阅读说明：本技术 一种检测中药材银杏叶中咖啡因含量的方法 (Method for detecting caffeine content in traditional Chinese medicine ginkgo leaves ) 是由 徐涛 王博然 刘春生 杨瑶珺 陈秀芬 于 2020-05-15 设计创作，主要内容包括：本发明涉及一种检测中药材银杏叶中咖啡因含量的方法,所述方法,步骤如下：1)供试品溶液的制备：取银杏叶样品适量,研成粉末,过四号筛,混匀,称取粉末1.0g,精密称定,置具塞锥形瓶中,精密加入甲醇8-12mL,密塞,称重,超声提取8-12min,放冷,再次称重,用甲醇补足减失的重量,摇匀,用0.2μm针筒式过滤器过滤,取续滤液,高速离心10-20min,转数10000-15000转/分钟,得上清液；2)对照品溶液的制备：取咖啡因对照品2.50mg,精密称定,置于5mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀,制成浓度为500μg/mL溶液；3)检测：取供试品溶液、对照品溶液,注入液质联用仪,以咖啡因对照品峰为参照峰,记录峰面积,根据峰面积可以计算出供试品溶液中的咖啡因含量。(The invention relates to a method for detecting caffeine content in traditional Chinese medicine ginkgo leaves, which comprises the following steps: 1) preparation of a test solution: taking a proper amount of a ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 8-12mL of methanol, sealing the plug, weighing, ultrasonically extracting for 8-12min, cooling, weighing again, complementing the loss weight by methanol, shaking uniformly, filtering by a 0.2 mu m syringe type filter, taking the subsequent filtrate, centrifuging at a high speed for 10-20min, and obtaining the supernatant at the rotation speed of 10000 plus materials/min; 2) preparation of control solutions: precisely weighing 2.50mg of caffeine reference substance, placing in a 5mL volumetric flask, dissolving with methanol, diluting to scale, shaking to obtain 500 μ g/mL solution; 3) and (3) detection: and (3) injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, taking the caffeine reference peak as a reference peak, recording the peak area, and calculating the caffeine content in the test solution according to the peak area.)

1. A method for detecting the content of caffeine in traditional Chinese medicinal material ginkgo leaves adopts an ultra high performance liquid chromatography-tandem mass spectrometry method, wherein a specific instrument is adopted, and a Waters acquisition UPLC I-Class ultra high performance liquid chromatograph is connected with a Xevo TQ-S micro triple quadrupole mass spectrometer in series for measurement.

2. The method of claim 1, comprising the steps of:

1) preparation of a test solution: extracting folium Ginkgo sample with methanol, filtering the extractive solution, and centrifuging the filtrate to obtain supernatant;

2) preparation of control solutions: dissolving caffeine control with methanol and diluting to scale;

3) detecting and taking a test solution and a reference solution, injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, recording peak areas by taking a caffeine reference peak as a reference peak, and calculating the caffeine content in the test solution according to the peak areas, wherein the adopted instrument for the chromatographic and mass spectrometric conditions of the liquid chromatograph-mass spectrometer is a Waters acquisition UPLC I-Class ultra high performance liquid chromatograph in series with a Xevo TQ-S micro triple quadrupole mass spectrometer.

3. The method of claim 2, wherein the liquid chromatography conditions are: a chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A; flow rate: 0.3 mL/min; the column temperature is 30 ℃; the sample volume is 2 mu L; mass spectrum parameter conditions: a positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy was 18.0V and 22.0V, respectively.

4. The method of claim 2, wherein the sample solution of step 1) is prepared by: taking a proper amount of ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of powder, precisely weighing, placing in a conical flask with a plug, precisely adding 8-12mL of methanol, sealing the plug, weighing, ultrasonically extracting for 8-12min, cooling, weighing again, supplementing the loss weight by using methanol, shaking uniformly, filtering by using a 0.2 mu m syringe type filter, taking the subsequent filtrate, centrifuging at a high speed for 10-20min, rotating at 10000 times and 15000 r/min to obtain a supernatant, namely the sample solution.

5. The method of claim 2, wherein the control solution of step 2) is prepared as follows: 2.50mg of caffeine control substance is precisely weighed, placed in a 5mL volumetric flask, dissolved by methanol and diluted to scale, shaken up to prepare a caffeine control substance solution with the concentration of 500 mug/mL.

6. The method of claim 2, wherein the sample solution of step 1) is prepared by the following method: taking a proper amount of ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of powder, precisely weighing, placing into a conical flask with a plug, precisely adding 10mL of methanol, sealing the plug, weighing, ultrasonically extracting for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering by a 0.2 mu m syringe filter, taking the subsequent filtrate, centrifuging at a high speed for 15min, and rotating at 12000 r/min to obtain a supernatant, namely the sample solution.

7. The method of claim 2, wherein the control solution of step 2) is prepared by the following method: 2.50mg of caffeine control substance is precisely weighed, placed in a 5mL volumetric flask, dissolved by methanol and diluted to scale, shaken up to prepare a caffeine control substance solution with the concentration of 500 mug/mL.

8. The method of claim 2, wherein the chromatographic and mass spectrometric conditions of step 3) are performed using a Waters Acquity UPLC I-Class hplc tandem Xevo TQ-S micro triple quadrupole mass spectrometer with the following parameters:

liquid chromatography conditions: a chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A; flow rate: 0.3 mL/min; the column temperature is 30 ℃; sample size 2 uL

Mass spectrum parameter conditions: a positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy was 18.0V and 22.0V, respectively.

9. The method according to any of claims 2-8, characterized by the steps of:

1) the preparation method of the test solution comprises the following steps: taking a proper amount of a ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 8-12mL of methanol, sealing the plug, weighing, ultrasonically extracting for 8-12min, cooling, weighing again, complementing the loss weight by methanol, shaking uniformly, filtering by a 0.2 mu m syringe filter, taking a subsequent filtrate, centrifuging at a high speed for 10-20min, rotating at 10000 + 15000 r/min to obtain a supernatant, namely a sample solution;

2) the control solution was prepared as follows: precisely weighing 2.50mg of caffeine reference substance, placing in a 5mL volumetric flask, dissolving with methanol, diluting to scale, shaking to obtain caffeine reference substance solution with concentration of 500 μ g/mL;

3) injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, recording the peak area by taking the caffeine reference peak as a reference peak, and calculating the caffeine content in the test solution according to the peak area;

wherein, the liquid chromatography conditions are as follows: a chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A; flow rate: 0.3 mL/min; the column temperature is 30 ℃; the sample volume is 2 mu L; mass spectrum parameter conditions: a positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy was 18.0V and 22.0V, respectively.

10. The method of claim 9, characterized by the steps of:

1) the preparation method of the test solution comprises the following steps: taking a proper amount of a ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of the powder, precisely weighing, placing into a conical flask with a plug, precisely adding 10mL of methanol, sealing the plug, weighing, ultrasonically extracting for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering by a 0.2 mu m syringe filter, taking the subsequent filtrate, centrifuging at a high speed for 15min, and rotating at 12000 r/min to obtain a supernatant which is a sample solution;

2) the control solution was prepared as follows: precisely weighing 2.50mg of caffeine reference substance, placing in a 5mL volumetric flask, dissolving with methanol, diluting to scale, shaking to obtain caffeine reference substance solution with concentration of 500 μ g/mL;

3) injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, recording the peak area by taking the caffeine reference peak as a reference peak, and calculating the caffeine content in the test solution according to the peak area;

wherein 3) the instrument adopted under the chromatographic and mass spectrometric conditions is a Waters Acquity UPLC I-Class ultra high performance liquid chromatograph connected with a Xevo TQ-S micro triple quadrupole mass spectrometer, and the specific parameters are as follows:

liquid chromatography conditions: a chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A; flow rate: 0.3 mL/min; the column temperature is 30 ℃; sample size 2 uL

Mass spectrum parameter conditions: a positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy was 18.0V and 22.0V, respectively.

Technical Field

The invention relates to a method for detecting related trace substances in medicinal materials, in particular to a method for detecting caffeine content in traditional Chinese medicinal material ginkgo leaves.

Background

Folium Ginkgo is dry leaf of Ginkgo biloba L. Collected in autumn when the leaves are still green, and dried in time. Has the effects of promoting blood circulation, removing blood stasis, dredging collaterals, relieving pain, astringing lung, relieving asthma, eliminating turbid pathogen, and reducing blood lipid. Can be used for treating blood stasis, obstruction of collaterals, thoracic obstruction, cardialgia, apoplexy, paralysis, cough and asthma due to lung deficiency, and hyperlipidemia.

It is stated in the Chinese pharmacopoeia of 2015 edition that ginkgo leaves contain total flavonol glycosides and terpene lactones, the latter mainly includes ginkgolide A (C)₂₀H₂₄O₉) Ginkgolide B (C)₂₀H₂₄O₁₀) Ginkgolide C (C)₂₀H₂₄O₁₁) And bilobalide (C)₁₅H₁₈O₈)。

Modern pharmacological research shows that ginkgo leaves have a plurality of pharmacological actions such as oxidation resistance, apoptosis resistance, cerebral blood flow improvement, nerve protection, platelet activity inhibition and the like, and can be used for treating cardiovascular and cerebrovascular diseases and the like.

Therefore, ginkgo biloba and ginkgo biloba preparations are widely used in clinical practice.

In recent years, high performance liquid chromatography-tandem mass spectrometry and ultra-high performance liquid chromatography-tandem mass spectrometry are successively used for detecting caffeine components in ginkgo biloba preparations and ginkgo biloba leaves respectively, and caffeine is used as a second class of psychotropic drugs, influences of adverse reactions such as palpitation, arrhythmia, central nervous system overexcitation and the like, particularly addiction of the drugs, and may cause harm to human bodies.

Because the caffeine content in the ginkgo leaves is very low, it is necessary to establish a simple and rapid detection method specially used for determining the caffeine content in the ginkgo leaves.

Methods for detecting caffeine have been reported, for example: the method is used for determining and detecting 33 illegal addition compounds such as sibutramine (containing caffeine) in a food (containing health food) matrix, and the obtained spectrum and data quality are good, the result is accurate and reliable, and the lower limit of a quantitative linear range reaches 0.1 ng/mL. However, the caffeine detected by the method is an exogenous substance and cannot be directly applied to the ginkgo leaves.

Disclosure of Invention

The invention provides a method for measuring the content of trace component caffeine in a ginkgo biloba leaf medicinal material, which is based on a BJS 201701 method (BJS 201701, short for document 1) for measuring compounds such as sibutramine in food, improves the detection conditions of the method to adapt to the ginkgo biloba leaf medicinal material, and simultaneously obtains particularly preferable conditions such as instruments, solvents, mobile phases, temperature, time and the like through screening, thereby being particularly suitable for the detection of the ginkgo biloba leaf medicinal material.

The detection method adopts an ultra high performance liquid chromatography-tandem mass spectrometry method, wherein a specific instrument, namely a Waters acquisition UPLC I-Class ultra high performance liquid chromatograph is connected with a Xevo TQ-S micro triple quadrupole mass spectrometer in series is adopted for measurement.

The method comprises the following steps:

1) preparation of a test solution:

2) preparation of control solutions:

3) and (3) detection: and (3) taking the test solution and the reference solution, injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, taking the caffeine reference peak as a reference peak, recording the peak area, and calculating the caffeine content in the test solution according to the peak area.

Preferably, the method of the present invention comprises the following steps:

1) preparation of a test solution: extracting folium Ginkgo sample with methanol, filtering the extractive solution, and centrifuging the filtrate to obtain supernatant;

2) preparation of control solutions: dissolving caffeine control with methanol and diluting to scale;

3) detecting and taking a test solution and a reference solution, injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, recording peak areas by taking a caffeine reference peak as a reference peak, calculating the caffeine content in the test solution according to the peak areas, wherein the chromatographic and mass spectrometric conditions of the liquid chromatograph-mass spectrometer adopt a Waters acquisition UPLC I-Class ultra high performance liquid chromatograph in series with a Xevo TQ-S micro triple quadrupole mass spectrometer,

wherein, the liquid chromatogram condition of the LC-MS is as follows: a chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A; flow rate: 0.3 mL/min; the column temperature is 30 ℃; the sample volume is 2 mu L; mass spectrum parameter conditions: a positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy was 18.0V and 22.0V, respectively.

Specifically, the method of the present invention, wherein,

step 1) the preparation method of the test solution is as follows: taking a proper amount of ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of powder, precisely weighing, placing in a conical flask with a plug, precisely adding 8-12mL of methanol, sealing the plug, weighing, ultrasonically extracting for 8-12min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering by a 0.2 mu m syringe type filter, taking the subsequent filtrate, centrifuging at a high speed for 10-20min, rotating at 10000 and 15000 r/min to obtain a supernatant, namely the sample solution.

Wherein, the preparation method of the reference substance solution in the step 2) is as follows: 2.50mg of caffeine control sample is precisely weighed, placed in a 5mL volumetric flask, dissolved and diluted to scale with methanol, and shaken up to prepare the caffeine control sample solution with the concentration of 500 mug/mL.

More specifically, the method of the present invention, wherein 1) the test solution is prepared as follows: taking a proper amount of a ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of powder, precisely weighing, placing into a conical flask with a plug, precisely adding 10mL of methanol, sealing the plug, weighing, ultrasonically extracting for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering by a 0.2 mu m syringe filter, taking a subsequent filtrate, centrifuging at a high speed for 15min, and rotating at 12000 rpm to obtain a supernatant which is a sample solution.

Wherein, the preparation method of the reference substance solution in the step 2) is as follows: 2.50mg of caffeine control sample is precisely weighed, placed in a 5mL volumetric flask, dissolved and diluted to scale with methanol, and shaken up to prepare the caffeine control sample solution with the concentration of 500 mug/mL.

Wherein the instrument adopted by the chromatographic and mass spectrometric conditions in the step 3) is a Waters Acquity UPLC I-Class ultra performance liquid chromatograph connected with a Xevo TQ-S micro triple quadrupole mass spectrometer, and the specific parameters are as follows:

liquid chromatography conditions: a chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A; flow rate: 0.3 mL/min; the column temperature is 30 ℃; sample size 2 uL

Mass spectrum parameter conditions: a positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy is 18.0V and 22.0V respectively

Particularly preferably, the method of the invention comprises the following steps:

1) the preparation method of the test solution comprises the following steps: taking a proper amount of a ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of the powder, precisely weighing, placing into a conical flask with a plug, precisely adding 8-12mL of methanol, sealing the plug, weighing, ultrasonically extracting for 8-12min, cooling, weighing again, complementing the loss weight by methanol, shaking uniformly, filtering by a 0.2 mu m syringe filter, taking a subsequent filtrate, centrifuging at a high speed for 10-20min, rotating at a speed of 10000 and 15000 r/min to obtain a supernatant, namely a sample solution;

2) the control solution was prepared as follows: precisely weighing 2.50mg of caffeine control, placing in a 5mL volumetric flask, dissolving with methanol, diluting to scale, shaking to obtain caffeine control solution with concentration of 500 μ g/mL;

3) injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, recording the peak area by taking the caffeine reference peak as a reference peak, and calculating the caffeine content in the test solution according to the peak area;

wherein, the liquid chromatography conditions are as follows: a chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A; flow rate: 0.3 mL/min; the column temperature is 30 ℃; the sample volume is 2 mu L; mass spectrum parameter conditions: a positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy was 18.0V and 22.0V, respectively.

Most preferably, the method of the present invention comprises the steps of:

1) the preparation method of the test solution comprises the following steps: taking a proper amount of a ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of the powder, precisely weighing, placing into a conical flask with a plug, precisely adding 10mL of methanol, sealing the plug, weighing, ultrasonically extracting for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering by a 0.2 mu m syringe filter, taking the subsequent filtrate, centrifuging at a high speed for 15min, and rotating at 12000 r/min to obtain a supernatant which is a sample solution;

2) the control solution was prepared as follows: precisely weighing 2.50mg of caffeine control, placing in a 5mL volumetric flask, dissolving with methanol, diluting to scale, shaking to obtain caffeine control solution with concentration of 500 μ g/mL;

3) injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, recording the peak area by taking the caffeine reference peak as a reference peak, and calculating the caffeine content in the test solution according to the peak area;

wherein 3) the instrument adopted under the chromatographic and mass spectrometric conditions is a Waters Acquity UPLC I-Class ultra high performance liquid chromatograph series Xevo TQ-S micro triple quadrupole mass spectrometer, and the specific parameters are as follows:

liquid chromatography conditions: a chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A; flow rate: 0.3 mL/min; the column temperature is 30 ℃; sample size 2 uL

Mass spectrum parameter conditions: a positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy was 18.0V and 22.0V, respectively.

The detection method is obtained by screening, and the screening process is as follows:

improvements over prior methods

The detection object of the BJS 201701 method (the determination of the sibutramine and other compounds in the food (BJS 201701, abbreviated as document 1) is a substance such as food (containing health-care food), when caffeine in ginkgo leaves is detected, the detection result is poor by directly using the document 1 method, the specific reasons are discussed previously, so far, the inventor improves the BJS 201701 method, and the detection object of the method is directed at ginkgo leaf crude drug samples.

1. Improvement of sample injection amount

The preparation of the test solution, the preparation of the reference solution, and the chromatographic mass spectrometry conditions in the document 1 are adopted.

And (3) screening the sample amount, and displaying the result: when the sample amount of the 0.1ng/mL caffeine control is 1 muL, the peak area is 361, the signal-to-noise ratio is 12.21, the S/N is only slightly more than the limit S/N which is 10, and the result is inaccurate because the calculation result of the signal-to-noise ratio has certain fluctuation range; when the sample amount is 2 muL, the peak area is 618, the signal-to-noise ratio is 20.24, which exceeds the limit of quantification by 2 times, and the method is used for more accurately quantifying the result. The sample injection amount is more than or equal to 2 mu L, the experimental requirements can be met, the sample consumption is saved by 2 mu L, and the pollution degree to instruments and chromatographic columns is smaller.

2. Modification of extraction vessel

The container of document 1 is a test tube with a plug, and because the shape of the test tube with the plug is long and narrow, ginkgo biloba powder is accumulated at the bottom of the test tube, and during ultrasonic extraction, the powder is mostly at the lower part and the solvent is mostly at the upper part, so that the contact degree of ginkgo biloba powder and the solvent in each batch is inconsistent, and the difference of results is large. And the conical bottle is plugged, so that the medicinal material powder is fully contacted with the extraction solvent during ultrasonic treatment, and the result stability is better.

The data (parallel experimental results of the same ginkgo biloba sample batch) using stoppered test tubes as extraction containers gave results of 255, 372 and 2443, respectively. The peak area had a large fluctuation and the RSD value was 123.28%.

The data (the results of the parallel experiments on the same ginkgo biloba sample batch) using the conical flask as the extraction container are 3029, 3057 and 3003 respectively. The peak area RSD value was 0.89%.

3. Improvements in sample handling

In document 1, food (including health food) is directly ground for use without removing impurities, sieving, and the like. Since the ginkgo leaf medicinal material contains impurities, the method in the literature 1 interferes the detection result due to the existence of the impurities, so that the detection result is inaccurate. The method comprises taking folium Ginkgo, removing impurities of non-folium Ginkgo medicinal parts, pulverizing whole folium Ginkgo with a pulverizer, and sieving with a fourth sieve for use.

In the method of the document 1, a microporous filter membrane is used for filtering, and a subsequent filtrate is taken and is properly diluted to a linear range according to the actual concentration for standby application, in the method, a 0.2-micron syringe type filter is used for filtering, the subsequent filtrate is taken and is centrifuged at a high speed for 15min, the rotation number is 12000 r/min, and a supernatant is taken and is used for standby application, the steps of centrifuging and taking the supernatant are added, so that impurities in a ginkgo leaf sample are further removed, the detection result is more accurate, and the pollution degree of the sample to a chromatographic column and an instrument is reduced.

Secondly, determining the extraction process conditions of the ginkgo leaves

A single-factor test is carried out by taking the methanol concentration, the extraction time and the feed-liquid ratio as the factor conditions, and the influence of each factor on the extraction rate of the caffeine in the ginkgo leaves is respectively examined, which is shown in Table 1.

Table 1: single factor test factors and levels

1. Influence of methanol concentration on caffeine extraction rate in folium Ginkgo

Weighing 1g of folium Ginkgo powder, extracting for 10min at a material-to-liquid ratio (g: mL) of 1:10, extracting caffeine from folium Ginkgo with 100%, 70% and 30% volume fraction methanol solution at 30 deg.C, and plotting with methanol concentration as abscissa and caffeine extraction rate (ng/g) as ordinate, as shown in figure 1-1.

As shown in fig. 1-1, the extraction rate was high when the methanol concentration was 100% and 70%, both of them were selected for extraction, and the recovery of the methanol solvent was facilitated by selecting 100% methanol in view of the convenience of solvent recovery, so that 100% methanol was selected as the extraction solvent in this experiment.

2. Influence of different extraction time on caffeine extraction rate in folium Ginkgo

Weighing 1g folium Ginkgo powder, with methanol concentration of 100%, material-to-liquid ratio (g: mL) of 1:10, and ultrasonic temperature of 30 deg.C, respectively extracting caffeine from folium Ginkgo by ultrasonic for 10min, 35min, and 60min, and plotting with extraction time as abscissa and caffeine extraction rate (ng/g) as ordinate, as shown in figure 1-2.

As can be seen from FIGS. 1-2, the extraction rate is highest when the extraction time is 10min, and the ultrasound extraction for 10min has the advantages of saving time and cost, so the extraction time is selected to be 10min in the experiment.

3. Influence of different feed liquid ratios on caffeine extraction rate in folium Ginkgo

Weighing 1g folium Ginkgo powder, extracting with methanol concentration of 100%, extracting for 10min, and ultrasonic temperature of 30 deg.C, respectively extracting caffeine with solvent amount of 1:10, 1:30, 1:50, and 1:100, with the ratio of material to liquid (g: mL) as abscissa and the extraction rate of caffeine (ng/g) as ordinate, and making into graph as shown in FIGS. 1-3.

As shown in fig. 1-3, the ratio of the liquid to the material is in the range of 1:10 to 1:100, and the extraction rate is higher and better as the ratio of the liquid to the material is increased, so that the ratio of the liquid to the material is in the range of 1:10 to 1:100, and when the ratio of the liquid to the material is 1:10, the peak area has the highest response value and the solvent is saved, so that the ratio of the liquid to the material selected in the experiment is 1:10 (namely, 1g of sample, 10mL of solvent is used).

Therefore, 100% methanol is adopted for ultrasonic extraction for 10min in the experiment, and the ratio of material to liquid is 1: 10.

third, the existing method is improved by second

Compared with the method of the prior document 'ultra high performance liquid chromatography-tandem mass spectrometry research on the transfer rule of caffeine in the extraction process of ginkgo biloba extract (Zhang Ping apple, etc., food industry science and technology, 22 th 2019, 34-49, hereinafter referred to as document 2)' the method of the invention

1. The extraction solvent is single, the solvent consumption is small, the extraction time is short, and the extraction method is simple.

(1) The extraction method of ginkgo leaves in the prior document 2 comprises the following steps: accurately weighing 1.0g (accurate to 0.0001) folium Ginkgo, placing in 250mL conical flask, adding 100mL 30% methanol solution, ultrasonic treating at 30 + -0.5 deg.C for 1 hr, diluting to linear range according to actual concentration, filtering with 0.22 μm filter membrane, bottling, and measuring with high performance liquid chromatography-tandem mass spectrometer.

(2) The method comprises the following steps: taking a proper amount of ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, and uniformly mixing. Weighing 1.0g of powder, precisely weighing, placing in a conical flask with a plug, precisely adding 10mL of methanol, sealing, weighing, ultrasonically extracting for 10min, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering with a 0.2 μm syringe type filter, taking the subsequent filtrate, centrifuging at high speed for 15min, rotating at 12000 r/min, and taking the supernatant for later use.

(3) And (3) comparison:

document 2 requires 30mL of methanol (the extraction solvent is 30% methanol solution, and the 100mL of solution contains 30mL of methanol), the method only requires 10mL of methanol, and the consumption of methanol is saved by 2 times.

b, the solvent extracted in the document 2 is 30% methanol water solution, and the solvent extracted by the method is pure methanol.

c is ultrasonic extraction, the extraction time of the document 2 is 1h, but the method only needs ultrasonic treatment for 10min, and the time is saved by 50 min.

The advantages are that: compared with the method in the prior document 2, the method has the advantages of solvent saving, single solvent, time saving and convenient recovery on the basis of detecting caffeine in ginkgo leaves, and can be used for quickly and simply detecting components such as caffeine in ginkgo leaves.

Fourthly, summarize

1. The method for detecting the caffeine in the ginkgo leaves has lower detection limit and quantitative limit than the existing method.

(1) The detection limit of the method of the prior document 2 is 5 mug/kg, and the quantification limit is 15 mug/kg.

(2) The method of the invention defines detection limit and quantification limit by S/N of 3 and S/N of 10 respectively, and the detection limit and the quantification limit are both below 0.1ng/ml (namely 1 mu g/kg).

2. The caffeine chromatographic peak obtained by the method has good peak shape, and the separation effect of the target peak and the impurity peak is obvious.

The graph obtained by the method of reference 2 is shown in FIG. 2-1

The figures obtained by the method of the invention are shown as the chromatograms of 2-2, 2-3 and 2-4 in a multi-reaction monitoring mode under the determination condition of a methanol blank solvent, a caffeine reference solution and a ginkgo leaf test solution respectively. The figure shows that under the measuring conditions of selected ion pairs 195.11 → 138.065 (quantitative) and 195.112 → 110.037 (qualitative), the determination of caffeine is not influenced by impurity in solvent, the chromatographic peak of caffeine in the caffeine control solution is not interfered by impurity, the peak shape is beautiful, the retention time of the peak corresponding to caffeine in the ginkgo leaf test solution is consistent with that of the control peak, and the peak shape is also consistent. The figure fully illustrates the effectiveness, reliability and maturity of the process.

The method provided by the invention has the following advantages:

1. method comparison

The method provided by the invention is mature, is established on the basis of a standard method of determination (BJS 201701) of sibutramine and other compounds in food, and is relatively difficult to detect caffeine in ginkgo leaves for the reasons that: 1) the content is very low (about 1.5260X 10)^-6g/kg～61.1070×10^-6g/kg); 2) the ginkgo leaf powder is easy to gather in a test tube with a plug and is not easy to disperse in a solvent; 3) caffeine in ginkgo leaves is an endogenous substance.

For the above reasons, in order to detect caffeine in ginkgo leaves, the inventors performed the following processes:

1) and (3) treatment of the test sample:

because the content of caffeine is too small, the impurities on and in the ginkgo leaves need to be removed in the treatment of the ginkgo leaves, and the invention adds two treatments: grinding A into powder, and sieving with a fourth sieve; B. filtering with 0.2 μm syringe filter, collecting the filtrate, centrifuging at high speed for 15min at 12000 rpm, and collecting the supernatant;

2) dissolving folium Ginkgo with pure methanol for 10min, saving time, and having optimal dissolution effect; the more suitable erlenmeyer flask with the plug is used, the ginkgo leaf medicinal material powder is accumulated at the bottom of the test tube in the conventional method, during ultrasonic extraction, the powder is mostly arranged at the lower part, and the solvent is mostly arranged at the upper part, so that the contact full degree of each batch of ginkgo leaf samples and the solvent is inconsistent, and the difference of results is larger. The conical bottle is plugged by the appliance, so that the medicinal material powder is fully contacted with the extraction container during ultrasonic treatment, and the result stability is good;

3) the change of the sample amount is related to the content of caffeine in the ginkgo leaves is too small, so the sample amount needs to be increased properly to reach the quantitative standard.

2. The method provided by the invention has more accurate, stable and reliable result.

Description of the drawings:

FIGS. 1-1, 1-2, and 1-3 show the effect of different concentrations of methanol, different extraction times, and different feed liquid ratios on the caffeine extraction rate from ginkgo leaves, respectively;

FIG. 2-1 is a chromatogram of caffeine in prior art method document 2;

FIG. 2-2 is a methanol blank solvent multiple reaction monitoring chromatogram (this is a methanol blank chromatogram);

FIGS. 2-3 are multi-reaction monitoring chromatograms of caffeine control (this figure is a chromatogram of caffeine control);

FIGS. 2-4 are multi-reaction monitoring chromatograms of folium Ginkgo test sample (the chromatogram is of folium Ginkgo medicinal material test sample);

FIG. 3 shows the results of the linear relationship examination.

Detailed Description

Example 1: a method for measuring the content of caffeine in ginkgo leaves comprises the following steps:

1. extraction method

(1) Preparation of test solution

Taking a proper amount of ginkgo leaf sample, grinding into powder, sieving by a fourth sieve, uniformly mixing, weighing 1.0g of powder, precisely weighing, placing into a conical flask with a plug, precisely adding 10mL of methanol, sealing the plug, weighing, ultrasonically extracting for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering by a 0.2 mu m syringe filter, taking the subsequent filtrate, centrifuging at a high speed for 15min, rotating at a speed of 12000 r/min, and taking the supernatant for later use.

(2) Preparation of control solutions

2.50mg of caffeine control sample is precisely weighed, placed in a 5mL volumetric flask, dissolved and diluted to the scale with methanol, shaken up to prepare a mother solution with the concentration of 500 mug/mL, and stored at-20 ℃.

2. Chromatographic and mass spectrum conditions

(1) Conditions of liquid chromatography

A chromatographic column: waters CORTECS T3 column (2.1X 100mm,2.7 μm); mobile phase: a is a 0.1% formic acid aqueous solution, B is a 0.1% formic acid acetonitrile solution, gradient elution, 0-5 min, 95% A; 5-22 min, 95% -2% A; 22-27 min, 2% A; 27-27.5 min, 2% -95% A; 27.5-32 min, 95% A. Flow rate: 0.3 mL/min; the column temperature is 30 ℃; the sample volume is 2 mu L;

wherein the 0.1% formic acid in acetonitrile is 1mL formic acid diluted with 1000mL acetonitrile.

(2) Conditions of Mass Spectrometry parameters

A positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 150 ℃; atomization temperature: 500 ℃; flow rate of the atomizer: 1000L/h; multiple reaction monitoring mode (MRM); ion pair 195.112 → 138.065 (quantitative), 195.112 → 110.037 (qualitative); the residence time is 0.165s and 0.165s respectively; the taper hole voltage is 4.0V and 4.0V respectively; the collision energy is 18.0V and 22.0V respectively

3. The detection method comprises the following steps: respectively preparing the test solution and the reference solution according to the preparation methods of the test solution and the reference solution, injecting the solutions into a liquid chromatograph-mass spectrometer for analysis according to the conditions of the chromatogram and the mass spectrum, and recording peak areas by taking the peak of the caffeine reference as a reference peak.

The chromatogram obtained by the method is shown in figure 2-3 as caffeine reference chromatogram, and figure 2-4 as folium Ginkgo drug test chromatogram.

Experimental example 1: methodology investigation

1. Investigation of linear relationships

Taking 500 mu g/ml of caffeine mother liquor, and respectively diluting the caffeine mother liquor by 5X 10⁴，1×10⁵，2×10⁵，5×10⁵，1×10⁶，5×10⁶And (5) performing sample injection analysis according to the conditions of the chromatogram and the mass spectrum. A standard curve is drawn by using the caffeine concentration as the abscissa and the peak area as the ordinate, and is shown in FIG. 3.

Figure 3 the results show: linear equation is y 4004.5x +483.89, R²＝0.999。

The results show that caffeine is in the range of 0.1ng/mL to 10ng/mL, and the linear relationship is good.

2. Detection limit and quantification limit

Taking the reference substance diluted in gradientInjecting the solution into a liquid chromatograph-mass spectrometer for analysis according to the conditions of the chromatogram and the mass spectrum, and determining that the quantification limit of the caffeine is 1 × 10 under the method by taking the signal-to-noise ratio equal to 10(S/N is 10) as the quantification limit^-4ng/mL, i.e. 1X 10^-3μ g/kg, the limit of detection (S/N-3) is less than this value.

3. Precision test

Weighing 1g folium Ginkgo (number YXY-10), processing according to the above extraction method, continuously introducing sample for 6 times according to chromatographic conditions, taking caffeine reference peak as reference peak, recording peak area and calculating Relative Standard Deviation (RSD).

Wherein:

s is standard deviation, and the calculation formula is

Is a mean value

The results are shown in table 2 for acceptable ranges of content and precision RSD.

Table 2: the content of the component to be measured in the sample, the acceptable range of precision RSD and the limit of recovery rate

The content of the component to be measured	Repeatability (RSD%)	Reproducibility (RSD%)	Recovery Limit (%)
				100％	1	2	98-101
10％	1.5	3	95-102
				1％	2	4	92-105
0.1％	3	6	90-108
				0.01％	4	8	85-110
10μg/g(ppm)	6	11	80-115
				1μg/g	8	16	75-120
10μg/kg(ppb)	15	32	70-125

The results in Table 2 calculated RSD value was 5.12%. The caffeine content in the sample is about 1ng/mL, namely 10 ng/g-10 mug/kg, and the acceptable precision range is within 15%.

The measurement result conforms to the regulation of 'Chinese pharmacopoeia' of 2015 edition, which shows that the precision of the instrument is good.

4. Repeatability test

Weighing 1g each of 6 parts of folium Ginkgo medicinal materials (numbered YXY-10) in parallel, operating according to the above extraction method and chromatographic conditions, recording peak area with caffeine reference peak as reference peak, and calculating to obtain RSD value of 5.87%.

The measurement result shows that: the method provided by the invention has good repeatability.

5. Stability test

Weighing 1 part of folium Ginkgo (numbered YXY-10)1g, processing according to the above method, detecting according to the above detection method at 0, 1, 2, 4, 8, and 12h after preparation of test solution, recording peak area with caffeine reference peak as reference peak, and calculating to obtain RSD value of 7.20%.

The measurement result shows that: the stability of the sample is better within 12 h.

6. Sample application recovery test

Weighing folium Ginkgo 6 parts (number YXY-10) 0.5g each, adding caffeine control 5ng, adding methanol 10mL precisely, extracting, detecting, etc. by the above method, recording peak area with caffeine control peak as reference peak, and calculating RSD value.

The results are shown in Table 2 (limit of recovery).

Table 2 the results show: the calculated RSD value is 6.24%, the average sample adding recovery rate is 83.29%, and the RSD value accords with the regulation of 'Chinese pharmacopoeia' of 2015 edition, which shows that the method has good recovery rate. The lowest recovery rate, the highest recovery rate and the average recovery rate are all in the required range.

Experimental example 2: sample assay

After the methodology investigation items are passed, quantitative research can be carried out on samples of which the caffeine in the ginkgo leaves is in the range of 0.1 ng/mL-10 ng/mL.

1. The method provided by the embodiment 1 of the invention and the BJS 201701 method (different in that the original sample is directly replaced by a ginkgo leaf sample, and other methods are the same) are adopted to detect the content of 10 ginkgo leaf samples, and the samples are from the Anhui Bozhou Chinese medicinal material transaction market.

The results are shown in Table 3

Table 3: measurement result of sample content

Table 3 the results show:

the content of caffeine in folium Ginkgo detected by the method of the invention is within the range of 0.1526 ng/mL-6.1107 ng/mL, namely the content of caffeine in folium Ginkgo is 1.5260 × 10^-6g/kg～61.1070×10^-6In the range of g/kg. The detection result of the BJS 201701 method is 0.2222-10.2530 ng/mL, namely the content of caffeine in folium ginkgo is 2.2220 x 10^-6g/kg～102.5300×10^-6In the range of g/kg.

In the method, the methodology investigation result shows that the data obtained by the method is accurate and reliable; the content of caffeine in the ginkgo leaves is completely determined according to the method of BJS 201701, the defects that response of a caffeine chromatographic peak is low and a parallel sample detection result is unstable are overcome, and in the experiment, the result is simply quantified by using an external standard method, so that the quantification accuracy is low.

In conclusion, the method provided by the invention is adopted for detection, and the result is more accurate, stable and reliable.

2. The method of BJS 201701 is combined with a folium ginkgo extraction method (ultra-high performance liquid chromatography-tandem mass spectrometry is adopted to study the transfer rule of caffeine in the extraction process of folium ginkgo extract (Zhangpi, etc., food industry science and technology, 22 years in 2019, 34-49), 21 batches of folium ginkgo are detected, and samples 1-10 and 11-20 are respectively from the trade markets of traditional Chinese medicinal materials in Anhuo, Bozhou and Hebei Anguo.

The results are shown in Table 4:

table 4: result of content detection by adopting BJS 201701 method

Table 4 the results show: the caffeine content in the ginkgo leaf detected by the combined method of the two documents is approximately between 0.0623 ng/mL and 10.2530ng/mL, namely the caffeine content in the ginkgo leaf is 0.6230X 10^-6g/kg～102.5300×10^-6In the g/kg range, the result has the defects of low response of the caffeine chromatographic peak and unstable detection result of a parallel sample, so that the quantitative accuracy is low.

The results show that: the method provided by the invention is adopted for detection, and the result is more accurate, stable and reliable.