CN117705971A - Method for identifying dendrobium candidum and dendrobium nobile - Google Patents

Method for identifying dendrobium candidum and dendrobium nobile Download PDF

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CN117705971A
CN117705971A CN202311705137.2A CN202311705137A CN117705971A CN 117705971 A CN117705971 A CN 117705971A CN 202311705137 A CN202311705137 A CN 202311705137A CN 117705971 A CN117705971 A CN 117705971A
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dendrobium
ion pair
dendrobium candidum
percent
candidum
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CN117705971B (en
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林涛
陈兴连
刘宏程
刘振环
魏茂琼
杜丽娟
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INSTITUTE OF QUALITY STANDARD AND DETECTION TECHNOLOGY YUNNAN ACADEMY OF AGRICULTURAL SCIENCES
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    • G01N30/02—Column chromatography
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Abstract

The invention belongs to the technical field of rapid identification of dendrobium, and discloses a method for identifying dendrobium candidum and dendrobium nobile. The method comprises the following steps: 1) Weighing Dendrobium officinale powder and Dendrobium officinale powder, respectively adding acetonitrile solution into the Dendrobium officinale powder and the Dendrobium officinale powder, mixing, and filtering to obtain Dendrobium officinale supernatant and Dendrobium officinale supernatant; 2) The dendrobium candidum supernatant and the dendrobium nobile supernatant are respectively measured by adopting an LC-MS/MS, a schedule-MRM mode is adopted during measurement, and the measurement conditions are set as follows: the detected ion pair is 579.2/271.0, wherein the parent ion pair is 579.2 and the child ion pair is 271.0; 3) By detection, the dendrobium candidum which cannot detect the ion pair 579.2/271.0 can detect the dendrobium candidum which is the ion pair 579.2/271.0. The method is simple and does not require extensive pretreatment.

Description

Method for identifying dendrobium candidum and dendrobium nobile
Technical Field
The invention relates to the technical field of rapid identification of dendrobium, in particular to a method for identifying dendrobium candidum and dendrobium nobile.
Background
Dendrobium officinale is known as the first part of Chinese Mesona herb, and is honored at home and abroad because of the good biological health care effect. Because of good activity, the product supply and demand are high, the market selling price is high, and the phenomenon of fake dendrobium candidum is common, and the dendrobium candidum is sold by adopting dendrobium candidum with low price and general health care function, such as dendrobium candidum. In general, the appearance difference between dendrobium candidum and dendrobium nobile is large, but when dendrobium candidum is dried or crushed into powder, the dendrobium candidum and dendrobium nobile are difficult to identify, and the molecular identification method is complex although the dendrobium candidum and dendrobium nobile can be identified in terms of molecular biology and species. Therefore, a method for rapidly and effectively identifying dendrobium candidum and dendrobium nobile is needed to be established in the field.
Disclosure of Invention
The invention aims to provide a method for identifying dendrobium candidum and dendrobium nobile, which aims to solve the problems of complex molecular biology and species identification in the prior art.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a method for identifying dendrobium candidum and dendrobium nobile, which comprises the following steps:
1) Weighing Dendrobium officinale powder and Dendrobium officinale powder, respectively adding acetonitrile solution into the Dendrobium officinale powder and the Dendrobium officinale powder, mixing, and filtering to obtain Dendrobium officinale supernatant and Dendrobium officinale supernatant;
2) The dendrobium candidum supernatant and the dendrobium nobile supernatant are respectively measured by adopting an LC-MS/MS, a schedule-MRM mode is adopted during measurement, and the measurement conditions are set as follows: the detected ion pair is 579.2/271.0, wherein the parent ion pair is 579.2 and the child ion pair is 271.0; the cluster removing voltage is-240 to-210V; the collision voltage is-55 to-30V; the temperature of the ion source is 350-550 ℃;
3) By detection, the dendrobium candidum serving as the ion pair 579.2/271.0 can not be detected, the dendrobium candidum serving as the ion pair 579.2/271.0 can be detected, and whether the dendrobium candidum and the dendrobium candidum are present or not can be effectively identified through the ion pair 579.2/271.0.
Preferably, the acetonitrile solution is an aqueous acetonitrile solution; in the acetonitrile solution, the volume ratio of acetonitrile to water is 7-9: 1 to 3.
Preferably, the mass volume ratio of the dendrobium candidum powder to the acetonitrile solution is 1-3 g:10mL; the mass volume ratio of the dendrobium nobile powder to the acetonitrile solution is 1-3 g:10mL.
Preferably, the mixing comprises the steps of firstly carrying out oscillating mixing, then carrying out ultrasonic mixing, and finally carrying out oscillating mixing; the time of the oscillation mixing is independently 3-15 min, and the speed of the oscillation mixing is 150-250 r/min; the ultrasonic mixing time is 1-3 min, and the ultrasonic mixing power is 20-30 kHz.
Preferably, the filtration is performed using an ultrafiltration membrane; the retention rate of the ultrafiltration membrane is more than or equal to 99.1 percent.
Preferably, in the step 2), the measurement conditions are set as follows: the mobile phase A is: methanol; the mobile phase B is: aqueous solution of ammonia water with mass fraction of 0.1-0.2%; the gradient elution conditions were: 0 to 2.5min,3 to 35 percent of A,2.5 to 5.5min,35 to 65 percent of A,5.5 to 8.5min,65 to 95 percent of A,8.5 to 9.8min and 95 percent of A;9.8 to 10min,95 to 3 percent of A; 10-12 min,3% A; the flow rate is: 180-220 mu L/min; the column temperature is: 35-45 ℃; the sample injection amount is as follows: 1.5 to 2.5 mu L.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for rapidly and effectively identifying dendrobium candidum and dendrobium nobile without complex pretreatment process, and only needs to identify through a single index.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard chromatogram of a 579.2/271.0 ion pair in an LC-MS/MS assay;
FIG. 2 is a chromatogram of the 579.2/271.0 ion pair in Dendrobium officinale in the LC-MS/MS assay of example 1;
FIG. 3 is a chromatogram of the 579.2/271.0 ion pair in Dendrobium nobile in the LC-MS/MS assay of example 1.
Detailed Description
The invention provides a method for identifying dendrobium candidum and dendrobium nobile, which comprises the following steps:
1) Weighing Dendrobium officinale powder and Dendrobium officinale powder, respectively adding acetonitrile solution into the Dendrobium officinale powder and the Dendrobium officinale powder, mixing, and filtering to obtain Dendrobium officinale supernatant and Dendrobium officinale supernatant;
2) The dendrobium candidum supernatant and the dendrobium nobile supernatant are respectively measured by adopting an LC-MS/MS, a schedule-MRM mode is adopted during measurement, and the measurement conditions are set as follows: the detected ion pair is 579.2/271.0, wherein the parent ion pair is 579.2 and the child ion pair is 271.0; the cluster removing voltage is-240 to-210V; the collision voltage is-55 to-30V; the temperature of the ion source is 350-550 ℃;
3) By detection, the dendrobium candidum serving as the ion pair 579.2/271.0 can not be detected, the dendrobium candidum serving as the ion pair 579.2/271.0 can be detected, and whether the dendrobium candidum and the dendrobium candidum are present or not can be effectively identified through the ion pair 579.2/271.0.
In the invention, the particle sizes of the dendrobium candidum powder and the dendrobium huoshanense powder are independently preferably 178-254 microns, and more preferably 180-220 microns.
In the present invention, the acetonitrile solution is preferably an aqueous solution of acetonitrile; in the acetonitrile solution, the volume ratio of acetonitrile to water is preferably 7-9: 1 to 3, more preferably 8:2.
in the invention, the mass volume ratio of the dendrobium candidum powder to the acetonitrile solution is preferably 1-3 g:10mL, more preferably 2 to 2.5g:10mL; the mass volume ratio of the dendrobium nobile powder to the acetonitrile solution is preferably 1-3 g:10mL, more preferably 2 to 2.5g:10mL.
In the invention, the mixing comprises the steps of firstly carrying out oscillating mixing, then carrying out ultrasonic mixing, and finally carrying out oscillating mixing; the independent time of the shaking mixing is preferably 3-15 min, more preferably 8-10 min; the independent speed of the oscillating mixing is preferably 150-250 r/min, more preferably 180-220 r/min; the ultrasonic mixing time is preferably 1 to 3 minutes, more preferably 2 to 2.5 minutes; the power of ultrasonic mixing is preferably 20 to 30kHz, more preferably 25 to 28kHz.
In the invention, the filtration is performed by using an ultrafiltration membrane; the rejection rate of the ultrafiltration membrane is preferably not less than 99.1%, and more preferably not less than 99.6%.
In the present invention, in the step 2), the measurement conditions are preferably set as follows: the mobile phase A is: methanol; the mobile phase B is: aqueous solution of ammonia water with mass fraction of 0.1-0.2%; the gradient elution conditions were: 0 to 2.5min,3 to 35 percent of A,2.5 to 5.5min,35 to 65 percent of A,5.5 to 8.5min,65 to 95 percent of A,8.5 to 9.8min and 95 percent of A;9.8 to 10min,95 to 3 percent of A; 10-12 min,3% A; the flow rate is: 180-220 mu L/min; the column temperature is: 35-45 ℃; the sample injection amount is as follows: 1.5 to 2.5 mu L. Further preferred are: the mobile phase A is: methanol; the mobile phase B is: aqueous solution of 0.1% ammonia water by mass fraction; the gradient elution conditions were: 0 to 2.5min,3 to 35 percent of A,2.5 to 5.5min,35 to 65 percent of A,5.5 to 8.5min,65 to 95 percent of A,8.5 to 9.8min and 95 percent of A;9.8 to 10min,95 to 3 percent of A; 10-12 min,3% A; the flow rate is: 200. Mu.L/min; the column temperature is: 40 ℃; the sample injection amount is as follows: 2. Mu.L.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The embodiment provides a method capable of rapidly and effectively identifying dendrobium candidum and dendrobium nobile, which comprises the following steps:
1) 2g of dendrobium candidum powder and dendrobium nobile powder are weighed, and 10mL of acetonitrile water solution (the volume ratio of acetonitrile to water is 8: 2) Oscillating for 10min at the speed of 200r/min, then carrying out ultrasonic treatment for 2min at the power of 25kHz, finally oscillating for 5min at the speed of 200r/min, and filtering by adopting an ultrafiltration membrane with the interception rate of 99.6% to obtain dendrobium candidum supernatant and dendrobium nobile supernatant.
2) LC-MS/MS determination is carried out on the dendrobium candidum supernatant and the dendrobium candidum supernatant respectively, and a schedule-MRM mode and an MRM detection window are adopted during determination: 30sec, anion scan, detected ion pair 579.2/271.0, with parent ion pair 579.2 and child ion pair 271.0; the cluster removing voltage is-210V, the collision voltage is-40V, and the ion source temperature is 450 ℃; the mobile phase A is: methanol; the mobile phase B is: aqueous solution of 0.1% ammonia water by mass fraction, gradient elution conditions: 0 to 2.5min,3 to 35 percent of A,2.5 to 5.5min,35 to 65 percent of A,5.5 to 8.5min,65 to 95 percent of A,8.5 to 9.8min and 95 percent of A;9.8 to 10min,95 to 3 percent of A; 10-12 min,3% A; flow rate: 200. Mu.L/min; column temperature: 40 ℃; sample injection amount: 2. Mu.L.
3) The peak time position corresponding to the 579.2/271.0 ion pair standard is shown in fig. 1 (retention time is 5.35 min), and according to the measurement, the dendrobium candidum has no 579.2/271.0 ion pair corresponding to the peak (shown in fig. 2), and the dendrobium candidum has 579.2/271.0 ion pair corresponding to the peak (shown in fig. 3).
Example 2
The embodiment provides a method capable of rapidly and effectively identifying dendrobium candidum and dendrobium nobile, which comprises the following steps:
1) 2g of dendrobium candidum powder and dendrobium nobile powder are weighed, and 10mL of acetonitrile water solution (the volume ratio of acetonitrile to water is 9: 1) Oscillating for 15min at 180r/min, then carrying out ultrasonic treatment for 2.5min at 20kHz, finally oscillating for 3min at 180r/min, and filtering by adopting an ultrafiltration membrane with a retention rate of 99.6% to obtain a dendrobium candidum supernatant and a dendrobium nobile supernatant.
2) LC-MS/MS determination is carried out on the dendrobium candidum supernatant and the dendrobium candidum supernatant respectively, and a schedule-MRM mode and an MRM detection window are adopted during determination: 30sec, anion scan, detected ion pair 579.2/271.0, with parent ion pair 579.2 and child ion pair 271.0; the cluster removing voltage is-220V, the collision voltage is-30V, and the ion source temperature is 480 ℃; the mobile phase A is: methanol; the mobile phase B is: aqueous solution of 0.1% ammonia water by mass fraction, gradient elution conditions: 0 to 2.5min,3 to 35 percent of A,2.5 to 5.5min,35 to 65 percent of A,5.5 to 8.5min,65 to 95 percent of A,8.5 to 9.8min and 95 percent of A;9.8 to 10min,95 to 3 percent of A; 10-12 min,3% A; flow rate: 200. Mu.L/min; column temperature: 40 ℃; sample injection amount: 2. Mu.L.
3) The peak time position corresponding to the 579.2/271.0 ion pair standard is shown in figure 1 (retention time is 5.35 min), and according to the measurement, the dendrobium candidum has no 579.2/271.0 ion pair corresponding to the peak, and the dendrobium candidum has 579.2/271.0 ion pair corresponding to the peak.
Example 3
The embodiment provides a method capable of rapidly and effectively identifying dendrobium candidum and dendrobium nobile, which comprises the following steps:
1) 2g of dendrobium candidum powder and dendrobium nobile powder are weighed, and 10mL of acetonitrile water solution (the volume ratio of acetonitrile to water is 8: 2) 2g of dendrobium candidum powder and dendrobium nobile powder are weighed, and 10mL of acetonitrile water solution (the volume ratio of acetonitrile to water is 9: 1) Oscillating for 15min at 150r/min, then carrying out ultrasonic treatment for 2.5min at 30kHz, finally oscillating for 3min at 150r/min, and filtering by adopting an ultrafiltration membrane with a retention rate of 99.2%, thereby obtaining dendrobium candidum supernatant and dendrobium candidum supernatant.
2) LC-MS/MS determination is carried out on the dendrobium candidum supernatant and the dendrobium candidum supernatant respectively, and a schedule-MRM mode and an MRM detection window are adopted during determination: 30sec, anion scan, detected ion pair 579.2/271.0, with parent ion pair 579.2 and child ion pair 271.0; the cluster removing voltage is-230V, the collision voltage is-50V, and the ion source temperature is 350 ℃; the mobile phase A is: methanol; the mobile phase B is: aqueous solution of 0.1% ammonia water by mass fraction, gradient elution conditions: 0 to 2.5min,3 to 35 percent of A,2.5 to 5.5min,35 to 65 percent of A,5.5 to 8.5min,65 to 95 percent of A,8.5 to 9.8min and 95 percent of A;9.8 to 10min,95 to 3 percent of A; 10-12 min,3% A; flow rate: 200. Mu.L/min; column temperature: 40 ℃; sample injection amount: 2. Mu.L.
3) The peak time position corresponding to the 579.2/271.0 ion pair standard is shown in figure 1 (retention time is 5.35 min), and according to the measurement, the dendrobium candidum has no 579.2/271.0 ion pair corresponding to the peak, and the dendrobium candidum has 579.2/271.0 ion pair corresponding to the peak.
Example 4
The embodiment provides a method capable of rapidly and effectively identifying dendrobium candidum and dendrobium nobile, which comprises the following steps:
1) 2g of dendrobium candidum powder and dendrobium nobile powder are weighed, and 10mL of acetonitrile water solution (the volume ratio of acetonitrile to water is 7: 3) Oscillating for 10min at the frequency of 250r/min, then carrying out ultrasonic treatment for 1.5min at the power of 30kHz, finally oscillating for 1min at the frequency of 250r/min, and filtering by adopting an ultrafiltration membrane with the interception rate of 99.1% to obtain the dendrobium candidum supernatant and the dendrobium candidum supernatant.
2) LC-MS/MS determination is carried out on the dendrobium candidum supernatant and the dendrobium candidum supernatant respectively, and a schedule-MRM mode and an MRM detection window are adopted during determination: 30sec, anion scan, detected ion pair 579.2/271.0, with parent ion pair 579.2 and child ion pair 271.0; the cluster removing voltage is-240V, the collision voltage is-55V, and the ion source temperature is 550 ℃; the mobile phase A is: methanol; the mobile phase B is: aqueous solution of 0.1% ammonia water by mass fraction, gradient elution conditions: 0 to 2.5min,3 to 35 percent of A,2.5 to 5.5min,35 to 65 percent of A,5.5 to 8.5min,65 to 95 percent of A,8.5 to 9.8min and 95 percent of A;9.8 to 10min,95 to 3 percent of A; 10-12 min,3% A; flow rate: 200. Mu.L/min; column temperature: 40 ℃; sample injection amount: 2. Mu.L.
3) The peak time position corresponding to the 579.2/271.0 ion pair standard is shown in figure 1 (retention time is 5.35 min), and according to the measurement, the dendrobium candidum has no 579.2/271.0 ion pair corresponding to the peak, and the dendrobium candidum has 579.2/271.0 ion pair corresponding to the peak.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (6)

1. The method for identifying dendrobium candidum and dendrobium nobile is characterized by comprising the following steps:
1) Weighing Dendrobium officinale powder and Dendrobium officinale powder, respectively adding acetonitrile solution into the Dendrobium officinale powder and the Dendrobium officinale powder, mixing, and filtering to obtain Dendrobium officinale supernatant and Dendrobium officinale supernatant;
2) The dendrobium candidum supernatant and the dendrobium nobile supernatant are respectively measured by adopting an LC-MS/MS, a schedule-MRM mode is adopted during measurement, and the measurement conditions are set as follows: the detected ion pair is 579.2/271.0, wherein the parent ion pair is 579.2 and the child ion pair is 271.0; the cluster removing voltage is-240 to-210V; the collision voltage is-55 to-30V; the temperature of the ion source is 350-550 ℃;
3) By detection, the dendrobium candidum serving as the ion pair 579.2/271.0 can not be detected, the dendrobium candidum serving as the ion pair 579.2/271.0 can be detected, and whether the dendrobium candidum and the dendrobium candidum are present or not can be effectively identified through the ion pair 579.2/271.0.
2. The method for identifying dendrobium candidum and dendrobium nobile according to claim 1, wherein the acetonitrile solution is an aqueous acetonitrile solution; in the acetonitrile solution, the volume ratio of acetonitrile to water is 7-9: 1 to 3.
3. The method for identifying dendrobium candidum and dendrobium nobile according to claim 2, wherein the mass-volume ratio of the dendrobium candidum powder to the acetonitrile solution is 1-3 g:10mL; the mass volume ratio of the dendrobium nobile powder to the acetonitrile solution is 1-3 g:10mL.
4. A method of identifying dendrobium candidum and dendrobium nobile according to any one of claims 1-3, wherein the mixing includes first performing shaking mixing, then performing ultrasonic mixing, and finally performing shaking mixing; the time of the oscillation mixing is independently 3-15 min, and the speed of the oscillation mixing is 150-250 r/min; the ultrasonic mixing time is 1-3 min, and the ultrasonic mixing power is 20-30 kHz.
5. The method of identifying Dendrobium officinale and Dendrobium nobile according to claim 4, wherein the filtration is performed by ultrafiltration membrane; the retention rate of the ultrafiltration membrane is more than or equal to 99.1 percent.
6. The method according to claim 5, wherein in the step 2), the measurement conditions are set as follows: the mobile phase A is: methanol; the mobile phase B is: aqueous solution of ammonia water with mass fraction of 0.1-0.2%; the gradient elution conditions were: 0 to 2.5min,3 to 35 percent of A,2.5 to 5.5min,35 to 65 percent of A,5.5 to 8.5min,65 to 95 percent of A,8.5 to 9.8min and 95 percent of A;9.8 to 10min,95 to 3 percent of A; 10-12 min,3% A; the flow rate is: 180-220 mu L/min; the column temperature is: 35-45 ℃; the sample injection amount is as follows: 1.5 to 2.5 mu L.
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CN103837511A (en) * 2014-01-26 2014-06-04 浙江工业大学 Rapid identification method for dendrobium officinale
CN105424850A (en) * 2015-11-05 2016-03-23 北京蓝标一成科技有限公司 Quality evaluation method for dendrobe medicinal material
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CN106521003A (en) * 2016-12-26 2017-03-22 三明市农业科学研究院 Method and PCR (polymerase chain reaction) primer for identifying dendrobium officinale
CN107941951A (en) * 2017-11-28 2018-04-20 安徽农业大学 A kind of method of Quercetin in efficient detection dendrobium candidum
CN109828053A (en) * 2019-03-15 2019-05-31 遵义医科大学 The raw HERBA DENDROBII of a kind of pair of stone and the method for setting raw HERBA DENDROBII progress chromatographic identification
CN115184497A (en) * 2022-07-18 2022-10-14 浙江省农业科学院 Method for measuring content of 2, 4-epibrassinolide in dendrobium officinale

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Publication number Priority date Publication date Assignee Title
CN102370891A (en) * 2010-08-18 2012-03-14 浙江中医药大学 Method for authenticating dendrobium officinale by using HPLC fingerprint
CN103837511A (en) * 2014-01-26 2014-06-04 浙江工业大学 Rapid identification method for dendrobium officinale
CN105424850A (en) * 2015-11-05 2016-03-23 北京蓝标一成科技有限公司 Quality evaluation method for dendrobe medicinal material
CN106521003A (en) * 2016-12-26 2017-03-22 三明市农业科学研究院 Method and PCR (polymerase chain reaction) primer for identifying dendrobium officinale
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CN115184497A (en) * 2022-07-18 2022-10-14 浙江省农业科学院 Method for measuring content of 2, 4-epibrassinolide in dendrobium officinale

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