mass spectrometry on the solution to be detected. The lower limit of the quantitative determination of the combined method of the invention to 20 alkaloids is 5.0 mug/kg; the addition recovery experiment of 3 concentration levels is carried out on four samples of milk powder, honey, oatmeal and complementary food, the average recovery rate is 81-109%, and the relative standard deviation is 1.6-8.3%. Please analyze the reason why the above patent cannot be used to detect 33 alkaloids in bees.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for detecting 33 alkaloids in honey.

33 alkaloids with obvious structural difference and very different chemical properties are selected as research objects in the research, and the liquid chromatography-tandem mass spectrometry determination method of various types of alkaloids in honey is established. The method is simple to operate, low in detection limit, high in sensitivity and good in stability, and can be used for quickly and accurately performing qualitative and quantitative analysis, so that a basis is provided for safety evaluation of toxic alkaloids in food. The 33 alkaloids are respectively: ephedrine hydrochloride, monocrotaline, sophoridine, chelerythrine, clofibrate, mesaconine, hypaconitine, brucine, nuciferine, podophylline, aconitine, arecoline, pilocarpine, gelsemine, physostigmine, solanine, D-tetrahydroxyjatrorrhizine, harmine, matrine, protopine, pilocarpine hydrochloride, laburnine, Senecine, scopolamine, tetrahydropalmatine, coptisine hydrochloride, benzoylaconitine, evodiamine, narciscine, strychnine, pseudoephedrine hydrochloride, camptothecin, atropine.

Compared with the scheme disclosed in CN110161143A, the method disclosed by the application has the advantages that the number of analysis targets is increased, so that a specific extraction and purification scheme is required, the extraction scheme disclosed by the application has a better effect, and the operation is simpler; by using the PSA powder, the substrate benefit can be effectively reduced.

The specific scheme is as follows:

a method for detecting 33 alkaloids in honey comprises the following steps:

sample pretreatment: accurately weighing 4-6g of honey into a 50mL centrifuge tube, adding 3-7mL of secondary water, 200 +/-20 mu L of ammonia water, adding 10 +/-2 mL of acetonitrile, and uniformly mixing; then adding sodium chloride to saturation, whirling for 1-2min, centrifuging, and taking the upper layer; adding 10mL of +/-2 acetonitrile into the lower-layer water phase, performing vortex for 1-3min, centrifuging, and combining the upper-layer organic phases; the combined upper organic phases were nitrogen blown dry and washed with 0.05M HCl: diluting methanol to 2mL (V/V) at a constant volume of 8:2 to obtain an extracting solution; adding 0.1-0.3g of N-propyl ethylenediamine powder into the extracting solution, uniformly mixing for 1-2min in a vortex manner, centrifuging for 2-5min at 10000-;

high performance liquid chromatography-tandem mass spectrometry detection: the pretreated sample is sent into a high performance liquid chromatography and a mass spectrum in sequence for analysis,

the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: agilent Eclipse Plus C18, mobile phase a: 0.1 plus or minus 0.05 percent of formic acid aqueous solution; mobile phase B: methanol; mobile phase elution procedure: 0.0-1.0 min, 40% B; 1.0-3.0 min, 40-60% B; 3.0-5.0 min, 60-70% B; 5.0-5.1 min, 70% -90% B; 5.10-10 min, 90% -100% B; 10.0-12.0 min 100% B; column temperature: 35-45 ℃; sample introduction amount: 10.0 +/-1.0 mu L; flow rate: 0.1-0.3 mL/min;

the conditions of the mass spectrum were as follows:

an ion source: electrospray ion source, positive ion scanning mode; parameters were adjusted to optimize the alkaloid target compound assay conditions: the temperature of atomizing gas is 250-;

and qualitatively judging the existence of 33 alkaloids in the honey according to the analysis result, or quantitatively calculating the contents of the 33 alkaloids in the honey:

the qualitative judgment method comprises the following steps of qualitatively judging 33 alkaloids in honey to be mesaconine, hypaconitine, brucine, nuciferine, podophylline, aconitine, arecoline, pilocarpine, gelsemine, physostigmine, solanine, D-tetrahydroxyjatrorrhizine, dehydropeganine, matrine, protopine, pilocarpine hydrochloride, laburnine, Senecine, scopolamine, tetrahydropalmatine, berberine hydrochloride, benzoylprotopine, evodiamine, colchicine, strychnine, pseudoephedrine hydrochloride, camptothecin, atropine, ephedrine hydrochloride, monocrotaline, sophoridine, chelerythrine and barbital, and qualitatively judging by adopting 4 identification points;

the 28 alkaloids in the honey are calculated quantitatively and are selected from mesaconine, hypaconitine, brucine, nuciferine, podophylline, aconitine, arecoline, pilocarpine, gelsemine, physostigmine, solanine, D-tetrahydroxyjatrorrhizine, harmine, matrine, protopine, pilocarpine hydrochloride, laburnine, Senecine, scopolamine, aconitine, berberine hydrochloride, benzoylprotopine, evodiamine, colchicine, strychnine, pseudoephedrine hydrochloride, camptothecin and atropine; the quantitative calculation method comprises the following steps: preparing a series of standard solutions, carrying out sample injection with the same volume according to the conditions of the high performance liquid chromatography, establishing a standard curve by taking the sample injection concentration as a horizontal coordinate and the peak area of a quantitative ion pair as a vertical coordinate, and carrying out quantification by an external standard curve method.

Further, the sample pretreatment comprises the following steps: accurately weighing 5g of honey into a 50mL centrifuge tube, adding 5mL of secondary water, 200 mu L of ammonia water and 10mL of acetonitrile, and uniformly mixing; then adding sodium chloride to saturation, whirling for 2min, centrifuging, and taking the upper layer; adding 10mL of acetonitrile into the lower-layer water phase, vortexing for 2min, centrifuging, and combining the upper-layer organic phases; the combined upper organic phases were nitrogen blown dry and washed with 0.05M HCl: diluting methanol to 2mL (V/V) at a constant volume of 8:2 to obtain an extracting solution; adding 0.2g N-propyl ethylenediamine powder into the extractive solution, mixing uniformly for 1min, centrifuging at 15000r/min for 3min, collecting supernatant, filtering with 0.22 μm microporous membrane, and testing.

Further, the conditions of the high performance liquid chromatography are as follows: a chromatographic column: agilent Eclipse Plus C18(3.0X 50mm, 1.8 μm), mobile phase A: 0.1% aqueous formic acid; mobile phase B: methanol; mobile phase elution procedure: 0.0-1.0 min, 40% B; 1.0-3.0 min, 40-60% B; 3.0-5.0 min, 60-70% B; 5.0-5.1 min, 70% -90% B; 5.10-10 min, 90% -100% B; 10.0-12.0 min 100% B; column temperature: 40 ℃; sample introduction amount: 10.0 μ L; flow rate: 0.2 mL/min.

Further, the conditions of the mass spectrum are as follows: an ion source: electrospray ion source (ESI), positive ion scanning mode; parameters were adjusted to optimize the alkaloid target compound assay conditions: the temperature of atomizing gas is 300 ℃, the flow rate of drying gas is 8.0L/min, the pressure of the atomizer is 35psi, the temperature of sheath flow gas is 400 ℃, the flow rate of sheath flow gas is 12.0L/min, the voltage of a capillary tube is 3500V, and the voltage of a nozzle is 500V.

Furthermore, the method for detecting 33 alkaloids in honey has good linear relation to 33 alkaloids within the concentration range of 0.5-50 mug/L.

Furthermore, the method for detecting 33 alkaloids in honey has a detection limit (signal-to-noise ratio S/N is 3) and a quantification limit (signal-to-noise ratio S/N is 10), wherein the detection limit of the 33 alkaloids is 0.33-6.7 mug/kg, and the quantification limit is 1.0-20 mug/kg.

Further, the method for detecting 33 alkaloids in honey comprises the steps of carrying out a labeling recovery test on honey matrix at the level of 2, 4 and 20 mu g/kg, and carrying out matrix matching curve correction on 28 alkaloids, namely, neoaconitine, hypaconitine, brucine, nuciferine, podophyllotoxin, aconitine, arecoline, pilocarpine, gelsemine, physostigmine, solanine, D-tetrahydroxyjatrorrhizine, dehydroharmine, matrine, protopine, pilocarpine hydrochloride, labelline, muricine, scopolamine, tetrahydropalmatine, berberine hydrochloride, benzoylaconitine, evodiamine, colchicine, strychnine, pseudoephedrine hydrochloride, camptothecin and atropine, wherein the recovery rate ranges from 64.5% to 100.2%, and the relative standard deviation ranges from 4.7% to 14.6%.

Has the advantages that: the method for detecting 33 alkaloids in honey is simple to operate, has high sensitivity, and can quickly perform qualitative and quantitative analysis, thereby providing a basis for safety evaluation of toxic alkaloids in honey.

Drawings

In order to illustrate the technical solution of the present invention more clearly, the drawings will be briefly described below, and it is apparent that the drawings in the following description relate only to some embodiments of the present invention and are not intended to limit the present invention.

FIG. 1 is a TIC total ion flux diagram of an alkaloid standard mixed solution (50. mu.g/L) provided in one embodiment 1 of the present invention;

FIG. 2 is a graph showing the effect of different extracts on alkaloid extraction according to example 2 of the present invention;

FIG. 3 is a graph showing the effect of different purification methods on alkaloid purification provided in example 3 of the present invention;

FIG. 4a is one of the MRM charts of monocrotaline provided in an embodiment 6 of the present invention;

FIG. 4b is a second MRM chart of monocrotaline provided in an embodiment 6 of the present invention;

FIG. 4c is a third MRM chart of monocrotaline provided in an embodiment 6 of the present invention;

FIG. 5a is one of the MRM profiles of the senecine provided in one embodiment 6 of the present invention;

FIG. 5b is a second MRM chart of the senecine provided in accordance with one embodiment 6 of the present invention;

FIG. 5c is a third MRM chart of kilometer base provided in an embodiment 6 of the present invention;

FIG. 6 is a graph of a mesaconitine standard curve provided in accordance with one embodiment 6 of the present invention;

FIG. 7 is a graph of a aconitine standard curve provided in accordance with one embodiment of the present invention;

fig. 8 is a graph of the brucine standard provided in example 6 of the present invention.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the following examples, "%" means weight percent, unless otherwise specified.