Method for analyzing and detecting five alpha-receptor blockers in plant extract

Abstract

The invention belongs to the technical field of analysis and detection, and relates to a method for analyzing and detecting five alpha-receptor blockers in a plant extract, which comprises the following steps: S1, uniformly mixing a plant sample with an organic solvent, and then carrying out ultrasonic extraction and centrifugal separation to obtain supernate, namely the plant extract; s2, carrying out purification treatment on the plant extract by adopting a solid-phase extraction method; s3, the purified plant extract is separated through a high performance liquid chromatography column, and then the content of the five alpha-receptor blockers in the plant extract is obtained through a mass spectrometer. By establishing an analysis and detection method of solid-phase extraction purification and ultrahigh liquid chromatography-tandem mass spectrometry, the method has the advantages of simplicity, convenience and rapidness in purification treatment, small interference, high accuracy and high sensitivity, and accurate quantitative detection of the five alpha-receptor blockers in the plant extract is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and relates to a method for analyzing and detecting 5 α-blockers in plant extracts. Background Art

[0002] Plant extracts refer to products formed by taking natural plants as raw materials, through physical and chemical extraction and separation processes according to the needs of the final product use, and directionally obtaining and concentrating certain or multiple effective components in the plants without changing the structures of the effective components. However, due to toxic and harmful substances such as pesticide residues and illegal additives, the quality of plant extracts has been affected, greatly influencing the product quality of plant extracts.

[0003] α-blockers are drugs that can selectively bind to α-receptors, competitively block the binding of neurotransmitters or α-receptor agonists to α-receptors, and thus produce an antagonistic effect on the α-receptor agonist phenomenon. Clinically, α-blockers are mainly used to treat diseases such as hypertension and prostatic hyperplasia, and the representative components mainly include phentolamine, tolazoline, prazosin, terazosin, and yohimbine, etc. However, the addition of α-blockers to non-drug products such as foods and health products is generally prohibited because long-term consumption of foods and health products containing α-blockers without awareness can lead to a sharp increase in blood pressure fluctuations or too rapid blood pressure reduction, causing liver damage and endangering life in severe cases. Therefore, the content of α-blockers in foods and health products should be detected.

[0004] Regarding the detection of 5 α-blockers such as phentolamine, tolazoline, prazosin, terazosin, and yohimbine, there are many reported methods, such as capillary electrophoresis, high-performance liquid chromatography, and spectrophotometry. References: "Determination of the content of yohimbine in yohimbe bark by high-performance capillary electrophoresis" (Li Kaijun, Chen Qinhua, Zhang Zhuo, etc.), "Determination of the content of phentolamine mesylate injection by high-performance liquid chromatography" (Liu Yubo, Che Hui, Liu Yan'e, etc.), and "Determination of the content of phentolamine mesylate injection by ultraviolet spectrophotometry" (Wang Lizhen). Although the existing publicly reported methods can detect the content of α-blockers in foods and health products, there are still the following problems: the existing detection methods are not applicable to the detection of 5 α-blockers in plant extracts because the matrix of plant extracts is complex and difficult to purify in the early stage, resulting in large interference, so the accurate qualitative and quantitative determination of α-blockers cannot be achieved. Summary of the Invention

[0005] Aiming at the technical problems that the detection of 5 α-blockers in existing plant extracts has a complex matrix, is difficult to purify in the early stage, resulting in large interference, and the accurate qualitative and quantitative determination of α-blockers cannot be achieved, the present invention provides a method for analyzing and detecting 5 α-blockers in plant extracts.

[0006] The present invention establishes an analytical detection method of solid-phase extraction purification and ultra-high performance liquid chromatography-tandem mass spectrometry, which has the advantages of simple and rapid purification treatment, small interference, high accuracy and sensitivity, and realizes the accurate quantitative detection of 5 α-receptor blockers in plant extracts.

[0007] An analytical detection method for 5 α-receptor blockers in plant extracts comprises the following steps:

[0008] S1. Obtain plant extracts

[0009] Mix a plant sample and an organic solvent evenly according to a mass-volume ratio of (0.5-5) g:(5-15) ml, then perform ultrasonic extraction and centrifugal separation to obtain a supernatant, which is the plant extract;

[0010] S2. Purification

[0011] Purify the plant extract in step S1 by using a solid-phase extraction method;

[0012] S3. High performance liquid chromatography-tandem mass spectrometry

[0013] For the purified plant extract, after separation by a high performance liquid chromatography column, the content of α-receptor blockers in the plant extract is obtained by a mass spectrometer; the α-receptor blockers include phentolamine, tolazoline, prazosin, terazosin and yohimbine.

[0014] Further defined, in step S1, the ultrasonic extraction time is 20-30 min, and the centrifugal separation conditions are: rotation speed 3000-6000 r / min, time 5-10 min.

[0015] Further defined, the plant extract is artichoke extract, ivy extract, white willow bark extract, white birch leaf extract, valerian extract, echinacea extract, hops extract, cherry powder extract, cranberry extract, strawberry extract, white kidney bean extract, hawthorn extract, ashwagandha extract, wolfberry extract or grape seed extract.

[0016] Further defined, the plant extract is obtained by extracting from different parts of a plant sample; the different parts are roots, stems, leaves, flowers, fruits or seeds;

[0017] Further defined, in step S2, the process of purifying the plant extract in step S1 by using a solid-phase extraction method is:

[0018] Add the plant extract to a pre-activated MCX solid-phase extraction column, wash it successively with formic acid aqueous solution and methanol, dry it by suction, elute it with ammonia-methanol solution, collect the eluate, dry it with nitrogen, re-dissolve it with methanol, mix well, and filter it through a 0.22 μm organic microporous filter membrane.

[0019] Further defined, the MCX solid-phase extraction column is activated successively with methanol and water; the washing solution is 3 mL to 10 mL of 2% formic acid aqueous solution and 3 mL to 10 mL of methanol; the eluent is 3 mL to 10 mL of 5% ammonia-methanol solution; add 1.00 mL to 5.00 mL of methanol for re-dissolution.

[0020] Further defined, in the step S3, the high-performance liquid chromatography column is: CAPCELL PAK C18 chromatography column;

[0021] The separation conditions are: mobile phase A is formic acid aqueous solution containing 0.1%, mobile phase B is acetonitrile; column temperature: 35 °C, flow rate 0.3 mL / min, injection volume 2 μL to 10 μL; gradient elution conditions: 0 - 1 min, 95% A; 1 - 3 min, 95% - 10% A: 3 min - 4.5 min, 10% A; 4.5 min - 5.0 min, 10% - 95% A; 5.0 min - 6.0 min, 95% A; flow rate: 0.3 mL / min.

[0022] Further defined, in the step S3, the conditions of the mass spectrometer are: ion source: electrospray ionization source, positive ion mode ESI+; scan mode: multiple reaction monitoring MRM; capillary voltage: 3500 °C; ion source temperature: 300 °C; ion transfer tube temperature: 320 °C; sheath gas flow rate: 11 L / min; auxiliary gas: 3 L / min;

[0023] The mass spectrometry parameters of the α-blocker are as follows:

[0024]

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. The present invention establishes an analytical detection method for determining 5 α-receptor blockers in plant extracts by solid-phase extraction purification and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). This analytical detection method has the advantages of simple and rapid purification treatment, small interference, high accuracy, and high sensitivity, and is suitable for the accurate quantitative detection of 5 α-receptor blockers added in plant extracts.

[0028] 2. The present invention better solves the technical problems of complex matrix and large interference in plant extracts through purification and ultra-high performance liquid chromatography separation, and improves the accuracy of detection results.

[0029] 3. Through verification, the present invention adopts the provided analytical detection method, and for 5 α-blockers, a good linear relationship is presented within the concentration range of 1.0 - 800 μg / L, and the correlation coefficients are all greater than 0.9923. This indicates that the analytical detection method has the advantages of good resolution, high sensitivity, and good repeatability, and is applicable to the determination of 5 α-blockers such as phentolamine in plant extracts. Description of the Drawings

[0030] Figure 1 It is the chromatogram of the T3 chromatographic column;

[0031] Figure 2 It is the chromatogram of the CAPCELL PAK C18 chromatographic column;

[0032] Figure 3 It is the chromatogram of the RPC18 chromatographic column;

[0033] Figure 4 It is the influence of the purification column on the recovery rates of 5 α-blockers. Detailed Embodiments

[0034] Now, the technical solutions protected by the present invention will be described in detail in combination with the embodiments. However, obviously, the described embodiments are a part of the embodiments of the present application, rather than all of the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present application.

[0035] By establishing an analytical detection method of solid-phase extraction (SPE) purification and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), the present invention can achieve the quantitative and rapid detection of 5 α-blockers in plant extracts. Moreover, the analytical detection method has the advantages of simple and rapid purification treatment, little interference, high accuracy, and high sensitivity, and is applicable to the analysis and detection of 5 illegally added α-blockers in plant extracts.

[0036] The following will describe in detail the analytical detection method provided by the present invention through embodiments.

[0037] The instruments, equipment, reagents, standards, and the preparation of the standards adopted in the following embodiments are specifically as follows.

[0038] 1. Instruments and Equipment

[0039] Liquid chromatography-tandem high-resolution mass spectrometer (Q-Exactive, Thermo Fisher Scientific); high-speed centrifuge (AllegraTM X-22R centrifuge, BECKMAN, Germany); vortex mixer ( VORTEX GENIUS3 (IKA, Germany); nitrogen blowing concentrator (Tuibo Vap LV, Biotage, China); oscillator ( -WAKER, IKA, Germany); Milli-Q ultrapure water purifier (Advantage-10 / Elix, Millipore, USA); pipettes: 100 μL, 200 μL, 1000 μL, 2.0 mL and 5.0 mL.

[0040] 2. Reagents and standards

[0041] Methanol, acetonitrile (chromatographic grade, TEDIA, USA); MCX purification column (60 mg, 3 mL).

[0042] The standards used in this invention: yohimbine, terazosin, phentolamine, prazosin, and tolazoline were all purchased from Anpu Company, and their purities were all ≥95.0%. Other reagents were all domestic analytical pure reagents.

[0043] All the plant extracts in this invention were from a local plant extract company in Shaanxi.

[0044] 3. Preparation of standards

[0045] Standard stock solution: Accurately weigh 10.0 mg of each target standard, dissolve it with methanol and prepare a 1.0 mg / mL standard stock solution, and store it at -18°C.

[0046] The target standards include phentolamine, prazosin, terazosin, yohimbine, and tolazoline. The phentolamine standard stock solution, prazosin standard stock solution, terazosin standard stock solution, yohimbine standard stock solution, and tolazoline standard stock solution were prepared respectively.

[0047] Mixed standard intermediate solution 1: Pipette a certain amount of the corresponding standard stock solution of each target standard, dilute it with methanol, and prepare a mixed standard solution containing 2 μg / mL of phentolamine, 2 μg / mL of prazosin, 1 μg / mL of terazosin, 1 μg / mL of yohimbine, and 8 μg / mL of tolazoline, and store it at -18°C.

[0048] Mixed standard intermediate solution 2: Pipette 1.0 mL of mixed standard intermediate solution 1 into 10.0 mL, dilute it with methanol, and prepare a mixed standard solution containing 0.2 μg / mL of phentolamine, 0.2 μg / mL of prazosin, 0.1 μg / mL of terazosin, 0.1 μg / mL of yohimbine, and 0.8 μg / mL of tolazoline, and store it at -18°C.

[0049] Matrix-mixed standard curve solution: After the plant is extracted and purified, take 2 mL of the purified extract, add an appropriate amount of mixed standard intermediate solution 2, and make up the volume with methanol to prepare a series of matrix-mixed standard curve solutions with terazosin and yohimbine in the range of 1 - 100 ng / mL, prazosin and phentolamine in the range of 2 - 200 ng / mL, and tolazoline in the range of 8 - 800 ng / mL. Prepare it freshly before use.

[0050] Example 1. Optimization of chromatographic mobile phase

[0051] According to the chemical structural formulas and properties of 5 α-receptor blockers, in this example, the effects of two organic phase reagents, methanol and acetonitrile, as mobile phases on the analytical detection results were investigated.

[0052] The analytical detection method for 5 α-receptor blockers in the plant extract provided in this example includes the following steps:

[0053] S1. Obtain the plant extract

[0054] Weigh 2 g (accurate to 0.01 g) of the plant sample into a 50 mL stoppered centrifuge tube, add 10.0 mL of methanol, vortex to mix evenly, ultrasonically extract for 20 min, centrifuge at 4200 r / min for 5 min, and transfer the supernatant for purification. The supernatant is the plant extract.

[0055] In this example, the plant sample refers to the plant to be extracted. The plant sample is Rhodiola rosea, and the obtained plant extract is Rhodiola rosea extract.

[0056] S2. Purification

[0057] Transfer 2.0 mL of the supernatant and load it onto an MCX solid-phase extraction column pre-activated with 5 mL of methanol and 5 mL of water. Wash it successively with 5 mL of 2% formic acid aqueous solution and 5 mL of methanol. After drying by suction, elute with 5 mL of 5% ammonia methanol solution, collect the eluate, blow the eluate to nearly dry with nitrogen, accurately add 2.00 mL of methanol to re-dissolve, vortex to mix evenly, and filter through a 0.22 μm organic microporous filter membrane for instrument detection.

[0058] S3. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) detection

[0059] (1) Chromatographic conditions

[0060] Chromatographic column: CAPCELL PAK C18 (2.0 mm × 150 mm, 5 μm); Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is acetonitrile or methanol. Column oven: 35 °C, flow rate 0.3 mL / min, injection volume 10 μL. Gradient elution conditions: 0 - 1 min, 95% A; 1 - 3 min, 95% - 10% A; 3 min - 4.5 min, 10% A; 4.5 min - 5.0 min, 10% - 95% A; 5.0 min - 6.0 min, 95% A. Flow rate: 0.3 mL / min.

[0061] (2) Mass spectrometry conditions

[0062] Ion source: electrospray ionization source, positive ion mode (ESI+); Scanning mode: multiple reaction monitoring (MRM); Capillary voltage: 3500 °C; Ion source temperature: 300 °C; Ion transfer tube temperature: 320 °C; Sheath gas flow rate: 11 L / min; Auxiliary gas: 3 L / min. The optimal mass spectrometry parameters for 5 target compounds (α - receptor blockers) are shown in Table 1.

[0063] Table 1 Chromatographic and mass spectrometric information of 5 α - blockers

[0064]

[0065]

[0066] The results showed that the response of the standard solution prepared with methanol in the methanol system was higher than that in acetonitrile, but in the matrix mixed standard curve solution and the plant extract spiked samples, the matrix effect in the methanol system was large and the interference peaks increased significantly. Therefore, in this example, acetonitrile was preferably used as the organic mobile phase; at the same time, the target compounds (α - receptor blockers) are weakly basic, and the aqueous phase uses 0.1% formic acid aqueous solution, and each target compound has good sensitivity and peak shape.

[0067] Example 2, Optimization of chromatographic column

[0068] According to the properties of the α - receptor blockers, in this example, three different specifications of C18 chromatographic columns were selected for comparison, namely WATERS ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.7 μm) chromatographic column, WATERS ACQUITY RPC18 (100 mm × 2.1 mm, 1.7 μm) chromatographic column and CAPCELL PAK C18 (2.0 mm × 150 mm, 5 μm) chromatographic column.

[0069] The analytical and detection method for 5 α - receptor blockers in the plant extract provided in this example includes the following steps:

[0070] S1. Plant extract sample

[0071] Weigh 2 g (accurate to 0.01 g) of plant sample into a 50 mL stoppered centrifuge tube, add 10.0 mL of methanol, vortex to mix evenly, extract ultrasonically for 20 min, centrifuge at 4200 r / min for 5 min, and transfer the supernatant for purification.

[0072] In this example, the plant sample refers to the plant to be extracted. The plant sample is Rhodiola rosea, and the obtained plant extract is Rhodiola rosea extract.

[0073] S2. Purification

[0074] Transfer 2.0 mL of the supernatant and load it onto an MCX solid-phase extraction column pre-activated with 5 mL of methanol and 5 mL of water. Wash it successively with 5 mL of 2% formic acid aqueous solution and 5 mL of methanol. After drying by suction, elute with 5 mL of 5% ammonia methanol solution, collect the eluate, blow the eluate to nearly dry with nitrogen, accurately add 2.00 mL of methanol to re-dissolve, vortex to mix evenly, and then pass through a 0.22 μm organic microporous filter membrane for instrument detection.

[0075] S3. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) detection

[0076] (1) Chromatographic conditions

[0077] Chromatographic column: Three different specifications of chromatographic columns; Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is acetonitrile. Column oven: 35 °C, flow rate 0.3 mL / min, injection volume 10 μL. Gradient elution conditions: 0 - 1 min, 95% A; 1 - 3 min, 95% - 10% A; 3 min - 4.5 min, 10% A; 4.5 min - 5.0 min, 10% - 95% A; 5.0 min - 6.0 min, 95% A. Flow rate: 0.3 mL / min.

[0078] (2) Mass spectrometry conditions

[0079] Ion source: Electrospray ionization source, positive ion mode (ESI+); Scanning mode: Multiple reaction monitoring (MRM); Capillary voltage: 3500 °C; Ion source temperature: 300 °C; Ion transfer tube temperature: 320 °C; Sheath gas flow rate: 11 L / min; Auxiliary gas: 3 L / min.

[0080] In this example, the separation effects of different chromatographic columns are shown in Figures 1 to 3 . Among them: a is tolazoline; b is phentolamine; c is yohimbine; d is prazosin; e is terazosin.

[0081] The results showed that under the same conditions, the other four compounds except tolazoline achieved good separation and sharp and symmetric peak shapes on both RPC18 and CAPCELL PAK C18 columns. However, on the T3 (100 mm × 2.1 mm, 1.7 μm) column, the phenomenon of two peaks for one compound occurred. Tolazoline had almost no retention on the RPC18 column and the T3 column, with an extremely early elution time of 0.86 min. But when using the CAPCELL PAK C18 column, the elution time of tolazoline was 3.51 min. Therefore, in this example, the CAPCELL PAK C18 (2.0 mm × 150 mm, 5 μm) column was preferably used to separate the five compounds.

[0082] Example 3: Optimization of injection volume

[0083] The analytical detection method for the 5 α - receptor blockers in the plant extract provided in this example referred to Example 1. The difference was that the injection volume was different, and the specific injection volume was 2 μL to 5 μL.

[0084] According to the principle of like dissolves like, the 5 α - receptor blockers were dissolved in organic solvents such as methanol and acetonitrile. When the proportion of water in the constant - volume solution was greater than 20%, the recovery rate of the target compounds was lower than 50%. Therefore, the standard working curve was prepared with 100% methanol.

[0085] When the injection volume ≥ 10 μL, due to the solvent effect, except for tolazoline, the peak shapes of other compounds were asymmetric and began to split. When the injection volume ≤ 5 μL, the peak shapes of each target compound were symmetric and had high responses. Considering the sample pretreatment matrix, the injection volume was further optimized subsequently.

[0086] Example 4: Optimization of mass spectrometry conditions

[0087] The analytical detection method for the 5 α - receptor blockers in the plant extract provided in this example referred to Example 1. The difference was that the mass spectrometry conditions were different.

[0088] In this experiment, the ESI+ scan mode was used for primary mass spectrometry analysis to determine the parent ions. The parameters such as the collision gas energy of the secondary mass spectrometry of each compound were optimized to obtain the secondary mass spectrometry diagram. Two pairs of product ions with stronger abundances and less interference were selected as qualitative and quantitative ions, and the mass spectrometry parameters of each compound were optimized in the MRM mode, as shown in Table 2.

[0089] Table 2 Mass spectrometry parameters of 5 α - blockers

[0090]

[0091] Example 5: Optimization of pretreatment conditions

[0092] 5.1 Optimization of extraction solvent

[0093] The analytical and detection method for 5 α-blockers in the plant extract provided in this example refers to Example 1. The difference lies in the different extraction solvents.

[0094] All 5 α-blockers are alkaloids and are weakly basic. According to the principle of similar solubility and referring to the standard "Determination of 5 α-blocker drugs in foods, BJS201808", this example examines the extraction effects of acetonitrile, methanol, methanol containing 1% formic acid, and acetonitrile containing 1% formic acid on the target compounds (α-blockers).

[0095] The results show that when acidified acetonitrile and methanol are used as extraction solvents, the recovery rates are relatively low. This may be because after adding formic acid, the matrix acidity increases, and the compounds exist in ionic form, resulting in a decrease in solubility in organic solvents. For phentolamine and tolazoline, the recovery rate is about 50% when acetonitrile is used as the extraction agent, and the recovery rate is greater than 80% when methanol is used as the extraction agent. This is because the polarity of methanol is greater than that of acetonitrile, and the extraction efficiency is higher. Therefore, this example preferably uses methanol as the extraction solvent.

[0096] 5.2 Optimization of purification method

[0097] The analytical and detection method for 5 α-blockers in the plant extract provided in this example refers to Example 1. The difference lies in the different solid-phase extraction columns used for purification.

[0098] The plant extract contains a large amount of impurities such as carbohydrates, organic acids, pigments, and tannins, resulting in great matrix interference in the determination of target compounds. To ensure the extraction efficiency of target compounds and reduce the interference of impurities such as pigments and polysaccharides, according to the weak basic chemical properties of 5 α-blockers, Prime HLB purification column (60mg, 3cc, solid-phase extraction column), Captiva EMR-Lipid purification column (60mg, 3cc, solid-phase extraction column), WAX purification column (60mg, 3cc, weak anion exchange column), and MCX purification column (60mg, 3cc, mixed strong cation exchange column) are respectively investigated.

[0099] The purification effect is examined by comparing the recovery rates, as shown in Figure 4 , and the recovery rate of the MCX purification column is between 88% and 96%, with good purification effect. Therefore, this example preferably uses the MCX solid-phase extraction column as the solid-phase extraction column.

[0100] 5.3 Optimization of volume-fixing solvent and injection volume

[0101] The analytical and detection method for 5 α-blockers in the plant extract provided in this example refers to Example 1. The difference lies in the different volume-fixing solvents and injection volumes.

[0102] There are differences in the dissolution efficiency of target compounds in the sample residue by methanol aqueous solutions with different volume fractions. In this study, the recoveries were investigated using pure methanol, 80% methanol-aqueous solution, and 20% methanol-aqueous solution as the constant volume solvents. It was found that the recovery decreased with the increase in the proportion of water, probably because the target compounds are easily soluble in organic solvents. Therefore, in this example, pure methanol is preferably used as the constant volume solvent.

[0103] When using pure methanol for constant volume, the volume effect needs to be considered. Experiments found that when the injection volume ≤ 5 μL, the peak shape is symmetric and the response is high. The recoveries of injection volumes of 2 μL and 5 μL were investigated. Experiments found that when injecting 2 μL, the target compounds are less affected by the matrix effect and the recovery is higher. Therefore, in this example, 2 μL is preferably used as the injection volume.

[0104] Through the above optimizations, when detecting 5 α-blockers in the rhodiola extract, the detection conditions for each step are preferably as follows:

[0105] (1) In the purification step, the extraction solvent is methanol and the constant volume solvent is pure methanol; the purification column is an MCX column;

[0106] (2) In high performance liquid chromatography: the chromatographic column is CAPCELL PAK C18 (2.0 mm × 150 mm, 5 μm); mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is acetonitrile. Column oven: 35 °C, flow rate 0.3 mL / min, injection volume 2 μL. Gradient elution conditions: 0 - 1 min, 95% A; 1 - 3 min, 95% - 10% A; 3 min - 4.5 min, 10% A; 4.5 min - 5.0 min, 10% - 95% A; 5.0 min - 6.0 min, 95% A. Flow rate: 0.3 mL / min.

[0107] (3) Mass spectrometry conditions Ion source: electrospray ionization source, positive ion mode (ESI+); scanning mode: multiple reaction monitoring (MRM); capillary voltage: 3500 °C; ion source temperature: 300 °C; ion transfer tube temperature: 320 °C; sheath gas flow rate: 11 L / min; auxiliary gas: 3 L / min. The optimal mass spectrometry parameters for 5 target compounds (α-blockers) are shown in Table 1.

[0108] In the above example, when obtaining the plant extract, the mixing mass-volume ratio of the plant sample and the organic solvent can also be arbitrarily selected and replaced within the range of (0.5 - 5) g : (5 - 15) mL; the ultrasonic extraction time can be arbitrarily selected and replaced within the range of 20 min - 30 min; the rotation speed of centrifugal separation can be arbitrarily selected and replaced within the range of 3000 r / min - 6000 r / min, and the centrifugal separation time can be arbitrarily selected and replaced within the range of 5 min - 10 min.

[0109] In the above embodiments, when the plant extract is replaced with artichoke extract, ivy extract, white willow bark extract, white birch leaf extract, valerian extract, echinacea extract, hops extract, cherry powder extract, cranberry extract, strawberry extract, white kidney bean extract, hawthorn extract, ashwagandha extract, wolfberry extract or grape seed extract, etc., which are plant extracts of roots, stems, leaves, flowers, fruits and seeds, the selected detection conditions are the same as those for the detection of 5 α - receptor blockers in rhodiola extract.

[0110] Example 6: Detection of plant extracts from different parts

[0111] Since the matrices of plant extracts obtained from different parts of plants are different. For example, flower and fruit extracts such as echinacea extract and wolfberry extract contain abundant sugars and have complex matrices, and extracts with more pigments such as cranberry extract also have relatively complex matrices. Therefore, the applicability of the analytical detection provided by the present invention for the detection of plant extracts extracted from different parts (roots, stems, leaves, flowers, fruits and seeds) is studied.

[0112] In this example, when performing the analytical detection, referring to the analytical detection method of Example 1, and the detection conditions for each step adopt the above - selected detection conditions, and finally the recoveries of plant extracts with different matrices such as roots, stems, leaves, flowers, fruits and seeds are investigated through detection.

[0113] This example selects rhodiola extract, artichoke extract, ivy extract, white willow bark extract, white birch leaf extract, valerian extract, echinacea extract, hops extract, cherry powder extract, cranberry extract, strawberry extract, white kidney bean extract, hawthorn extract, ashwagandha extract, wolfberry extract or grape seed extract as the objects of investigation. The specific results are shown in Table 3.

[0114] Table 3 Recoveries of different plant extracts

[0115]

[0116]

[0117] As can be seen from the results in Table 3, when detecting 5 α - receptor blockers in plant extracts from different parts by using the method provided by the present invention, the recoveries are all greater than 80%. This indicates that the analytical detection provided by the present invention has good applicability for the detection of 5 α - receptor blockers in plant extracts extracted from different parts (roots, stems, leaves, flowers, fruits and seeds).

[0118] Example 7: Determination of actual samples

[0119] Randomly purchase nearly 20 batches of plant extracts (Rhodiola rosea extract) from the market, and use the analytical detection method of the present invention to detect the contents of 5 α-receptor blockers in the plant extracts.

[0120] For the analytical detection method of 5 α-receptor blockers in the plant extracts provided in this example, refer to Example 1.

[0121] S1. Purification

[0122] Transfer 2.0 mL of the supernatant to an MCX solid-phase extraction column pre-activated with 5 mL of methanol and 5 mL of water, wash successively with 5 mL of 2% formic acid aqueous solution and 5 mL of methanol, dry by suction, elute with 5 mL of 5% ammonia methanol solution, collect the eluate, blow the eluate to nearly dry with nitrogen, accurately add 2.00 mL of methanol for reconstitution, vortex and mix evenly, and pass through a 0.22 μm organic microporous filter membrane for instrument detection.

[0123] S2. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) detection

[0124] (1) Chromatographic conditions

[0125] Chromatographic column: CAPCELL PAK C18 (2.0 mm × 150 mm, 5 μm); mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is acetonitrile. Column temperature: 35 °C, flow rate: 0.3 mL / min, injection volume: 10 μL. Gradient elution conditions: 0 - 1 min, 95% A; 1 - 3 min, 95% - 10% A; 3 min - 4.5 min, 10% A; 4.5 min - 5.0 min, 10% - 95% A; 5.0 min - 6.0 min, 95% A. Flow rate: 0.3 mL / min.

[0126] (2) Mass spectrometry conditions

[0127] Ion source: electrospray ionization source, positive ion mode (ESI+); scanning mode: multiple reaction monitoring (MRM); capillary voltage: 3500 °C; ion source temperature: 300 °C; ion transfer tube temperature: 320 °C; sheath gas flow rate: 11 L / min; auxiliary gas: 3 L / min. The mass spectrometry parameters are shown in Table 1.

[0128] Through detection, it was found that yohimbine was detected in two batches of Rhodiola rosea extracts, with contents of 19.9 μg / kg and 16.69 μg / kg respectively, and 5 α-receptor blockers were not detected in the other batches of Rhodiola rosea plant extracts.

[0129] In order to verify the accuracy of the analytical detection method of the present invention, the following tests were carried out.

[0130] Test 1

[0131] 1.1 Matrix effect

[0132] Plant extracts have a complex matrix, and their components mainly come from the roots, stems and leaves of plants, containing impurities such as sugars, pigments, and saponins, which interfere with the determination of target compounds. According to the matrix effect calculation formula:

[0133] Matrix effect = (slope of matrix-matched curve / slope of standard curve - 1) × 100%

[0134] A negative matrix effect indicates the presence of matrix suppression effect; a positive value indicates the presence of matrix enhancement effect. Experiments have found that there are significant matrix effects for other target compounds (α-receptor blockers) in plant extracts except phentolamine, and tolazoline is most affected by the matrix effect. Therefore, in this invention, matrix-matched curves are used for the determination of content.

[0135] 1.2 Linear range, detection limit and quantification limit

[0136] Take a total of 2 mL of purified blank plant extract matrix and blank extract solution, and divide them into 5 portions, which are placed in 5 different 10 mL centrifuge tubes respectively; add 0.1 mL, 0.2 mL and 1.0 mL of 10 ng / mL mixed standard intermediate solution, and 0.4 mL and 1.0 mL of 100 ng / mL mixed standard intermediate solution to the 5 centrifuge tubes respectively, and blow to near dryness with nitrogen, then dissolve and make up the volume to 2.00 mL with methanol respectively to prepare matrix-matched mixed standard solutions for determination. The mixed standard intermediate solution is prepared from the standard stock solution, and the preparation method refers to the preparation process of mixed standard intermediate solution 1 and mixed standard intermediate solution 2. Using the mass of 5 target compounds (α-receptor blockers) as the abscissa and the corresponding peak area as the ordinate, draw a standard curve (matrix-matched curve), and calculate the corresponding linear regression equation and correlation coefficient (R 2 ).

[0137] Add matrix-matched mixed standard solutions with different concentrations to negative samples (plant extracts to be analyzed and detected without pesticide residues, that is, plant extracts to be analyzed and detected), analyze according to the above purification method and chromatographic conditions, and determine the method detection limit (LOD) and quantification limit (LOQ) with 3 times and 10 times signal-to-noise ratio (S / N) respectively. The specific results are shown in Table 4.

[0138] Table 4 Detection limit, quantification limit and linear relationship of 5 target compounds

[0139] Target compound Standard curve <![CDATA[Correlation coefficient (R 2 )]]> LOD (μg / kg) LOQ (μg / kg) Tolazoline <![CDATA[Y = 3.197×10 4 x - 1.747×10 2 > 0.9950 15 40 Phentolamine <![CDATA[Y = 1.725×10 4 x - 1.65×10 4 > 0.9950 3 10 Yohimbine <![CDATA[Y = 4.225×10 4 x - 9.883×10 2 > 0.9949 2 5 Prazosin <![CDATA[Y = 3.212×10 4 x - 1.576×10 3 > 0.9963 3 10 Terazosin <![CDATA[Y = 4.042×10 4 x - 1.585×10 3 > 0.9923 2 5

[0140] The results showed that the limits of detection (LOD) of the 5 target compounds, namely phentolamine, tolazoline, prazosin, terazosin, and yohimbine, were 3 μg / kg, 15 μg / kg, 3 μg / kg, 2 μg / kg, and 2 μg / kg, respectively; the limits of quantification (LOQ) were 10 μg / kg, 40 μg / kg, 10 μg / kg, 5 μg / kg, and 5 μg / kg, respectively; it could meet the detection of 5 α-blockers at low concentrations; the 5 α-blockers had a good linear relationship within the linear range, and the square of the linear correlation coefficient (R 2 ) was greater than 0.9923, which could meet the accuracy requirements of quantitative analysis, and the detection results were more accurate.

[0141] Experiment 2, Recovery Rate and Precision

[0142] Plant extracts are mainly composed of the roots, stems, leaves, flowers, fruits, seeds, etc. of plants, and the contents of impurities such as sugars and pigments in different parts of plants are not the same. Therefore, rhodiola extract, artichoke extract, ivy extract, white willow bark extract, white birch leaf extract, valerian extract, echinacea extract, hops extract, cherry powder extract, cranberry extract, hawthorn extract, withania somnifera extract, wolfberry extract, and grape seed extract were used for recovery rate and precision analysis.

[0143] The test method was as follows: After the above-mentioned various plant extracts were purified, low, medium, and high contents of the 5 target compounds were added respectively to form three low, medium, and high-level mixed standard solutions for the determination of the spiked recovery rate (n = 6); the determination results are shown in Table 5.

[0144] The low content was: 10 μg / kg for both yohimbine and terazosin, 20 μg / kg for both phentolamine and prazosin, and 80 μg / kg for tolazoline.

[0145] The medium content was: 20 μg / kg for both yohimbine and terazosin, 40 μg / kg for both phentolamine and prazosin, and 160 μg / kg for tolazoline.

[0146] The high content was: 50 μg / kg for both yohimbine and terazosin, 100 μg / kg for both phentolamine and prazosin, and 400 μg / kg for tolazoline.

[0147] The results in Table 5 showed that the average spiked recovery rate of the 5 target compounds was 70.0% - 106.0%, and the relative standard deviation (RSD) was 0.2% - 13.4%.

[0148] Table 5 Average Spiked Recovery Rates of 5 α-Blockers in Plant Extracts

[0149]

[0150]

[0151] The present invention uses solid-phase extraction purification - ultra-high performance liquid chromatography tandem mass spectrometry to establish a detection method for five α-receptor blockers, namely phentolamine, prazosin, terazosin, yohimbine, and tolazoline, in plant extracts. By using an MCX solid-phase extraction column to purify the plant extracts before detection, the problem of difficult purification in the detection of current plant extracts is solved. After verification, this method has good resolution, high sensitivity, and good repeatability, and is applicable to the determination of five α-receptor blockers such as phentolamine in plant extracts.

[0152] The specific embodiments described above further elaborate on the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for analyzing and detecting 5 α - receptor blockers in a plant extract, characterized in that, It includes the following steps: S1. Obtain a plant extract Mix a plant sample with an organic solvent in a mass-to-volume ratio of (0.5 - 5) g : (5 - 15) ml evenly, then perform ultrasonic extraction and centrifugal separation to obtain the supernatant, which is the plant extract; S2. Purification Purify the plant extract in step S1 by using the solid-phase extraction method; S3. High-performance liquid chromatography-tandem mass spectrometry For the purified plant extract, first separate it through a high-performance liquid chromatography column, and then obtain the content of α-receptor blockers in the plant extract by using a mass spectrometer; the α-receptor blockers include phentolamine, tolazoline, prazosin, terazosin, and yohimbine.

2. The analytical detection method for five α-receptor blockers in the plant extract according to claim 1, characterized in that, In step S1, the ultrasonic extraction time is 20 min - 30 min, and the centrifugal separation conditions are: rotation speed 3000 r / min - 6000 r / min, time 5 min - 10 min.

3. The analytical detection method for five α-blockers in the plant extract according to claim 1, characterized in that, The plant extract is artichoke extract, ivy extract, white willow bark extract, white birch leaf extract, valerian extract, echinacea extract, hops extract, cherry powder extract, cranberry extract, strawberry extract, white kidney bean extract, hawthorn extract, ashwagandha extract, wolfberry extract, or grape seed extract.

4. The analytical detection method for five α-receptor blockers in the plant extract according to claim 1, characterized in that, The plant extract is obtained by extracting from different parts of the plant sample; the different parts include roots, stems, leaves, flowers, fruits, or seeds.

5. The analytical detection method for five α-receptor blockers in the plant extract according to claim 1, characterized in that, In step S2, the process of purifying the plant extract in step S1 by using the solid-phase extraction method is as follows: Add the plant extract to a pre-activated MCX solid-phase extraction column, wash it successively with formic acid aqueous solution and methanol, after drying, elute it with ammonia-methanol solution, collect the eluate, dry it with nitrogen, add methanol for reconstitution, mix well, and then pass through a 0.22 μm organic microporous filter membrane.

6. The analytical detection method for five α-receptor blockers in the plant extract according to claim 5, characterized in that, The MCX solid-phase extraction column is activated successively with methanol and water; the washing solutions are 3 mL - 10 mL of 2% formic acid aqueous solution and 3 mL - 10 mL of methanol; the elution solution is 3 mL - 10 mL of 5% ammonia-methanol solution; 1.00 mL - 5.00 mL of methanol is added for reconstitution.

7. The method for analyzing and detecting five α-receptor blockers in the plant extract according to claim 1, characterized in that, In step S3, the high-performance liquid chromatography column is: CAPCELL PAK C18 chromatographic column; The separation conditions are: mobile phase A is an aqueous solution containing 0.1% formic acid, mobile phase B is acetonitrile; column temperature: 35 °C, flow rate 0.3 mL / min, injection volume 2 μL - 10 μL; gradient elution conditions: 0 - 1 min, 95% A; 1 - 3 min, 95% - 10% A; 3 min - 4.5 min, 10% A; 4.5 min - 5.0 min, 10% - 95% A; 5.0 min - 6.0 min, 95% A; flow rate: 0.3 mL / min.

8. The analytical detection method for five α-receptor blockers in the plant extract according to claim 1, characterized in that, In step S3, the conditions of the mass spectrometer are as follows: Ion source: electrospray ionization source, positive ion mode ESI+; scanning mode: multiple reaction monitoring MRM; capillary voltage: 3500 °C; ion source temperature: 300 °C; ion transfer tube temperature: 320 °C; sheath gas flow rate: 11 L / min; auxiliary gas: 3 L / min; The mass spectrometry parameters of the α-receptor blocker are as follows: 。

Images