CN114414679B - Method for detecting belladonna alkaloid in animal tissues - Google Patents
Method for detecting belladonna alkaloid in animal tissues Download PDFInfo
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- CN114414679B CN114414679B CN202111614723.7A CN202111614723A CN114414679B CN 114414679 B CN114414679 B CN 114414679B CN 202111614723 A CN202111614723 A CN 202111614723A CN 114414679 B CN114414679 B CN 114414679B
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Abstract
The invention provides a method for detecting belladonna alkaloids in animal tissues, which adopts a dispersive solid-phase extraction-high performance liquid chromatography triple quadrupole mass spectrometry method, and adopts later martropine as an internal standard substance, and simultaneously detects anisodamine, racanisodamine, anisodamine, atropine, scopolamine, tropinic acid, noratropine and the like in animal tissues, thereby optimizing pretreatment and separation detection conditions. The target compound has good linearity in the range of 0.5ng/L-200ng/L, the detection limit of the method is 0.05-0.1 mug/kg, the quantitative limit of the method is 0.2-0.5 mug/kg, the matrix blank standard adding recovery rate is 80.2-119%, and the relative standard deviation of parallel measurement is 1.13-14.5%. The method can accurately measure the belladonna alkaloid in animal tissues, has accurate and reliable measurement results, provides reliable data for the measurement of the belladonna alkaloid, has obvious economic benefit and has wide market prospect. Has good reference value for guiding the measurement of drug residues.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for detecting belladonna alkaloid in animal tissues.
Background
Toxic Solanaceae plants such as herba Daturae, semen hyoscyami, and belladonna contain various toxic alkaloids such as hyoscyamine, atropine, scopolamine, anisodamine, and anisodamine. These alkaloids are collectively referred to as belladonna alkaloids (be l l adonna a l ka loids), also known as tropane alkaloids (tropane a l ka loids). Belladonna alkaloid can interfere cerebral cortex to generate central inhibition, can competitively block the excitation action of M choline receptor on respiratory center, can cause the phenomena of dry mouth, pupil expansion, hoarseness, heart beat acceleration, vasodilation, intermittent convulsion or spasm, and the like, can lead the central system to generate disorder, mania, dizziness, illusion and general dosage to feel tired, enter dreamless sleep, and lead a user to generate mental confusion, absentmince of consciousness, symptoms such as illusion, coma and the like if the user excessively uses, and finally generate respiratory failure to die. Anisodamine and atropine excite medulla oblongata and brain, and the patients are very manic and accompanied with symptoms of language disorder, and can also have symptoms of inhibition, so that the patients are unconscious, and the patients can die if the patients are inhibited from breathing. The medicine residue enters the human body through the food chain to reach a certain concentration, and symptoms such as nausea, vomit, dizziness, weakness, limb numbness and the like can appear, and serious patients can die due to respiratory failure.
In conclusion, the establishment of the efficient and reliable drug detection method has very important significance for finding potential risks of belladonna alkaloid original drugs and metabolites and developing related researches.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting belladonna alkaloids in animal tissues, which aims to solve the defects in the prior art.
In order to achieve the above object, the present invention provides a method for detecting belladonna alkaloid in animal tissue, comprising:
preparing a sample, cleaning the unfrozen animal tissue to be tested, cutting into pieces, mashing, subpackaging in a sample container, and preserving for later use;
Extracting a sample, namely weighing 5g of a sample, accurately obtaining 0.01g of the sample, adding 50 mu L of internal standard working solution into a 50mL centrifuge tube, mixing for 30s by vortex, standing for 10min, adding 15mL of acidified acetonitrile, mixing for 1min by vortex, extracting for 10min by ultrasonic at 35-55 ℃, taking out, cooling to room temperature, centrifuging for 5min at 4500r/min, and transferring the supernatant into a 100mL chicken heart bottle;
Extracting the solids in the centrifuge tube after removing the supernatant twice to obtain a supernatant of the second time, combining the supernatants of the second time, and performing rotary evaporation to near dryness at 45 ℃ to obtain residues;
Sample purification, namely adding 50 mu L of methanol and water into the residues, dissolving the residues, transferring the residues into a 2mL centrifuge tube, fixing the volume to 1.0mL by using water, adding an adsorbent, carrying out vortex oscillation for 30S, adding 1mL of normal hexane, carrying out vortex oscillation for 10S, carrying out 12000r/min for 5min, discarding an upper grease layer, repeatedly purifying once, freezing to-20 ℃ for 30min, carrying out 12000r/min for 5min, and filtering the lower clear liquid for liquid use;
sample detection, namely detecting the filtered supernatant by a machine, wherein detection conditions comprise:
Chromatographic column: i nfin ityLabPoroshe l l 120HPH-C18 (2.1 mm. Times.100 mm,1.9 μm); flow rate: 0.2mL/min; mobile phase: phase A is 5mM ammonium formate+0.1% formic acid; the phase B is methanol, and the gradient elution is carried out; column temperature: 40 ℃, sample injection amount: 10. Mu.L; quantitative determination by an internal standard method;
mass spectrum ion source, ESI; the temperature of the ion source is 100 ℃; the dry air flow is 10L/h; capillary voltage 4000V; the temperature of the air flow is 350 ℃; the atomization gas pressure is 40ps i; the collision gas type is nitrogen; positive ions; the detection mode is MRM.
According to one embodiment of the invention, the adsorbent comprises 10mg psa,10mg NH2, 20mg neutral alumina.
According to one embodiment of the invention, the gradient elution conditions are in volume fraction at a flow rate of 0.2ml/min; in 0-1 min, the phase A of the mobile phase is 95% and the phase B of the mobile phase is 5%; during 2-4 min, the mobile phase A phase is 90% and the mobile phase B phase is 10%; at 5min, mobile phase A was 50% and mobile phase B was 50%; at 10min, mobile phase A was 20% and mobile phase B was 80%; at 10-15 min, the mobile phase A phase is 95% and the mobile phase B phase is 5%.
According to one embodiment of the invention, the secondary extraction of the solids after removal of the supernatant in the centrifuge tube comprises: shaking the solid, adding 15mL of acidified acetonitrile, shaking up and down by hand for 30s, mixing by vortex, and adding ultrasound to obtain a supernatant of the second time.
According to one embodiment of the invention, the filtering of the supernatant comprises: the supernatant was filtered through a 0.22 μm filter.
According to one embodiment of the invention, the animal tissue comprises: animal muscle, meat, heart, liver, kidney, lung.
According to one embodiment of the invention, the detection method further comprises:
standard stock solution: respectively weighing proper amounts of anisodamine, racanisodamine, anisodamine hydrobromide, atropine sulfate, hyoscyamine, scopolamine, after hydrobromic acid, tropinic acid and noratropine, wherein the contents of anisodamine, racanisodamine, anisodamine, atropine, hyoscyamine, scopolamine, rear-equinox, tropinic acid and noratropine are all 10mg, and methanol is used for fixing the volume into a 10mL brown volumetric flask to prepare a standard stock solution with the mass concentration of 1000 mug/mL;
Transferring anisodamine, racanisodamine, anisodamine, atropine, hyoscyamine, scopolamine, back-horse tropine, tropinic acid and noratropine standard solution, and fixing volume in 10mL brown volumetric flask with methanol to obtain mixed intermediate solution with mass concentration of 1000 ng/mL;
10ng/mL of mixed standard solution 20 mu L, 50 mu L, 100 mu L, 0.5mL and 2mL are respectively added into a blank matrix, 1000ng/mL of mixed standard solution 50 mu L, 100 mu L and 200 mu L are added, the mixture is treated according to the same extraction and purification steps as the sample, 8 mass concentration standard solutions of 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 200ng/mL are prepared, and a post-martrope is used as an internal standard, and a working curve is drawn by an internal standard method.
The detection method of the embodiment of the invention has at least the following beneficial effects:
According to the method for detecting the belladonna alkaloids in the animal tissues, disclosed by the embodiment of the invention, the belladonna alkaloids in the animal tissues can be accurately detected, the detection result is accurate and reliable, reliable data is provided for the measurement of the belladonna alkaloids, and the method has remarkable economic benefits and wide market prospects. Has good reference value for guiding the measurement of drug residues.
Drawings
FIG. 1 is a mass-correlation spectrum of anisodamine characteristic ions in pork matrix of the present invention;
FIG. 2 is a mass-correlation spectrum of racanisodamine characteristic ions in pork matrix of the present invention;
FIG. 3 is a mass correlation spectrum of anisodine characteristic ions in pork matrix according to the present invention;
FIG. 4 is a mass correlation spectrum of noratropine characteristic ions in pork matrix according to the present invention;
FIG. 5 is a graph of the mass correlation of atropine characteristic ions in pork matrix according to the present invention;
FIG. 6 is a graph of the mass correlation of post-captopril characteristic ions in pork matrix according to the invention;
FIG. 7 is a graph of the correlation of the ion mass of scopolamine characteristic in the pork matrix of the present invention;
FIG. 8 is a mass correlation spectrum of scopolamine characteristic ions in pork matrix of the present invention;
FIG. 9 is a graph of the correlation of the mass of SKF-525A characteristic ions in pork matrix according to the present invention;
FIG. 10 is a graph of the mass correlation of tropina-characteristic ions in pork matrix according to the present invention;
FIG. 11 is a mass spectrum of the target compound characteristic ion superposition in the pork matrix of the present invention.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The instrument and the device of the invention:
6410B liquid chromatograph-tandem quadrupole mass spectrometer, company Agi lent, USA;
XS205 analytical balance, mett ler company, switzerland;
TE212-L electronic balance, sartor ius, germany;
an adjustable range pipette, company VI TLAB, germany;
TDL-40B desk-top centrifuge, shanghai's pavilion scientific instrument factory.
The material of the invention is as follows:
TABLE 1 list of standard substances
| Standard substance name | CAS number | Standard value | Manufacturing factories |
| Anisodamine | 55869-99-3 | 10mg，≥99.5％ | German HPC-68205 |
| Raanisodamine | 17659-49-3 | 100mg，≥99.8％ | Center institute 100249-201303 |
| Anisodine hydrobromide | 76822-34-9 | 100mg，≥99.8％ | Center check institute 100399 |
| Atropine sulfate | 5908-99-6 | 100mg，≥99.6％ | Altar ink quality test 75801-100mg |
| Hyoscyamine | 101-31-5 | 20mg，≥99.5％ | U.S. CATOCCPE901375 |
| Scopolamine | 51-34-3 | 50mg，≥98.6％ | U.S. CATOCCPE901273 |
| Tropine acid | 529-64-6, | 50mg，≥99.0％ | Chinese food and drug verification research |
| Noratropine | 16839-98-8 | 5mg，≥99.8％ | U.S. PANPHY |
| Hydrobromic acid post-ma tuo pin | 51-56-9 | 50mg，≥99.7％ | Canadian TRC |
Acetonitrile, methanol and n-hexane are all chromatographic purities, company Fi sher in the united states;
formic acid (LC/MS), company Fi sher, U.S.;
Ammonium formate (LC/MS), sigma-Aldr ich, USA;
Amino (NH 2) SPE bulk adsorbent, U.S. Agi lent;
Bondes i l-PSA bulk adsorbent (40 um), us Agi lent;
the test water meets the requirements of GB/T6682 primary water, and the rest reagents are all of domestic analytical purity.
The selection and detection process of parameters and technical indexes in the present invention will be described below with reference to specific embodiments.
Preparation of (one) Standard stock solution
Standard stock solution: and respectively weighing proper amounts of anisodamine, racanisodamine, anisodamine hydrobromide, atropine sulfate, scopolamine, after hydrobromic acid, tropinic acid and noratropine, wherein the contents of anisodamine, racanisodamine, anisodamine, atropine, scopolamine, post-scopolamine, tropinic acid and noratropine are all 10mg, and methanol is used for constant volume into a 10mL brown volumetric flask to prepare the standard stock solution with the mass concentration of 1000 mug/mL.
Transferring anisodamine, racanisodamine, anisodamine, atropine, hyoscyamine, scopolamine, post-ma tropine, tropinic acid and noratropine standard solution, and fixing volume in 10mL brown volumetric flask with methanol to obtain mixed intermediate solution with mass concentration of 1000ng/mL, and freeze-preserving at-16-20deg.C in dark place. When in use, the mixture is gradually diluted to the concentration of 100ng/mL and 10ng/mL by methanol, and the mixture is prepared on site.
10Ng/mL of mixed standard solution (20 mu L, 50 mu L, 100 mu L, 0.5mL and 2 mL) and 1000ng/mL of mixed standard solution (50 mu L, 100 mu L and 200 mu L) are respectively added into a blank matrix, the mixture is treated according to the same extraction and purification steps as the samples, 8 standard solutions (0.2, 0.5, 1, 5, 20, 50, 100 and 200 ng/mL) with different mass concentrations are prepared, and a post-martrope is used as an internal standard, and a working curve is drawn by an internal standard method.
(II) sample preparation
After the cold fresh livestock and poultry muscle products are cleaned, removing hair, skin, blood stasis, tendons and bones, and preparing the cold fresh livestock and poultry muscle products. Thawing frozen livestock meat at room temperature, slightly thawing and softening the sample, removing hair, skin, tendons and bones when the interior is just thawed and the frozen water is not flowing out, preparing, and cleaning by-products such as pig heart, liver, kidney and lung with experimental water. The sample is cut into small pieces of 1cm multiplied by 1cm, uniformly mixed, reduced to 500g by a quartering method, smashed by a smashing machine, uniformly mixed and packaged into a plurality of sample containers for standby. Pretreating viscera of livestock and fowl, dicing or cutting, mashing with a masher, mixing, and packaging into sample containers. After the fish sample is cleaned, removing scales, taking edible skin and meat, cutting into small pieces of 1cm multiplied by 1cm, dividing into 500g by a quartering method, mashing, homogenizing, mixing uniformly, and sub-packaging for later use.
(III) sample preservation
After the sample is prepared, if the sample cannot be detected in time, the sample should be frozen and stored immediately, so that the sample cannot be melted and deteriorated before detection. The test sample tested on the same day can be stored in a temporary refrigeration way for no more than 8 hours. The frozen sample should be subjected to test after thawing at 45 ℃ or below for no more than 15min, or 2-5 ℃ for no more than 18 h.
(IV) extraction
Weighing 5g of sample, accurately obtaining 0.01g, adding 50 mu L of internal standard working solution into a 50mL centrifuge tube, vortex mixing for 30s, standing for 10min, adding 15mL of acidified acetonitrile, vortex mixing for 1min, ultrasonic extracting for 10min at 35-55 ℃, taking out, cooling to room temperature, centrifuging for 5min at 4500r/min, and transferring the supernatant into a 100mL chicken heart bottle. Shaking to disperse solid, adding 15mL of acidified acetonitrile, shaking up and down for 30s by hand, mixing uniformly by vortex, adding ultrasonic to repeatedly extract for 1 time, and combining the two supernatants; rotary evaporation to near dryness at 45 ℃.
(V) purification
Adding 50 mu L of methanol, a small amount of water and dissolving residues, transferring to a 2mL centrifuge tube, fixing the volume to 1.0mL by using water, adding an adsorbent (10 mgPSA,10mg NH2 and 20mg neutral alumina), carrying out vortex oscillation for 30S, adding 1mL of n-hexane, carrying out vortex oscillation for 10S, centrifuging for 5min by 12000r/min, discarding an upper grease layer, repeatedly purifying once, freezing for 30min at-20 ℃, centrifuging for 5min by 12000r/min, and filtering the lower supernatant by a 0.22 mu m filter membrane for liquid quality use.
Sixth, on-machine detection
Detection conditions were measured using a 6410B liquid chromatograph-tandem quadrupole mass spectrometer, company Agi lent, usa:
chromatographic column: i nfin ity Lab Poroshe l l 120HPH-C18 (2.1 mm. Times.100 mm,1.9 μm); flow rate: 0.2mL/min; mobile phase: phase A is 5mM ammonium formate+0.1% formic acid; the phase B is methanol, the gradient elution is carried out, and the specific setting conditions are shown in Table 2; column temperature: 40 ℃; sample injection amount: 10. Mu.L; and (5) quantifying by an internal standard method.
TABLE 2 gradient elution conditions for mobile phases
Mass spectrometry ion source: the temperature of the ESI ion source is 100 ℃; the dry air flow is 10L/h; capillary voltage 4000V; the temperature of the air flow is 350 ℃; the atomization gas pressure is 40ps i; the collision gas type is nitrogen; positive ions; the detection mode is MRM. Wherein, the mass spectrum acquisition parameter conditions are shown in Table 3.
Table 3 conditions for mass spectrum acquisition parameters
(Seventh) results and analysis
In the test process, in order to obtain the best experimental result, a great amount of tests and comparisons are carried out on the selection of the extractant, the extraction mode, the extraction time of the feed liquid ratio and the like, and the result is analyzed. The analysis procedure of the results is specifically described below:
1. Spectrogram analysis
The target compounds such as anisodamine, racanisodamine, anisodamine, atropine, hyoscyamine, scopolamine, tropinic acid and noratropine are analyzed according to chromatographic conditions, the anisodamine and the racanisodamine can be effectively separated, but the atropine and the anisodamine cannot be separated, other 6 target compounds are completely separated from interfering substances, and the peak shape is good, and the detailed descriptions are shown in table 4 and figures 1-11. In view of the fact that scopolamine is a levorotatory body and atropine is a mixed rotatory body, the scopolamine can change after a period of time under normal conditions to generate an optically active body so as to be converted into atropine, and thus the atropine is selected as a target compound; anisodamine is alkaloid extracted from anisodamine tangutica, which is a special plant in China, is a levo natural product, and racanisodamine is a racemic body which is synthesized by two pairs of enantiomers artificially and exists, namely, the anisodamine is separated from the racanisodamine, namely, the dextro body is effectively separated from the levo body, and the related pharmacological actions of the anisodamine and the racanisodamine are obviously different, so that the effective separation has very important significance in practical application, such as analyzing the source approach of target compounds and the like. Thus anisodamine, racanisodamine, anisodamine, atropine, scopolamine, tropinic acid and noratropine are selected as target compounds.
TABLE 4 target compound retention time and relative abundance of next strongest fragment ions
| Target compound | Retention time min | The relative abundance of the second-strongest fragment ions% |
| Anisodamine | 8.922 | 55 |
| Raanisodamine | 9.278 | 43 |
| Anisodine | 7.558 | 36 |
| Noratropine | 9.458 | 59 |
| Atropine | 9.152 | 51 |
| Rear horse riding product | 9.024 | 67 |
| Hyoscyamine | 9.088 | 53 |
| Scopolamine | 9.074 | 45 |
| SKF-525A | 9.047 | 40 |
| Tropine acid | 4.624 | 30 |
2. Pretreatment condition optimization
(1) Selection of extractant
The extraction effects of acetone, methanol, acetonitrile and acidified acetonitrile (0.1% formic acid) on 7 target compounds are respectively examined, the extraction interference of the acetone is more, the subsequent separation on an upper machine is not facilitated, the standard recovery rate of the target compounds is not obviously influenced by the methanol, the acetonitrile and the acidified acetonitrile, but after the acidified acetonitrile is extracted, the peak area response value of characteristic ion fragments of each target compound is obviously higher than that of other extractants, the absolute recovery rate is higher than that of other reagents, the analysis recovery rate is not obviously influenced, and the main reason is that the internal standard method counteracts the loss of the seed in the extraction process, so that the acidified acetonitrile is preferably used as the extractant.
3. Selection of extraction mode, time and temperature
The meat, liver, heart, lung and kidney samples have different complexity, and the tissue and body fluid components and proportions are different, so that the anhydrous sodium sulfate and the anhydrous magnesium sulfate are used for removing water by comparison, and the anhydrous sodium sulfate or the anhydrous magnesium sulfate is needed to be more for the pork liver and the pork lung by comparison, the anhydrous magnesium sulfate has better water removing effect than the anhydrous sodium sulfate, needs less quality, and has a certain adsorption effect on target compounds. However, through repeated experiments, the moisture content of different samples of the same substrate still has difference, and a small amount of moisture can still exist in the respective samples after the water is removed, and the time of spin-steaming dry consumption is long. In view of the main purpose of water removal in the test process, the method is to unify the proportion of the water phase and the organic phase of each sample finally fed into the machine, reduce the solvent effect, and after comprehensive consideration, unify the proportion of the water phase and the organic phase of the final fed into the machine in a mode of constant volume after transferring without adding a water removing agent when rotary steaming to near dryness.
Pork, pork liver, pork heart, pork lung and pork kidney are different in caking and wall attachment degree in the extraction process, modes such as shaking by a large number of tests, vortex mixing, rotary shaking, ultrasonic extraction and the like are compared, the extraction mode of vortex mixing and ultrasonic combination is found to be capable of fully extracting target compounds, the samples are easy to agglomerate and wall attachment after centrifugation for the first extraction, the samples cannot be effectively dispersed by vortex and ultrasonic after the extractant is directly added, so that the extraction efficiency is affected, the samples can be dispersed by adopting a simple mechanical shaking mode before the reagent is added, and the conditions of the pork lung, the pork liver and the pork kidney sample wall attachment can be effectively improved by an up-down shaking mode after the reagent is added. Comprehensively considering the extraction effect and the working efficiency, selecting an extraction mode of vortex for 1min and ultrasonic for 10 min. In addition, the proper temperature can effectively accelerate the protein deterioration and improve the purification efficiency, but the temperature is too high to easily cause the decomposition of target compounds, and the ultrasonic temperature is selected to be 35-55 ℃ after a large number of experiments.
4. Selection of concentration mode
The two sample concentration modes of nitrogen blowing and rotary evaporation are examined, and rotary evaporation is preferred because of more extraction solvent. In the test process, the pressure and time required by rotary evaporation are greatly different due to different substrates of pork, pork liver, pork heart, pork lung and pork kidney samples, more manpower is required to be adjusted at any time according to different substrates, and in consideration of the fact that the samples are easy to be subjected to the bumping when being evaporated to near dryness, 5mL of isopropanol is added by rotary evaporation to 4-5 mL, so that the problems are improved, and the samples are reduced in bumping and are easy to be evaporated to near dryness.
5. Selection of the purification conditions
The purification effect of the adsorption purifying agents such as C 18, PSA, neutral alumina, GBC, diatomite, NH 2 and the like on pork, pork liver, pork heart, pork lung, pork kidney and other different matrix samples is examined, the adsorption rate of GBC on each target compound is 70-95%, C18 has a certain adsorption effect on atropine and scopolamine, diatomite has a certain adsorption effect on atropine, PSA and NH 2、 neutral alumina has a small adsorption effect on the target compound, and obvious purification effect on the samples, but if the use amount is large, the adsorption effect on the target compound is a certain degree, after the experiment, 10mgPSA, 10mgNH 2 and 20mg neutral alumina are preferable for purifying, and 1mL normal hexane is added for freezing and degreasing.
6. Selection of detection conditions
(1) Conditions for liquid phase separation
A plurality of mobile phase proportions such as methanol and acetonitrile are examined, an aqueous phase is water, 0.1% formic acid, 5mmo of ammonium acetate (0.1% formic acid water) solution, 5mmo of ammonium formate (0.1% formic acid water) solution, 0.01% ammonia water-0.1% ammonia water and the like, a large number of experiments show that the separation effect of each target compound is better than acetonitrile when the organic phase is methanol, and when the aqueous phase is water, 0.1% formic acid and 5mmo of ammonium acetate (0.1% formic acid water) solution, the scopolamine signal response is low and only has response when the concentration is higher. When the aqueous phase is ammonia water, scopolamine has good response, but tropine has low response and unstable mobile phase, wherein the ammonia water is relatively volatile, so that a test system is unstable, the peak time and the peak of a target compound are easy to change, and the high-throughput analysis is not facilitated. The response value of 7 target compounds of 5mmol ammonium formate (0.1% formic acid water) is relatively good, and the peak type is good, and the influence of interference is avoided. Through adjusting gradient elution condition test, anisodamine and its dextroisomer are separated, all target compounds peak within 10min, and the rapid and efficient test requirement is satisfied.
(2) Mass spectrometry conditions
Through a large number of experiments, various mass spectrum parameters are tuned, the response degree of each fragment ion is comprehensively compared, and the mass spectrum ion source conditions, the fragmentation voltage, the collision voltage, the qualitative and quantitative ions are determined as shown in table 3.
7. Selection of internal standard
The synchronous response degree of the back martropne and the SKF-525A to the target compounds is examined, and compared, the back martropne has better synchronous response to 7 compounds in different matrixes, and can be selected as a reference internal standard, the SKF-525A has lower polarity, the peak-out time is longer, the synchronous response of the tolperisoic acid and the noratropine is correspondingly better, and other target compounds have certain deviation, so that the back martropne is not selected as an internal standard.
8. Verification of methods
(1) Evaluation of matrix Effect
Since matrices often have a significant disturbance to the analysis process of target analytes and affect the accuracy of the analysis results, these effects and disturbances are referred to as matrix effects. And (3) adopting an extraction and addition method (Matuszewski and the like), and examining the matrix effect of the target compound after being added into blank matrixes of pork, pork liver, pork heart, pork lung and pork kidney by calculating the ionic response intensity of the target compound in the solvent and the target compound in the matrix extracting solution, wherein the calculation method of the matrix effect is shown in a formula (1).
Matrix effect
Wherein: a is the response value of the target in the animal edible tissue matrix; b is the response value of the target in the reagent. If A/B > 1, this indicates that the response of the matrix to the analyte produces an enhancing effect; if A/B < 1, it indicates that the response of the matrix to the analyte produces an inhibitory effect; if a/b=1, this indicates that no matrix effect is present. When A/B is between 0.8 and 1.2, the interference degree of the matrix is low; when 0.5 < A/B < 0.8 or 1.2 < A/B < 1.5, a moderate matrix interference effect is exhibited; when A/B < 0.5 or A/B > 1.5, the interference indicating the matrix effect is strong.
Taking 50ng/mL to-be-measured concentration as an example, the matrix effect evaluation result shows that the pig liver, pig lung and pig kidney matrixes have stronger matrix enhancement effect on 7 target compounds, and measures are needed to be taken in the detection process to reduce the influence of the matrix effect. The method adopts the method of improving the purification efficiency and using an internal standard method and a method of preparing a matrix standard solution to improve the matrix effect.
(2) Evaluation of relative matrix Effect
And drawing a solvent standard solution working curve and different matrix solution standard adding working curves by using an internal standard method respectively, and observing the relative matrix effect between the solvent internal standard method and the matrix internal standard method according to a formula 2.
Where Sm and Ss represent the slopes of the matrix matching standard curve and the solvent standard curve, respectively. Negative results indicate matrix inhibition effects and positive results indicate matrix enhancement effects. When ME is-20%, the effect is weak matrix; the ME is medium matrix effect when being-50% to-20% or 20% -50%; a strong matrix effect is obtained when ME is < -50% or > 50%.
After calculation, the internal standard method is adopted, the matrix effect of various matrixes on the target compound is improved to different degrees, and the medium-weak or medium-strong enhancement effect is mainly adopted, wherein the quality effects of tolypic acid, noratropine, atropine and camphor Liu Jianji are obvious, and the detection result is still influenced, so that a standard working curve of matrix solution is necessary to be used in the detection process. In consideration of the actual test, the simultaneous drawing of standard working curves of various matrix solutions can significantly increase the test workload, which is unfavorable for the actual operation, so that the use of a representative matrix for drawing the curves is considered. Through investigation, when the pig heart is used as a matrix to draw a working curve, the relative matrix effect of 7 target compounds of various other matrixes is basically kept at a weak matrix effect level, and the method is simple and easy to operate while considering the matrix effect, so that the actual needs can be met.
(3) Linear range, detection limit and quantification limit
To the blank substrate, 50. Mu.L, 100. Mu.L, 0.5mL and 1mL of a mixed standard solution of 10ng/mL were added, 50. Mu.L, 100. Mu.L and 200. Mu.L of a mixed standard solution of 1000ng/mL were added, and the mixture was treated according to the above steps of sample extraction and purification to prepare 7 standard solutions (0.5, 1, 5, 10, 50, 100 and 200 ng/mL) of different mass concentrations, and an internal standard method was used to draw a working curve. Standard and standard samples were measured, the standard deviation of the measurement results of 7 target compounds was calculated by measuring 7 times the sample added at the lowest acceptable concentration of 0.1 μg/kg, the limit of detection was calculated as lod=0+3s, and the limit of quantification was calculated as loq=0+10s. The linear regression equation and the linear correlation are shown in tables 5 and 6, and the detection limit and the quantitative limit of the method are shown in Table 7.
Table 5 7 matrix linear regression equations for the target compounds
TABLE 67 linear correlation coefficients for 5 substrates of target compounds
| Target compound | Heart shape | Liver | Lung (lung) | Kidney and kidney | Meat product |
| Noratropine | 0.99989301 | 0.99989301 | 0.99989301 | 0.9991217 | 0.99937346 |
| Anisodine | 0.99924751 | 0.99924751 | 0.99924751 | 0.99949468 | 0.9992622 |
| Anisodamine | 0.99980799 | 0.99980799 | 0.99980799 | 0.99960388 | 0.99933294 |
| Rabdosia anisodamine | 0.99957601 | 0.99931270 | 0.99938299 | 0.99951746 | 0.99926444 |
| Tropine acid | 0.99952551 | 0.99952551 | 0.99952551 | 0.99978111 | 0.9997966 |
| Scopolamine | 0.99963698 | 0.99963698 | 0.99963698 | 0.99947054 | 0.99935892 |
| Atropine | 0.99966066 | 0.99966066 | 0.99966066 | 0.99948379 | 0.99948448 |
Table 7 7 target compounds 5 substrates detection limit and quantitative limit
(4) Method precision and recovery
Respectively testing 3 parts of positive samples with concentration level of 10.0 mug/kg in different animal edible tissues such as pork, pork liver, pig kidney, pig heart, pig lung, sheep leg meat, chicken breast meat, beef, fish meat and the like, wherein the test concentration of the positive samples on the machine is 50ng/mL. The recovery rate of the noratropine in different tissues is 81.6-107%, and the corresponding average relative standard deviation is 2.11-9.51%; the recovery rate of the anisodine in different tissues is 80.2 percent to 119 percent, and the corresponding average relative standard deviation is 1.39 percent to 12.7 percent; the recovery rate of anisodamine in different tissues is 80.2% -119%, and the corresponding average relative standard deviation is 1.57-14.5%; the recovery rate of the racanisodamine in different tissues is 87.8 percent to 115 percent, and the corresponding average relative standard deviation is 1.50 percent to 6.95 percent; the recovery rate of the tropinic acid in different tissues is 80.8-118%, and the corresponding average relative standard deviation is 1.13-9.01%; the recovery rate of scopolamine in different tissues is 90.0-119%, and the corresponding average relative standard deviation is 1.17-11.3%; the recovery rate of atropine in different tissues is 82.6-111%, and the corresponding average relative standard deviation is 2.95-7.08%, and the detailed results are shown in tables 8-9.
TABLE 87 recovery of target Compounds in different matrix samples% recovery
Table 97 precision test results RSD% of target Compounds in different matrix samples
| Target compound | Heart shape | Liver | Lung (lung) | Kidney and kidney | Meat product | Sheep leg meat | Beef | Chicken chest meat | Fish meat |
| Noratropine | 2.92 | 4.71 | 2.98 | 6.34 | 2.69 | 2.11 | 9.51 | 2.62 | 2.95 |
| Anisodine | 2.94 | 3.66 | 3.25 | 3.28 | 5.30 | 1.39 | 5.12 | 12.7 | 5.70 |
| Anisodamine | 1.57 | 4.25 | 2.84 | 4.87 | 4.48 | 14.5 | 14.1 | 7.46 | 3.22 |
| Rabdosia anisodamine | 3.18 | 1.71 | 2.76 | 1.50 | 6.13 | 4.86 | 3.32 | 6.95 | 3.08 |
| Tropine acid | 6.19 | 6.12 | 5.65 | 2.61 | 7.16 | 2.19 | 5.35 | 9.01 | 1.13 |
| Scopolamine | 4.16 | 1.17 | 2.56 | 4.68 | 4.07 | 3.64 | 11.3 | 11.3 | 2.36 |
| Atropine | 2.95 | 4.78 | 3.80 | 4.49 | 5.69 | 4.83 | 4.92 | 4.93 | 7.08 |
(5) Stability of
Pork samples with the same standard concentration level (10.0 mug/kg) are respectively measured by different operators at intervals of 0 day, 10 day and 30 day, the average measurement result RSD of 3 times is calculated, the repeatability of 8 target compounds in 5 different matrixes is 3.92% -11.3%, the repeatability is less than 15%, and the results show that the method is reliable and stable and can be reproduced, and the detailed table 10 is shown.
Table 10 results of pork sample recovery and precision test
While the foregoing has been disclosed in the specification and drawings, it will be apparent to those skilled in the art that various substitutions and modifications may be made without departing from the spirit of the invention, and it is intended that the scope of the invention be limited not by the specific embodiments disclosed, but by the appended claims.
Claims (6)
1. A method for detecting belladonna alkaloid in animal tissues is characterized by comprising the following steps:
Preparing a sample, cleaning the unfrozen animal tissue to be tested, cutting, mashing, subpackaging in a sample container, and preserving for later use, wherein the animal tissue to be tested comprises muscle of an edible pig and viscera of the pig;
Extracting a sample, namely weighing 5g of a sample, accurately obtaining 0.01g of the sample, adding 50 mu L of internal standard working solution into a 50mL centrifuge tube, mixing for 30s by vortex, standing for 10min, adding 15mL of acidified acetonitrile, mixing for 1min by vortex, performing ultrasonic extraction for 10min at 35-55 ℃, taking out, cooling to room temperature, centrifuging for 5min at 4500r/min, and transferring the supernatant into a 100mL chicken heart bottle;
Extracting the solids in the centrifuge tube after removing the supernatant twice to obtain a supernatant of the second time, combining the supernatants of the second time, and performing rotary evaporation to near dryness at 45 ℃ to obtain residues;
Sample purification, namely adding 50 mu L of methanol and water into the residues, dissolving the residues, transferring the residues into a 2mL centrifuge tube, fixing the volume to 1.0mL by using water, adding an adsorbent, carrying out vortex oscillation for 30S, adding 1mL of normal hexane, carrying out vortex oscillation for 10S, carrying out 12000r/min for 5min, removing an upper grease layer, repeatedly purifying once, freezing at the temperature of minus 20 ℃ for 30min, carrying out 12000r/min for 5min, and filtering a lower clear liquid for liquid use, wherein the adsorbent comprises 10mgPSA,10mg NH2 and 20mg neutral alumina;
sample detection, namely detecting the filtered supernatant by a machine, wherein detection conditions comprise:
Chromatographic column: infinityLabPoroshell120 HPH-C18 with an inner diameter of 2.1mm, a column length of 100 mm and a filler particle size of 1.9 mu m; flow rate: 0.2 mL/min; mobile phase: phase A is 5mM ammonium formate+0.1% formic acid; the phase B is methanol, and the gradient elution is carried out; column temperature: 40. degree C, sample injection amount: 10. 2, L; quantitative determination by an internal standard method;
Mass spectrum ion source, ESI; the temperature of the ion source is 100 ℃; the dry air flow is 10L/h; capillary voltage 4000V; the temperature of the air flow is 350 ℃; atomization gas pressure 40psi; the collision gas type is nitrogen; positive ions; the detection mode is MRM;
the belladonna alkaloid is anisodamine, racanisodamine, anisodamine, atropine, hyoscyamine, scopolamine, secomatropine, tropinic acid and noratropine.
2. The method according to claim 1, wherein the gradient elution condition is a volume fraction at a flow rate of 0.2ml/min; at 0-1 min, 95% of mobile phase A, 5% of mobile phase B, at 2-4 min, 90% of mobile phase A, 10% of mobile phase B, at 5min, 50% of mobile phase A, 50% of mobile phase B, at 10min, 20% of mobile phase A, 80% of mobile phase B, at 10-15 min, 95% of mobile phase A and 5% of mobile phase B.
3. The method of claim 1, wherein the secondary extraction of the supernatant-removed solids in the centrifuge tube comprises: shaking the solid, adding 15mL of acidified acetonitrile, shaking up and down by hand for 30s, mixing by vortex, and adding ultrasound to obtain a supernatant of the second time.
4. The method of claim 1, wherein the filtering of the supernatant comprises: and filtering the supernatant through a 0.22 mu m filter membrane.
5. The method of claim 1, wherein the viscera of the pig comprise: heart, liver, kidney and lung.
6. The method of detecting according to claim 1, further comprising:
Standard stock solution: respectively weighing proper amounts of anisodamine, racanisodamine, anisodamine hydrobromide, atropine sulfate, hyoscyamine, scopolamine, after hydrobromic acid, tropinic acid and noratropine, wherein the content of anisodamine, racanisodamine, anisodamine, atropine, hyoscyamine, scopolamine, after calculation, the content of the anisodamine, tropinic acid and noratropine is 10mg, and the anisodamine is fixed to a 10 mL brown volumetric flask by methanol to prepare a standard stock solution with the mass concentration of 1000 mug/mL;
Transferring anisodamine, racanisodamine, anisodamine, atropine, hyoscyamine, scopolamine, back-martrope, tropinic acid and noratropine standard solution, and fixing volume in 10 mL brown volumetric flask with methanol to obtain mixed intermediate solution with mass concentration of 1000 ng/mL;
10 ng/mL of mixed standard solution 20 [ mu ] L, 50 [ mu ] L, 100 [ mu ] L, 0.5 mL and 2mL are respectively added into a blank matrix, 1000ng/mL of mixed standard solution 50 [ mu ] L, 100 [ mu ] L and 200 [ mu ] L are added, the mixture is treated according to the same extraction and purification steps as the sample, 8 mass concentration standard solutions of 0.2 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL are prepared, and a post-horse riding product is used as an internal standard, and a working curve is drawn by an internal standard method.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1595059A (en) * | 1976-11-10 | 1981-08-05 | Saint Firmin A R | Tropane alkaloids |
| CN108896694A (en) * | 2018-07-05 | 2018-11-27 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of remaining LC-QToF-MS Screening analysis method of pesticide in animal food |
| CN109633048A (en) * | 2019-02-27 | 2019-04-16 | 梧州市食品药品检验所 | The rapid detection method of belladonna alkaloids in a kind of animal body |
| CN109828071A (en) * | 2019-02-20 | 2019-05-31 | 宁波检验检疫科学技术研究院 | Method that is a kind of while detecting 9 kinds of water filling class medicament residues in pork |
| CN110849990A (en) * | 2019-11-13 | 2020-02-28 | 中国肉类食品综合研究中心 | Method for detecting veterinary drug residues in animal-derived food and application thereof |
| CN111875600A (en) * | 2020-08-10 | 2020-11-03 | 中国科学院西北高原生物研究所 | Preparation method of tropane alkaloids in anisodamine |
| CN111961044A (en) * | 2020-08-10 | 2020-11-20 | 中国科学院西北高原生物研究所 | Preparation method of tropane alkaloids in hippophae rhamnoides |
-
2021
- 2021-12-27 CN CN202111614723.7A patent/CN114414679B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1595059A (en) * | 1976-11-10 | 1981-08-05 | Saint Firmin A R | Tropane alkaloids |
| CN108896694A (en) * | 2018-07-05 | 2018-11-27 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of remaining LC-QToF-MS Screening analysis method of pesticide in animal food |
| CN109828071A (en) * | 2019-02-20 | 2019-05-31 | 宁波检验检疫科学技术研究院 | Method that is a kind of while detecting 9 kinds of water filling class medicament residues in pork |
| CN109633048A (en) * | 2019-02-27 | 2019-04-16 | 梧州市食品药品检验所 | The rapid detection method of belladonna alkaloids in a kind of animal body |
| CN110849990A (en) * | 2019-11-13 | 2020-02-28 | 中国肉类食品综合研究中心 | Method for detecting veterinary drug residues in animal-derived food and application thereof |
| CN111875600A (en) * | 2020-08-10 | 2020-11-03 | 中国科学院西北高原生物研究所 | Preparation method of tropane alkaloids in anisodamine |
| CN111961044A (en) * | 2020-08-10 | 2020-11-20 | 中国科学院西北高原生物研究所 | Preparation method of tropane alkaloids in hippophae rhamnoides |
Non-Patent Citations (3)
| Title |
|---|
| Indirect competitive enzyme-linked immunosorbent assay based on a broadspectrum monoclonal antibody for tropane alkaloids detection in pig urine, pork and cereal flours;Zile Wang等;《Food Chemistry》;第337卷;第1-8页 * |
| LC －MS /MS 法测定氢溴酸东莨菪碱在大鼠体内的组织分布;夏天等;《药物分析杂志》;第32卷(第1期);第26-29页 * |
| 夏天等.LC －MS /MS 法测定氢溴酸东莨菪碱在大鼠体内的组织分布.《药物分析杂志》.2012,第32卷(第1期),第26-29页. * |
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