CN112903836B - Method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder - Google Patents
Method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder Download PDFInfo
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Abstract
The invention provides a method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder, which comprises the following steps: weighing an in-vitro cultured bear gall powder sample, putting the in-vitro cultured bear gall powder sample into water according to the proportion of 50-200mg/5ml, and then oscillating for 8-15 minutes until the sample is completely dissolved; loading the solution obtained by the treatment on an activated hydrophilic lipophilic balance solid phase extraction column, washing, eluting, collecting eluent, filtering, and taking a subsequent filtrate to obtain a test sample solution; and injecting the test solution into a liquid chromatography-mass spectrometer for detection. The determination method provided by the invention has the advantages of good intermediate precision, high accuracy and good repeatability by adjusting and optimizing the conditions and parameters in the purification and detection steps, and in addition, the concentration diluted by 10 times is taken as the detection limit of the method, and the detection limit of the method is 0.68mg/kg, so that a reliable and easy-to-operate method is provided for detecting the IPTG residual quantity in the in-vitro cultured bear gall powder, and the safety control of the in-vitro cultured bear gall powder is promoted.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder.
Background
Fel Ursi is prepared by drying bile of gallbladder of Ursidae animal such as black bear or brown bear, and has effects of clearing heat, suppressing hyperactive liver, and improving eyesight. Bear gall is one of four major precious animal medicines, has more than 2000 years of medicine taking history, and a large number of prescriptions contain bear gall components. The annual bear gall powder yield of China is about 30 tons, and the requirement of human health cannot be met. The contradiction between high-end bear gall powder supply and demand is increasingly prominent, and the market demand of the bear gall powder for low-end raw materials is continuously increased, so that the price of the bear gall powder is gradually increased, and the market price is over 5000 yuan/kg. What is more serious is that the long-term resistance of animal protection organizations at home and abroad to the gall-taking of live bears brings unprecedented resistance to the production of bear gall powder at home and the development of related industries. Therefore, the acceleration of the research and development of the artificial bear gall is the key for promoting the sustainable development of the bear gall industry. The prior patent with the application number of 201410588581.5 discloses an artificial bear gall powder and a preparation method thereof, and specifically produces the in-vitro cultivated (biotransformation) bear gall powder, which takes poultry gall powder as a raw material, performs biotransformation on the poultry gall powder by simulating bile acid liver-intestine circulation in vitro and adopting a two-step enzymatic method and a double-enzyme combined circulation transformation technology, has wide and easily-obtained raw material sources, does not need to add other chemical substances, and can promote the healthy development of the bear gall industry.
However, the key enzyme thallus, namely the enzyme thallus A and the enzyme thallus B, required in the preparation process of the bear gall powder cultured in vitro are obtained by transforming two recombinant plasmids, namely 7 alpha-HSDH and 7 beta-HSDH, into a host bacterium E.coli BL21 (DE 3), carrying out resistance screening and carrying out multi-stage culture. IPTG (isopropyl-beta-D-thiogalactopyranoside) is an inducer for inducing the expression of exogenous genes by the A enzyme thallus/B enzyme thallus. In order to enhance the safety control of the bear gall powder cultured in vitro (biotransformation), the content limit of main additives in the fermentation process, particularly IPTG, needs to be checked, but no method for detecting the IPTG residual quantity in the bear gall powder cultured in vitro exists at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for determining isopropyl-beta-D-thiogalactopyranoside in bear gall powder cultured in vitro.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder, which comprises the following steps:
step one, dissolving a sample: weighing an in-vitro cultured bear gall powder sample, putting the in-vitro cultured bear gall powder sample into water according to the proportion of 50-200mg/5ml, and then oscillating for 8-15 minutes until the sample is completely dissolved;
step two, purification treatment: loading the solution obtained by the first step on an activated hydrophilic lipophilic balance solid phase extraction column, washing, eluting, collecting eluent, filtering, and taking a subsequent filtrate to obtain a test sample solution;
and step three, injecting the test solution into a liquid chromatogram-mass spectrometer for detection.
Further, the solid phase extraction column is a hydrophilic lipophilic balance solid phase extraction column or a strong anion exchange reversed phase adsorption packing solid phase extraction column.
More preferably, the solid phase extraction column is a Waters Oasis HLB solid phase extraction column having a specification of 500mg/6cc.
Further, in the second step, methanol and water are sequentially adopted to activate the hydrophilic and lipophilic balance solid-phase extraction column; the volume ratio of methanol to water was 1:1.
And further, in the second step, washing is carried out by adopting water, and elution is carried out by adopting a methanol solution.
Further preferably, the elution is performed twice in step two using a 20% methanol solution.
Further, in the third step, a Waters CORTECS T3 column with a size of 100 mm. Times.2.1 mm,2.7 μm was used for the detection.
Further, the mobile phase used in the liquid chromatography was water at a flow rate of 0.30ml/min.
Further, the conditions for mass spectrometric detection are: a triple quadrupole mass spectrometry detector is adopted, and Multiple Reaction Monitoring (MRM) is carried out in an electrospray ionization (ESI) negative ion mode; the ion pairs and mass spectrum parameters for multiple reaction monitoring were as follows:
by adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the determination method of isopropyl-beta-D-thiogalactopyranoside in the in vitro cultured bear gall powder provided by the invention has the advantages of good intermediate precision, high accuracy and good repeatability by adjusting and optimizing conditions and parameters in the purification and detection steps, and in addition, the concentration diluted by 10 times is taken as the detection limit of the method, and the detection limit of the method is 0.68mg/kg, so that a reliable and easy-to-operate method is provided for the detection of IPTG residual quantity in the in vitro cultured bear gall powder, and the safety control of the in vitro cultured (biotransformation) bear gall powder is promoted.
Drawings
FIG. 1 is a MRM chromatogram of isopropyl-beta-D-thiogalactopyranoside.
Detailed Description
Aiming at the artificial bear gall powder in the prior patent with the application number of 201410588581.5, the invention provides a determination method of isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder, which comprises the following steps:
step one, dissolving a sample: weighing a sample, placing the sample in water, and then oscillating until the sample is completely dissolved;
step two, purification treatment: loading the solution obtained by the first step on an activated hydrophilic lipophilic balance solid phase extraction column, washing, eluting, collecting eluent, filtering, and taking a subsequent filtrate to obtain a test sample solution;
and step three, injecting the test solution into a liquid chromatogram-mass spectrometer for detection.
In a preferred embodiment of the invention, the ratio of sample to water in step one is 50-200mg/5ml; more preferably 100mg/5ml.
In a preferred embodiment of the present invention, the time of shaking in the first step is 8 to 15 minutes; preferably 8-10 minutes; more preferably 10 minutes.
In a preferred embodiment of the present invention, the solid phase extraction column is a hydrophilic lipophilic balance solid phase extraction column or a strong anion exchange reverse phase adsorption packing solid phase extraction column; preferably a hydrophilic lipophilic balance solid phase extraction column; more preferably a Waters Oasis HLB solid phase extraction column, which is 500mg/6cc in size.
In a preferred embodiment of the present invention, in the second step, methanol and water are sequentially used to activate the hydrophilic lipophilic balance solid phase extraction column; the volume ratio of methanol to water was 1:1.
In a preferred embodiment of the invention, the second step is washed with water and eluted with methanol solution; preferably, the elution is performed twice with 20% methanol solution.
In a preferred embodiment of the invention, the detection in step three is performed by using a Waters CORTECS T3 chromatographic column with the specification of 100mm multiplied by 2.1mm and 2.7 μm.
In a preferred embodiment of the invention, the liquid chromatography detection uses water as a mobile phase, and after all the components to be detected peak, acetonitrile-water (98; the flow rate was 0.30ml/min.
In a preferred embodiment of the present invention, the mass spectrometric detection conditions are: a triple quadrupole mass spectrometry detector is adopted, and Multiple Reaction Monitoring (MRM) is carried out in an electrospray ionization (ESI) negative ion mode; the ion pairs and mass spectral parameters for multiple reaction monitoring are shown in table 1 below:
TABLE 1 ion Pair for Mass Spectrometry monitoring and Mass Spectrometry parameters
The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1
In this example, conditions and parameters in the determination method of isopropyl- β -D-thiogalactopyranoside in vitro cultured bear gall powder are explored, and the specific strategy is as follows:
1. extraction method
Taking 100mg of in vitro cultured (biotransformation) bear gall powder, precisely weighing, placing in a 15ml centrifuge tube, adding 5ml of water, and shaking for 10 minutes until completely dissolving.
2. Purification method
The solution is directly injected for measurement, and the recovery rate is about 60 percent. The reason is presumed to be that the bear gall powder has a complex matrix component and is liable to generate a matrix effect, thereby affecting the response of the target compound. Therefore, two kinds of solid phase extraction columns are examined by reducing the influence of the matrix through the purification mode of the solid phase extraction column: (1) Using the pH difference between the substance to be detected (neutral) and the bear gall powder matrix (acidic) and adopting a strong anion exchange reverse phase adsorption filler solid phase extraction column (Waters Oasis MAX); (2) The polarity difference between the substance to be detected (strong polar compound) and the bear gall powder matrix is utilized to adopt a hydrophilic lipophilic balance solid phase extraction column (Waters Oasis HLB).
(1) Purifying by a strong anion exchange reversed phase adsorption packing solid phase extraction column: all of the above solutions were loaded onto a Waters Oasis MAX solid phase extraction column (specification 500mg/6 cc) previously activated with 4ml of methanol and 4ml of water in sequence; washing the centrifugal tube containing the extracting solution by 2ml of 5% ammonia water, loading the washing solution, and removing the washing solution; adding 4ml methanol for elution, collecting eluent, filtering, taking the subsequent filtrate, and injecting sample for determination. As a result, the recovery rate of the sample was 59%.
(2) Purifying by a reversed-phase solid-phase extraction column: the determination method comprises the following steps of repeatedly searching for a solid phase extraction sample loading, cleaning and eluting solvent: all of the above solutions were loaded onto a Waters Oasis HLB solid phase extraction column (specification 500mg/6 cc) previously activated with 4ml methanol and 4ml water in sequence; washing the centrifugal tube containing the extracting solution by using 2ml of deionized water, loading the washing solution, and removing the washing solution; adding 20% methanol twice and eluting with 6ml, collecting eluate, filtering, collecting filtrate, and determining by sample injection. As a result, the recovery rate of sample application was 76%.
Therefore, the Waters Oasis HLB solid-phase extraction column is finally adopted for purification treatment, and the sample solution is diluted to reduce the matrix effect and further improve the recovery rate.
In addition, sample sampling was examined. Four samples were compared: 100mg, 250mg, 500mg and 1000mg, and as a result, the recovery rate of only 100mg samples is better, and the recovery rates of other large samples are all lower than 10%. The specification of the solid phase extraction column can only bear 100mg of bear gall powder, and overload is caused when the sampling quantity is increased; if the specification of the solid phase extraction column is further improved, the detection cost is too high. The final sample was taken at 100mg.
3. Chromatographic mass spectrometry conditions
3.1 chromatographic conditions
Methanol-water (50); when water was used as the mobile phase, the IPTG retention time was 5.958min and the peak shape was good. Therefore, a Waters CORTECS T3 (100 mm. Times.2.1mm, 2.7 μm) column was selected; taking water as a mobile phase; the flow rate was 0.30ml/min.
When large-batch sample injection is carried out, the repeatability of part of samples is found to be poor, the existence of part of components which are remained in the chromatographic column strongly and are supposed to be present in the samples is estimated, and the components are co-distilled with the subsequent samples to generate a matrix enhancement effect, so that after water is taken as a mobile phase for elution to the peak of the component to be detected, the chromatographic column is fully cleaned by acetonitrile-water (98.
3.2 Mass Spectrometry conditions
A triple quadrupole mass spectrometer was used for Multiple Reaction Monitoring (MRM) in electrospray ionization (ESI) negative ion mode, and the monitored ion pairs and parameters are shown in table 1. The MRM chromatogram of IPTG is shown in FIG. 1.
Example 2
This example provides a method for determining isopropyl- β -D-thiogalactopyranoside based on example 1, comprising the steps of:
step one, preparing a reference substance solution: taking a proper amount of isopropyl-beta-D-thiogalactopyranoside reference substance, precisely weighing, and adding water to prepare a series of solutions of 200ng/ml, 100ng/ml, 50ng/ml, 20ng/ml and 10ng/ml as reference substance solutions;
step two, preparing a test solution: sampling 0.1g of sample powder, precisely weighing, placing in a centrifuge tube, adding 5ml of water, oscillating for 10 minutes until the sample powder is completely dissolved, loading the solution on a hydrophilic-lipophilic balance solid phase extraction column (a Waters Oasis HLB solid phase extraction column is recommended, the specification is 500mg/6cc, and the hydrophilic-lipophilic balance solid phase extraction column is activated by 4ml of methanol and 4ml of water in sequence in advance), cleaning by 2ml of water, discarding a cleaning solution, eluting twice by 20% of methanol, eluting by 3ml each time, combining the eluates into a 10ml measuring flask, diluting by water to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a sample solution;
and step three, precisely sucking 5 mu l of each of the reference solution and the test solution, injecting the reference solution and the test solution into a liquid chromatogram-mass spectrometer, and measuring, wherein the conditions of the chromatogram and the mass spectrum are set in the embodiment 1.
If a chromatographic peak is detected in the sample solution at the same time as the retention time of the reference substance, and the ion abundance ratio of the selected ion abundance ratio and the ion abundance ratio of the reference substance solution at the corresponding concentration are as defined in table 2 below, it can be determined that the sample solution has the component.
TABLE 2 comparison of selected ion abundance ratios to ion abundance ratios of control solutions of comparable concentrations
And (3) drawing a standard curve by taking the peak area of the reference substance quantitative ion pair as a vertical coordinate and the reference substance concentration as a horizontal coordinate, reading the concentration of the isopropyl-beta-D-thiogalactopyranoside in the test solution from the standard curve, and calculating to obtain the test solution.
Example 3
This example performs a methodological validation of the method provided in example 2, with the following validated performance parameters and methods:
(1) Specificity
Because IPTG is detected by in vitro cultured (biotransformation) bear gall powder and no blank matrix sample exists, the blank solution is prepared by taking the deionized water with the same volume according to the preparation method of the test solution, and the result is not interfered when the sample injection is measured.
(2) Linearity
A proper amount of IPTG reference substances are precisely weighed, and water is added to prepare 242.4520ng/ml, 121.2260ng/ml, 60.6130ng/ml, 24.2452ng/ml and 12.1226ng/ml series solutions as reference substance solutions. Respectively and precisely sucking 5 mu l of a control solution, injecting the control solution into a liquid phase mass spectrometer, recording peak areas, drawing a standard curve by taking the peak areas as ordinate and the concentrations as abscissa, wherein the result shows that the standard curve has a good linear relationship in the range of 12.1226-242.4520 ng/ml, y = 133.7238x +358.760359, and r =0.9984.
(3) Precision of the instrument
Precisely sucking 5 μ l of control solution (242.4520 ng/ml), continuously injecting sample for 6 times, recording peak area, calculating RSD, and finding out the result shown in Table 3.
TABLE 3 results of parallel tests of control solutions
(4) Stability of
Taking the same sample solution, respectively standing for 0h, 8h and 15h after preparation, and performing sample injection determination. The results show that the test solutions are substantially stable over 15 hours, and the results are shown in Table 4.
TABLE 4 stability test results
(5) Repeatability of
The same sample was sampled and run as above in 6 replicates to prepare 6 test solutions, which were then injected and measured to calculate concentration/sample weight, and the results are shown in Table 5.
TABLE 5 results of the repeatability tests
According to the general rule of the four parts of the China pharmacopoeia 2020 edition <9101>, when the content of the component to be measured in the sample is in the level of 10ppm, the repeatability limit is 6%. Thus, the method provided in example 2 is highly reproducible.
(6) Accuracy of
Taking 50mg of in vitro cultured (biotransformed) bear gall powder (the content is 7.2392 mg/kg), precisely weighing in six copies, precisely adding 1ml of a reference substance solution (232.4560 ng/ml) and 4ml of water respectively, and carrying out the same preparation operation on the test substance solution to obtain a sample adding solution. The sample injection amount is 5 mul, and the sample injection recovery rate is calculated, and the result is shown in table 6.
TABLE 6 accuracy test results
According to the general rule <9101> of the four parts of the Chinese pharmacopoeia 2015 edition, when the content of the component to be measured in the sample is in the level of 10ppm, the recovery rate limit is 80-115%, so the recovery rate of the test meets the requirement.
(7) Detection limit
Because the IPTG content in the bear gall powder cultured in vitro (biotransformation) provided by the market is higher, the test solution with the concentration of 6.7587ng/ml is diluted by 2 times, 5 times and 10 times and still detected. The concentration diluted by 10 times is taken as the detection limit of the method, and the detection limit of the method is 0.68mg/kg.
(8) Intermediate precision
In order to examine the influence of random variation factors on precision, the content of 3 batches of samples was determined by different analysts on different dates and different instruments according to the proposed method, and the results are shown in table 7.
TABLE 7 results for intermediate precision
The results show that: the results measured by different analysts on different dates and different instruments are basically consistent, and the intermediate precision of the method is good.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (5)
1. The method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder is characterized by comprising the following steps of:
step one, dissolving a sample: weighing an in-vitro cultured bear gall powder sample, putting the in-vitro cultured bear gall powder sample into water according to the proportion of 50-200mg/5ml, and then oscillating for 8-15 minutes until the sample is completely dissolved;
step two, purification treatment: loading the solution obtained by the first step on an activated hydrophilic lipophilic balance solid-phase extraction column, sequentially activating the hydrophilic lipophilic balance solid-phase extraction column by adopting methanol and water, washing by adopting water, eluting by adopting a methanol solution, collecting an eluent, filtering, and taking a subsequent filtrate to obtain a sample solution;
injecting the test solution into a liquid chromatogram-mass spectrometer for detection;
in the third step, a Waters CORTECS T3 chromatographic column is adopted for detection, the specification of the chromatographic column is 100mm multiplied by 2.1mm, and the diameter is 2.7 mu m; the mobile phase used in liquid chromatography was water with a flow rate of 0.30ml/min.
2. The assay of claim 1, wherein the solid phase extraction column is a Waters Oasis HLB solid phase extraction column having a specification of 500mg/6cc.
3. The method according to claim 1, wherein the hydrophilic lipophilic balance solid phase extraction column is activated by methanol and water in a volume ratio of 1:1 in step two.
4. The method according to claim 1, wherein the elution in the second step is performed twice using a 20% methanol solution.
5. The assay method according to claim 1, wherein the conditions for mass spectrometric detection are: monitoring multiple reactions in an electrospray ionization negative ion mode by using a triple quadrupole mass spectrometer; the ion pair and mass spectrum parameters for multiple reaction monitoring are as follows:
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