CN108444985B - Kidney sperm ICP-MS identification method and application - Google Patents
Kidney sperm ICP-MS identification method and application Download PDFInfo
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Abstract
The invention relates to an identification method of animal calculi traditional Chinese medicines, in particular to an ICP-MS identification method of kidney sperms and application thereof; carrying out wet digestion on samples of known primary renal sperms and renal sperms of different batches to be detected, and carrying out ICP-MS (inductively coupled plasma-mass spectrometry) to obtain data of 18 elements including Li, Na, Mg, Al, P, S, K, Ca, Ti, V, Mn, Co, Ni, Cu, Zn, In, Ba and U; then the measured data is imported into SPSS 22.0 software for cluster analysis, and a tree-shaped cluster map is generated by using a 'group connection' clustering method, so that whether the renal sperms to be measured and the known primitive renal sperms belong to the same species or not is distinguished; or the data is imported into SIMCA-P14.1 software for PCA analysis to obtain a PCA scatter diagram, and the uniform stability of the renal sperms of different batches to be detected is judged according to the aggregation degree in the scatter diagram; the species source of the renal sperms is identified from the aspect of inorganic element composition, and the method can also be used for identifying the uniform stability of different batches of renal sperms, and is simple and rapid.
Description
Technical Field
The invention relates to an identification method of animal calculi traditional Chinese medicines, in particular to an ICP-MS identification method of kidney sperms and application thereof.
Background
The problem of the uniformity of the quality of the traditional Chinese medicine preparation is always a hot point problem, and the uniformity of the quality of the traditional Chinese medicine preparation directly influences the safety and the effectiveness of clinical medication and is also related to the health and the safety of patients. However, there are many factors that affect the uniformity of the quality of the Chinese medicinal preparation, wherein the diversity of the sources of the Chinese medicinal materials, "one medicine with many bases", and the uniform stability of the Chinese medicinal materials in different batches all affect the uniformity of the Chinese medicinal preparation prepared by using the same as the raw and auxiliary materials.
Kidney essence of cattle family Bos taurus domesticusBos taurus domesticus GmelinWater buffaloBubalus bubalisLinnaeusOr a porcine animalSus scrofa domestica BrissonThe bladder stones are distributed all over the country. According to the literature, the kidney sperm has the effects of promoting water and reducing swelling, the traditional Chinese medicine is clinically used for treating uroschesis, and the single medicine use of the kidney sperm is also provided with the special effect in the liver soothing and stasis removing formula taught by Zhang Hei professor, but the kidney sperm does not have the quality standard at present, the market of kidney sperm medicinal materials is disordered and has good quality, an effective and reliable quality monitoring means is lacked, and the quality control of the medicinal materials is not facilitated. There is no effective identification method and monitoring means for uniform stability of different batches of renal spermatozoa and species origin of renal spermatozoa.
Disclosure of Invention
The invention aims to solve the technical problem that the species source of the renal sperm is lack of an effective identification method, and provides an ICP-MS (inductively coupled plasma-mass spectrometry) identification method and application of the renal sperm.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a method for identifying renal sperms by ICP-MS is characterized by comprising the following steps:
(1) carrying out wet digestion on samples of known primitive renal sperms and renal sperms of different batches to be detected;
(2) digesting the known primary kidney sperm and kidney sperm to be detected In different batches, diluting the digested primary kidney sperm and the digested kidney sperm to be detected, and carrying out ICP-MS (inductively coupled plasma-mass spectrometry) determination on the digested primary kidney sperm and the kidney sperm to be detected to obtain data of 18 elements including Li, Na, Mg, Al, P, S, K, Ca, Ti, V, Mn, Co, Ni, Cu, Zn, In, Ba and U;
(3) importing the data measured in the step (2) into SPSS 22.0 software for clustering analysis, and generating a dendriform clustering chart by using a 'group connection' clustering method so as to distinguish whether the renal sperms to be measured and the known primitive renal sperms belong to the same species or not;
(4) and (3) importing the data measured in the step (2) into SIMCA-P14.1 software to perform PCA analysis to obtain a PCA scatter diagram, wherein the different varieties of renal sperms have obvious separation tendency, and the chemical components of the various varieties of renal sperms have certain difference and different composition structure trends, so that whether the renal sperms to be measured and the known primitive renal sperms belong to the same species or not is distinguished.
Another object of the present invention is to provide an identification method for the problem of lack of an effective identification method for uniform stability of different batches of renal sperm, comprising performing the above steps (1) and (2), introducing the data obtained in step (2) into SIMCA-P14.1 software for PCA analysis to obtain a PCA scattergram, and determining the uniform stability of different batches of renal sperm to be tested according to the aggregation degree in the scattergram.
In the above steps, the ICP-MS measurement refers to inductively coupled plasma mass spectrometry. The reason why the data of 18 elements including Li, Na, Mg, Al, P, S, K, Ca, Ti, V, Mn, Co, Ni, Cu, Zn, In, Ba and U are selectively measured In the step (2) is that after statistical analysis, it is determined that the 18 elements have the inorganic element strength higher than 10000 In the kidney sperm. Among them, the SPSS 22.0 software and the SIMCA-P14.1 software are known analysis software.
Further, when performing wet digestion in the step (1), respectively and precisely weighing 0.1g of powder of the known primary kidney sperm and the kidney sperm of different batches to be detected, placing the powder in a beaker, respectively adding 10 mL of 65% nitric acid and 1mL of perchloric acid, placing the beaker on an electric hot plate for standing overnight, performing wet digestion at 180 ℃ until the liquid is clear and transparent, then driving up the acid on the electric hot plate until the residual 1mL of the liquid is obtained, and adding ultrapure water to fix the volume to a colorimetric tube of 50 mL.
Further, the wet-digested renal spermatozoa in step (1) are diluted 20 times before the ICP-MS measurement in step (2); in the step (2), the ICP-MS determination conditions are as follows: testing the sensitivity of a sample front tuning instrument and testing the positioning of tuning liquid; by adopting a KED mode, the purity of argon is 99.99 percent, the purity of helium is 99.99 percent, the flow rate of an atomizer is 1.0386L/min, the flow rate of a cooler is 14.00L/min, the flow rate of auxiliary gas is 0.8L/min, the sampling depth is 5mm, the plasma power is 1550W, and the pump speed is 40 rpm.
Furthermore, the difference between the two kinds of kidney sperms of pig origin and bovine origin is Li, Na, Mg, P, S, K, Ca, Ti, V, Zn, In and U.
Another objective of the invention is to apply a method for identifying renal sperm ICP-MS to identify the uniform stability of renal sperm of different batches and species sources of renal sperm.
Compared with the prior art, the invention has the following beneficial effects:
the ICP-MS method for identifying the renal sperms can simultaneously represent 18 inorganic elements including Na, Mg, P, S, K and other main elements, can comprehensively reflect the chemical components and quality characteristics of the renal sperms, can identify the species source of the renal sperms from the aspect of inorganic element composition, can also be used for identifying the uniform stability of different batches of renal sperms, and is simple and rapid.
Drawings
FIG. 1 is a dendrogram of different varieties of renal sperm cells from example 1.
FIG. 2 is a PCA scattergram of different varieties of renal sperms of example 1.
FIG. 3 is a scattergram of different species of renal sperm OPLS-DA of example 1.
FIG. 4 is a fitting graph of OPLS-DA of different varieties of renal sperm of example 1.
FIG. 5 is a graph of VIP values of different species of renal sperm in example 1.
FIG. 6 is a diagram of different varieties of renal sperm S-plots of example 1.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
The invention relates to a construction process of an ICP-MS (inductively coupled plasma-mass spectrometry) identification method for renal sperms.
Collecting commercially available 2 different varieties of kidney sperm in 12 batches (6 batches for each variety, wherein 1-6 are bovine (N); 7-12 are porcine (Z))
Firstly, measuring inorganic element spectrums of 12 batches of different types of kidney sperms.
(1) Respectively and precisely weighing 0.1g of the kidney sperm powder in a beaker, respectively adding 10 mL of 65% nitric acid and 1mL of perchloric acid, covering a glass dish, standing overnight, placing on an electric heating plate for wet digestion at 180 ℃, digesting until the liquid is clear and transparent, then driving acid on the electric heating plate until about 1mL of the liquid remains, adding ultrapure water, and fixing the volume to a 50mL colorimetric tube.
(2) And (3) diluting the sample by 20 times before testing, and testing by using an inductively coupled plasma mass spectrometer to obtain the data of each element of the kidney sperm ICP-MS.
(3) And (3) testing conditions are as follows: the sensitivity of the instrument was tuned before testing the sample and tuning solution positioning was tested. By adopting a KED mode, the purity of argon is 99.99 percent, the purity of helium is 99.99 percent, the flow rate of an atomizer is 1.0386L/min, the flow rate of a cooler is 14.00L/min, the flow rate of auxiliary gas is 0.8L/min, the sampling depth is 5mm, the plasma power is 1550W, and the pump speed is 40 rpm.
(4) Performing statistical analysis on ICP-MS data of the renal sperms, and determining that the elements with inorganic element strength higher than 10000 in the renal sperms are as follows: li, Na, Mg, Al, Si, P, S, K, Ca, Ti, V, Mn, Co, Ni, Cu, Zn, In, Ba, U total 18 elements.
(5) And importing the data into SPSS 22.0 software for cluster analysis. The clustering method of "connection between groups" was used to generate a dendrogram, as can be seen from FIG. 1, samples 1-6 are of the first type, and samples 7-12 are of the second type.
And secondly, comparing inorganic elements of 12 batches of kidney sperms of different varieties obtained by measurement.
The data obtained above were imported into SIMCA-P14.1 software for PCA analysis. As can be seen from PCA scatter diagrams (shown in figure 2) of different varieties of renal sperms, the renal sperms of different varieties have obvious separation tendency, which shows that the chemical components of the renal sperms of different varieties have certain difference and have different composition structure tendency, and the method is proved to be capable of well distinguishing two different primitive renal sperms; the aggregation degree of the pig-derived nephrosperms and the bovine-derived nephrosperms in the group is better, which indicates that the quality of the nephrosperms of the variety is uniform and stable.
The inorganic element spectra of different types of kidney sperms are compared and analyzed, the OPLS-DA scatter diagram (figure 3) of different types of kidney sperms shows that the relationship among the kidney sperm groups of different types is clear, the chemical components of the kidney sperms of different types have certain difference, the model fitting is established (figure 4), and the difference foreign matters between the two types of kidney sperms are Li, Na, Mg, P, S, K, Ca, Ti, V, Zn, In and U (figure 6) by combining the VIP value (figure 5) and the data of t-Test P which is less than 0.05. From this, it can be seen that the ICP-MS spectrum of inorganic elements is used for kidney sperm quality control, and the content and homogeneity of inorganic elements can be compared.
Example 2
A method for identifying renal sperms by ICP-MS comprises the following steps:
(1) carrying out wet digestion on samples of known primitive renal sperms and different batches of renal sperms to be tested: accurately weighing 0.1g of powder of known primary kidney sperms and kidney sperms of different batches to be detected respectively, adding 10 mL of 65% nitric acid and 1mL of perchloric acid respectively, standing the mixture in a glass dish for one night, placing the mixture on an electric heating plate for wet digestion at 180 ℃, digesting the mixture until the liquid is clear and transparent, driving acid on the electric heating plate until 1mL of the liquid remains, and adding ultrapure water to fix the volume to a 50mL colorimetric tube.
(2) Digesting the known primary kidney sperm and the kidney sperm to be detected In different batches, diluting the digested primary kidney sperm by 20 times, and carrying out ICP-MS (inductively coupled plasma-mass spectrometry) determination to obtain data of 18 elements including Li, Na, Mg, Al, P, S, K, Ca, Ti, V, Mn, Co, Ni, Cu, Zn, In, Ba and U; the specific determination conditions are that the sensitivity of the tuning instrument before the sample is tested and the tuning liquid is tested for positioning; by adopting a KED mode, the purity of argon is 99.99 percent, the purity of helium is 99.99 percent, the flow rate of an atomizer is 1.0386L/min, the flow rate of a cooler is 14.00L/min, the flow rate of auxiliary gas is 0.8L/min, the sampling depth is 5mm, the plasma power is 1550W, and the pump speed is 40 rpm.
(3) And (3) importing the data measured in the step (2) into SPSS 22.0 software for clustering analysis, and generating a dendriform clustering diagram by using a 'group connection' clustering method so as to distinguish whether the renal sperms to be measured and the known primitive renal sperms belong to the same species.
(4) And (3) importing the data measured in the step (2) into SIMCA-P14.1 software to perform PCA analysis to obtain a PCA scatter diagram, wherein different varieties of renal sperms have obvious separation tendency, and the chemical components of the renal sperms of different varieties have certain difference and different composition structure trends, so that whether the renal sperms to be detected and the known primitive renal sperms belong to the same species or not is distinguished, and meanwhile, the uniform stability of the renal sperms of different batches to be detected is judged according to the aggregation degree in the scatter diagram.
Claims (5)
1. An ICP-MS identification method for renal sperms is characterized by comprising the following steps:
(1) respectively carrying out wet digestion on samples of known primitive renal sperms and renal sperms of different batches to be detected;
(2) digesting the known primary kidney sperm and kidney sperm to be detected In different batches, diluting the digested primary kidney sperm and the digested kidney sperm to be detected, and carrying out ICP-MS (inductively coupled plasma-mass spectrometry) determination on the digested primary kidney sperm and the kidney sperm to be detected to obtain data of 18 elements including Li, Na, Mg, Al, P, S, K, Ca, Ti, V, Mn, Co, Ni, Cu, Zn, In, Ba and U;
(3) importing the data measured in the step (2) into SPSS 22.0 software for clustering analysis, and generating a dendriform clustering chart by using a 'group connection' clustering method so as to distinguish whether the renal sperms to be measured and the known primitive renal sperms belong to the same species;
(4) or the data measured in the step (2) is imported into SIMCA-P14.1 software for PCA analysis to obtain a PCA scatter diagram, and because different varieties of renal sperms have the obvious separation tendency, the chemical components of the renal sperms of all varieties have certain difference and have different composition structure trends, the renal sperms to be measured and the known primitive renal sperms belong to the same species or not are distinguished;
and (3) importing the data measured in the step (2) into SIMCA-P14.1 software to perform PCA analysis to obtain a PCA scatter diagram, and meanwhile, judging the uniform stability of the renal sperms of different batches to be measured according to the aggregation degree in the scatter diagram.
2. The ICP-MS identification method for kidney sperms according to claim 1, wherein in the wet digestion in the step (1), 0.1g of powder of known primary kidney sperms and kidney sperms of different batches to be detected are precisely weighed in a beaker, 10 mL of 65% nitric acid and 1mL of perchloric acid are respectively added, the beaker is covered with a glass dish and placed on an electric heating plate for wet digestion at 180 ℃ after being placed overnight, the solution is digested until the solution is clear and transparent, then acid is removed on the electric heating plate until 1mL of the solution remains, and ultrapure water is added to fix the volume to a 50mL colorimetric tube.
3. The ICP-MS method for identifying renal spermatozoa according to claim 1, wherein the wet-digested renal spermatozoa in step (1) are diluted 20-fold before ICP-MS measurement in step (2); in the step (2), the ICP-MS determination conditions are as follows: testing the sensitivity of a sample front tuning instrument and testing the positioning of tuning liquid; by adopting a KED mode, the purity of argon is 99.99 percent, the purity of helium is 99.99 percent, the flow rate of an atomizer is 1.0386L/min, the flow rate of a cooler is 14.00L/min, the flow rate of auxiliary gas is 0.8L/min, the sampling depth is 5mm, the plasma power is 1550W, and the pump speed is 40 rpm.
4. The ICP-MS method for identifying renal spermatozoa according to claim 1, wherein the difference between the two renal spermatozoa of porcine origin and bovine origin is Li, Na, Mg, P, S, K, Ca, Ti, V, Zn, In, U.
5. The use of the ICP-MS method of identifying renal sperm of claim 1, wherein said method is used to identify the species origin of renal sperm and the uniform stability of renal sperm in different batches.
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