阅读说明：本技术 一种银杏叶提取物产品中5种银杏酸的超痕量检测方法 (Ultra-trace detection method for 5 ginkgolic acids in ginkgo leaf extract product ) 是由 梅秀明 张驰 吴肖肖 蒋迪尧 纪晗旭 杨淼 张宇 乔玲 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种银杏叶提取物产品中5种银杏酸的超痕量检测方法,属于检测技术领域。该方法是将待测样品经超声提取后采用液相色谱串联质谱进行检测,通过对提取溶剂和提取次数的考察,以及超声时间的确定,建立了一种前处理简单易操作、提取效率高,定性、定量结果准确的高灵敏检测方法,可以满足银杏叶提取物产品中银杏酸含量的精准检测,能够全面监测银杏叶提取物产品中银杏酸含量,对保障银杏叶提取物产品的安全性具有重要的作用。(The invention discloses an ultra-trace detection method for 5 ginkgoic acids in a ginkgo leaf extract product, and belongs to the technical field of detection. The method is characterized in that a sample to be detected is subjected to ultrasonic extraction and then is detected by adopting liquid chromatography-tandem mass spectrometry, and through the investigation of an extraction solvent and extraction times and the determination of ultrasonic time, a high-sensitivity detection method with simple pretreatment, easy operation, high extraction efficiency and accurate qualitative and quantitative results is established, so that the accurate detection of the content of ginkgolic acid in the ginkgo biloba extract product can be met, the content of ginkgolic acid in the ginkgo biloba extract product can be comprehensively monitored, and the method has an important effect on ensuring the safety of the ginkgo biloba extract product.)

1. An ultra-trace detection method for 5 kinds of ginkgolic acids in a ginkgo leaf extract product is characterized by comprising the following steps: the method comprises the following steps:

step 1, pretreatment: crushing and sieving a sample to be detected, adding an extraction solvent, carrying out ultrasonic extraction, standing and cooling, filtering and separating by using filter paper, extracting the filtered residue once by using the extraction solvent, combining the two filtrates, and fixing the volume to obtain a sample solution to be detected;

step 2, detection: detecting the sample liquid to be detected obtained in the step 1 by using a liquid chromatography tandem mass spectrometry, and performing qualitative determination through the abundance ratio of secondary ion fragments and quantitative determination through an external standard method;

the 5 kinds of ginkgolic acids are ginkgolic acids, hydroginkgolic acids, heptadecadienoic ginkgolic acids and heptadecadienoic ginkgolic acids;

the liquid chromatography conditions were as follows:

the chromatographic column is C₁₈Chromatographic column

Mobile phase: the mobile phase A is 0.2% acetic acid water solution, the mobile phase B is a mixture of isopropanol and acetonitrile, and the volume ratio of the isopropanol to the acetonitrile is 40: 60

Gradient program: 0-3.0 min, 60-92% B, 3.0-6.0 min, 92% B, 6.0-8.0 min, 92-95% B; and (3) post-operation: 2min

Sample introduction amount: 2 muL; flow rate: 0.2 mL/min; column temperature: 30 ℃;

the mass spectrometry conditions were as follows:

ionization mode: electrospray dual-spray ion source, negative ion mode

Mass spectrum scanning mode: multiple reaction monitoring mode

Atomizer pressure: 40psi

Gas temperature: 300 ℃, gas flow rate: 10L/min

Temperature of sheath gas: 300 ℃; flow rate of sheath gas: 11L/min

Capillary voltage: 3000V

Fragmentation voltage: 0V.

2. The detection method according to claim 1, characterized in that: the sample to be detected is a product of ginkgo biloba extract.

3. The detection method according to claim 1, characterized in that: the extraction solvent in step 1 is methanol.

4. The detection method according to claim 1, characterized in that: the extraction method in step 1 is ultrasonic, and the time is 30 min.

5. The detection method according to claim 1, characterized in that: and 2, extracting the filtered residue in the step 1 with an extraction solvent for one time, namely adding methanol into the residue, performing ultrasonic extraction, filtering with filter paper, and combining the two filtrates.

6. The detection method according to claim 1, characterized in that: the specific process of the step 1 is as follows: weighing a certain mass of crushed and sieved sample, adding an extraction solvent for ultrasonic extraction, standing and cooling, filtering with filter paper, extracting filter residue with the extraction solvent once, filtering with filter paper, combining filtrates of the two times, transferring into a constant volume bottle for constant volume, and filtering with an organic filter membrane to obtain the sample solution to be detected.

Technical Field

The invention belongs to the technical field of detection, and particularly relates to an ultra-trace detection method for 5 ginkgoic acids in a ginkgo leaf extract product.

Background

The folium Ginkgo extract is prepared by drying folium Ginkgo of Ginkgoaceae, has effects of promoting blood circulation for removing blood stasis, dredging channels and collaterals, and can be made into health product, medicine, etc., for improving microcirculation and treating ischemic cardiovascular and cerebrovascular diseases. Ginkgolic acids are 2-hydroxy-6-alkyl-benzoate compounds present in leaves, seeds, roots and bark of ginkgo biloba, and are the most major toxic substances in ginkgo biloba extracts, and 5 common ginkgolic acids are ginkgolic acid (C13:0), ginkgolic acid (C15:1), hydroginkgolic acid (C15:0), heptadecene ginkgolic acid (C17:1) and heptadecadiene ginkgolic acid (C17:2), respectively. Ginkgolic acid has sensitization, mutagenesis and strong cytotoxicity, and can cause anaphylactic reaction, gene mutation and nerve injury, thereby causing adverse reactions such as nausea, heartburn, anaphylactic shock, allergic purpura, tetany, paralysis and the like. At present, ginkgolic acid is used as a key index for controlling the quality of ginkgo products in all countries in the world.

The method for measuring ginkgolic acid in ginkgo leaves is multiple, the TLC method is rapid, cheap and simple and convenient to operate, but the method is only suitable for qualitative analysis due to large error and limited precision; the determination of ginkgolic acids by GC and GC-MS requires derivatization, is relatively complicated, and is not easy to judge the result, because the determination is carried out on ginkgolic acid derivatives, but not on ginkgolic acids per se. The liquid chromatography-mass spectrometry combined technology has the advantages of high efficiency, high speed, high sensitivity and the like, is suitable for measuring trace, difficult to separate or variable components, and provides rich information such as retention time, molecular weight, characteristic component structure fragments and the like of a sample.

In the existing ginkgolic acid substance detection, only one ginkgolic acid is quantified and then converted into the content of the total ginkgolic acid according to an empirical coefficient, but the content of the total ginkgolic acid in the product is difficult to accurately judge only by measuring 1 or 2 ginkgolic acids, and the method has certain risk for evaluating the safety problem of the ginkgolic acid products. Therefore, the establishment of the detection method of 5 ginkgolic acids has great significance.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the ultra-trace detection method for 5 kinds of ginkgolic acids in the ginkgo leaf extract product, the method has simple and convenient pretreatment and high extraction efficiency, and the liquid chromatography-tandem mass spectrometry can better separate the 5 kinds of ginkgolic acids and has stable and reliable detection result and high sensitivity.

In order to achieve the purpose, the invention adopts the following technical scheme:

an ultra-trace detection method for 5 ginkgolic acids in a ginkgo biloba extract product comprises the following steps:

step 1, pretreatment: crushing and sieving a sample to be detected, adding an extraction solvent, carrying out ultrasonic extraction, standing and cooling, filtering and separating by using filter paper, extracting residues after filtering once by using the extraction solvent, combining filtrates obtained in two times, fixing the volume by using a volume fixing bottle, and filtering by using an organic filter membrane to obtain a sample liquid to be detected;

step 2, detection: detecting the sample liquid to be detected obtained in the step 1 by using a liquid chromatography tandem mass spectrometry, and performing qualitative determination through the abundance ratio of secondary ion fragments and quantitative determination through an external standard method;

the 5 kinds of ginkgolic acids are ginkgolic acids, hydroginkgolic acids, heptadecadienoic ginkgolic acids and heptadecadienoic ginkgolic acids;

the liquid chromatography conditions were as follows:

the chromatographic column is C₁₈Chromatographic column (1.8 μm, 2.1X 50mm)

Mobile phase: the mobile phase A is 0.2% acetic acid water solution, the mobile phase B is a mixture of isopropanol and acetonitrile, and the volume ratio of the isopropanol to the acetonitrile is 40: 60

Gradient program: 0-3.0 min, 60% -92% B; 3.0-6.0 min, 92% B; 6.0-8.0 min, 92% -95% B; and (3) post-operation: 2min

Sample introduction amount: 2 mu L of the solution; flow rate: 0.2 mL/min; column temperature: 30 ℃;

the mass spectrometry conditions were as follows:

ionization mode: electrospray Dual spray ion source, negative ion mode (Dual AJS ESI)^-)

Mass spectrum scanning mode: multiple Reaction Monitoring (MRM)

Atomizer pressure: 40psi

Gas temperature: 300 ℃, gas flow rate: 10L/min

Sheath gas (N)₂) Temperature: 300 ℃; sheath gas (N)₂) Flow rate: 11L/min

Capillary voltage: 3000V

Fragmentation voltage: 0V.

Further, the sample to be detected is a product of ginkgo biloba extract.

Further, the extraction solvent in step 1 is methanol.

Further, the extraction mode in the step 1 is ultrasonic, and the time is 30 min.

Further, the extraction of the residue filtered in step 1 with an extraction solvent is carried out by adding methanol to the residue, carrying out ultrasonic extraction, filtering with filter paper, and combining the two filtrates.

Mass spectrometry monitoring of parent ions, quantitative daughter ions, qualitative daughter ion mass-to-charge ratio, collision energy, fragment voltage are as follows, and are marked as quantitative ions:

has the advantages that:

the invention provides an ultra-trace detection method for accurately and stably measuring 5 kinds of ginkgoic acids in a ginkgo leaf extract product, which establishes a high-sensitivity detection method with high extraction efficiency and accurate qualitative and quantitative results by optimizing conditions such as an extraction solvent, extraction times, ultrasonic time and the like, wherein the quantitative limit of the 5 kinds of ginkgoic acids in the invention is 0.05-0.1 mu g/kg, a standard curve is 0.5-100 mu g/L, the linear relation is good, the correlation coefficient is more than 0.999, and the standard recovery detection result is as follows: the daily recovery rate of 5 ginkgoic acids is 90.75-111.69%, RSD is 0.15-5.60%, the daytime recovery rate is 91.33-105.69%, RSD is 1.02-4.36%, and the sensitivity and accuracy are high.

The detection method can be used for accurately detecting the content of trace ginkgoic acid in the ginkgo leaf extract product, can ensure the safety of the ginkgo leaf extract product and ensure the life health and safety of consumers.

Drawings

Fig. 1 is a total ion flow diagram of 5 ginkgolic acids on a liquid chromatography tandem mass spectrometry detection apparatus, wherein: 1. c13:0, neoacid ginkgo; 2. c15:1, ginkgolic acid; 3. c17:2, heptadecadienylginkgolic acid; 4. c15:0, hydroginkgolic acid; 5. c17:1, heptadecadienylginkgolic acid.

FIG. 2 is an ion chromatogram of 5 ginkgolic acids in a liquid phase detection system, wherein: 1. c13:0, neoacid ginkgo; 2. c15:1, ginkgolic acid; 3. c17:2, heptadecadienylginkgolic acid; 4. c15:0, hydroginkgolic acid; 5. c17:1, heptadecadienylginkgolic acid.

Detailed Description

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

Example 1

Using methanol as solvent to prepare 5 kinds of standard solutions of ginkgolic acid with concentration of 0.5-100 mug/L, performing liquid chromatography-tandem mass spectrometry to establish standard curve of external standard method.

Weighing 0.1g of sample, adding 5 kinds of standard solutions of ginkgoic acid, wherein the concentrations are 0.5ng/g, 1ng/g and 2ng/g respectively, adding 10mL of methanol, carrying out ultrasonic extraction for 30min, standing for 10min, filtering and separating by using filter paper, taking filtrate, extracting residues once by using methanol, combining the two filtrates, fixing the volume by using a volume fixing bottle, then passing through a microporous filter membrane, detecting by using liquid chromatography-tandem mass spectrometry, respectively measuring the daily and daytime standard addition recovery results, and calculating the standard addition recovery rate and the relative standard deviation.

And (3) detection results: the standard curve has good linear relation in the concentration range of 0.5-100 mug/L, and the correlation coefficient is more than 0.999.

TABLE 1 test result table for recovery with mark

The daily recovery rate of 5 kinds of ginkgoic acid is 90.75-111.69%, the RSD is 0.15-5.60%, the daytime recovery rate is 91.33-105.69%, and the RSD is 1.02-4.36%. The accuracy and precision of the detection method are high as proved by the standard recovery rate and the relative standard deviation data.

Example 2

The standard sample was diluted with the extract to a certain low concentration, and the detection limit of the corresponding target was determined when the signal-to-noise ratio (S/N) was 3 according to the detection conditions in example 1.

And (3) detection results: when the sample weight is 0.1g and the constant volume is 25mL, the detection limits of 5 ginkgolic acids are as follows: 0.01ng/g of neoacid ginkgo (C13: 0); ginkgolic acid (C15:1), 0.02 ng/g; hydroginkgolic acid (C15:0), 0.02 ng/g; heptadecadienylginkgolic acid (C17:1), 0.05 ng/g; heptadecadienylginkgolic acid (C17:2)0.02 ng/g.

Example 3

Weighing 0.1g of No. 1 sample after crushing and sieving, respectively adding 10mL of methanol, acetonitrile and ethanol, performing ultrasonic treatment for 30min, standing for 10min, filtering with filter paper, fixing volume, filtering the filtrate with an organic filter membrane (0.22 μm), transferring into a sample bottle, and detecting with a computer.

TABLE 2 content of ginkgolic acids extracted with different extraction solvents (μ g/g)

As shown in Table 2, methanol was used as the solvent for extracting ginkgolic acids because of its best effect.

Example 4

Weighing 0.1g (accurate to 0.0001g) of No. 1 sample after crushing and sieving to a 15mL graduated test tube, adding 10mL of methanol, performing ultrasonic treatment for 10min, 20min, 30min, 40min and 50min respectively, standing for 10min, performing filtration by using filter paper, fixing the volume, taking the filtrate, filtering the filtrate by using an organic filter membrane (0.22 mu m), transferring the filtrate into a sample injection bottle, and detecting.

TABLE 3 Effect of ultrasound time on ginkgolic acid extraction (. mu.g/g)

As shown in Table 3, along with the extension of the ultrasonic time, especially 10min-30min, the extraction efficiency of ginkgolic acid is gradually improved, after 30min, the rising trend of ginkgolic acid content is slowed down, and ultrasonic treatment is selected for 30min in order to save the extraction time.

Example 5

Weighing a test tube with a scale of 15mL for 0.1g (accurate to 0.0001g) of a No. 1 sample after crushing and sieving, adding 10mL of methanol, carrying out ultrasonic treatment for 30min, standing for 10min, carrying out filter paper filtration, and carrying out constant volume, wherein the measured total ginkgoic acid content is 2.10 mug/g, considering that the residue also contains a certain amount of ginkgoic acid, 10mL of methanol is added into the filter residue, carrying out ultrasonic extraction for 30min, carrying out filter paper filtration, and carrying out constant volume, taking the filtrate to pass through an organic filter membrane, then transferring the filtrate into a sample injection bottle, detecting that the obtained ginkgoic acid content is 0.25 mug/g, and improving the extraction efficiency of two times by 11.90% compared with that of one time, thus determining secondary extraction.

And (3) detection results: the secondary extraction mode is determined as the extraction method of 5 kinds of micro ginkgolic acid in the ginkgo leaf extract product.

Example 6

Comparison of liquid chromatography-tandem mass spectrometry with liquid chromatography for detection of ginkgolic acids: mixing 5 kinds of ginkgolic acid standard solution with sample liquid chromatography detection system, wherein the concentration of 5 kinds of ginkgolic acid in the standard mixture is 10 μ g/mL, and Kromasil 100-5C is adopted₁₈Column (250X 4.6mm), sample size 20. mu.L, mobile phase 0.2% acetic acid aqueous solution and methanol. Liquid phase color of 5 kinds of ginkgolic acidsThe peak appearance in the spectral detection system is shown in fig. 2. The detection results of the two detection devices show that: the liquid chromatography-tandem mass spectrometry detection system has the advantages of fast bee emergence time, small sample amount, detection limit obviously lower than that of the liquid chromatography detection system, and great risk on the control of ginkgolic acid content in the product because ginkgolic acid in many ginkgo leaf extract products is not detected in the liquid chromatography detection system. The liquid chromatography-tandem mass spectrometry detection method established by the invention has obvious superiority in detecting the content of the trace ginkgoic acid.