CN103940935A - Concentration determining method of arecoline hydrobromide in animal blood plasma - Google Patents
Concentration determining method of arecoline hydrobromide in animal blood plasma Download PDFInfo
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- CN103940935A CN103940935A CN201310023215.0A CN201310023215A CN103940935A CN 103940935 A CN103940935 A CN 103940935A CN 201310023215 A CN201310023215 A CN 201310023215A CN 103940935 A CN103940935 A CN 103940935A
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Abstract
The invention relates to a concentration determining method of arecoline hydrobromide in animal blood plasma. The method applies high performance liquid chromatography-tandem mass spectrum technology for concentration determination of the arecoline hydrobromide in the animal blood plasma. Beta-pinene is adopted as an internal standard substance, and the concentration of the arecoline hydrobromide in the animal blood plasma can be accurately determined. The high performance liquid chromatography-tandem mass spectrum process which is rapid, accurate and sensitive is adopted for the concentration determination of the arecoline hydrobromide in the animal blood plasma. The method is high in specificity and good in separation effect. Linearity, stability, reproducibility, and recovery tests of the method satisfy good technical requirements. The method lays methodology foundations for research of pharmacokinetics and bioequivalence of the medicine inside animals.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, relate to the method for Arecoline hydrobromide content in a kind of quantitative measurement animal blood plasma.
Background technology
[0002] arecaline is one of active component in Chinese medicine betel nut, is mainly used in treating tapeworm, ox glandular stomach is slow on veterinary clinic, and the livestock and poultry disease such as equus dilatation of the stomach, Large intestinal obstruction, enteroparalysis.In addition, utilize the water-snail eradication synergy of arecaline, can significantly reduce the dosage of chemical molluscacide, reduced therewith pollution on the environment.Have obvious spinoff because arecaline is applied to animal, the use on important economic animal and pet is restricted.Its hydrobromate of arecaline has the good effect of promotion gastrointestinal motility and anthelmintic action, Arecoline hydrobromide has expeling effect to multiple parasite, can replace at present conventional clinically traditional product, especially can reach 100% to Taenia elliptica anthelminthic effect, clinical effectiveness is remarkable, have wide popularizing application prospect, belong to efficient, safety, pollution less, residual agriculture input little, inexpensive, easy to use, be conducive to solve the problem of preventing and treating of parasitic disease in current livestock breeding industry.Due to Arecoline hydrobromide ultraviolet maximum absorption wavelength 215nm, belonging to end absorbs, uv absorption is very weak, and high performance cation exchange chromatography method and the sensitivity of high performance liquid chromatography-uv detection method are not high, and the current report without Arecoline hydrobromide detection method in animal blood plasma.In order to solve Arecoline hydrobromide assay problem in animal blood plasma, need be in analysis and detection technology seeking breakthrough, to improve sensitivity and the accuracy of detection.
HPLC-MS/MS(liquid chromatography-tandem mass spectrometry) coupling technique is current more novel technology, because of its efficient separating power and mass spectrographic accurate qualitative ability that combines liquid chromatography, becomes in recent years the most reliable and practical analysis means.This analytical technology needs that sample amount is few, detectability is low, is particularly useful for analyte or the structural research of metabolin and the variation of amount that medicine produces under the effect of the factors such as organism metabolism.
Summary of the invention
In view of above-mentioned, the object of the present invention is to provide a kind of assay method that detects Arecoline hydrobromide content in animal blood plasma.The method is applied to high performance liquid chromatography-tandem mass coupling technique the assay of Arecoline hydrobromide in animal blood plasma, taking nopinene as interior mark, and the content of Arecoline hydrobromide in energy Accurate Determining animal blood plasma.
Object of the present invention is achieved through the following technical solutions:
In animal blood plasma, an assay method for Arecoline hydrobromide content, the steps include:
A, get on an empty stomach animal vein blank plasma, add Arecoline hydrobromide standard solution, inner mark solution, vortex mixed, adds the AgNO of 0.1M
3, vortex 30-40s, adds NaCl, vortex 2-3min, adds sherwood oil, vortex 2-3min, centrifugal 30-40min, separate organic layer and be placed in another centrifuge tube, in 40 DEG C of water-baths, nitrogen dries up, and adds ultrapure water dissolved residue, obtain standard plasma solutions, cross 0.45 μ m or 0.22 μ m miillpore filter, obtain standard plasma solutions, to be measured;
B, get the animal blood plasma after oral administration, add inner mark solution, vortex mixed, adds the AgNO of 0.1M
3, vortex 30-40s, adds NaCl, vortex 2-2.5min, add sherwood oil, vortex 2-2.5min, centrifugal 30-40min, separate organic layer and be placed in another centrifuge tube, in 40-50 DEG C of water-bath, nitrogen dries up, and adds ultrapure water dissolved residue, crosses 0.45 μ m or 0.22 μ m miillpore filter, obtain need testing solution, to be measured;
Standard plasma solutions and the need testing solution that a, b step obtain measured respectively in c, high performance liquid chromatography-tandem mass coupling, the condition of measuring is that the ammonium formate aqueous solution ratio that mobile phase acetonitrile and pH are 4 is 8: 92 by the fixing phase chromatographic column of carbon octadecyl silane; Flow velocity 0.4mLmin
-1; Electron spray ESI source; Spray voltage Is is 4kv; Atomization temperature: 350 DEG C; Atomization gas flow: l1 L/min; Collision airshed: 10 Lmin
-1; Capillary outlet terminal voltage: 50v; Collision energy: 20v; Deta EMV+:180v; Detection mode is positive ion multiple-reaction monitoring (MRM), for the arecaline mother/daughter ion of quantitative test to being m/z 156.2-53.0;
D. the standard plasma solutions and the need testing solution that a, b step are obtained, injecting respectively high performance liquid chromatography-tandem mass instrument detects, obtain total ion current figure and mass spectrogram, measure peak area, with arecaline and interior target peak area ratio (Y), arecaline concentration (X) in blood plasma is carried out to linear regression, obtain linear relationship and regression equation, calculate testing concentration.
Above-mentioned Arecoline hydrobromide standard solution, inner mark solution are respectively precision to take Arecoline hydrobromide standard items appropriate, and water dissolves and constant volume obtains Arecoline hydrobromide standard solution; It is appropriate that precision takes nopinene, obtains inner mark solution with anhydrous alcohol solution constant volume.
Animal described above can be the beast of prey and the birds etc. such as dog.
Advantage of the present invention:
1, sample treatment of the present invention is simple and easy to operate, and the method recovery is in 95% left and right, and RSD (%) is less than 3%, meets analyzing and testing requirement.
2, adopt the present invention HPLC-MS/MS technology to be applied to the detection of Arecoline hydrobromide in animal blood plasma, tested component detection sensitivity improves, low (1 ngmL of detectability
-1), Artesunate, qinghaosu and dihydroartemisinine peak degree of separation are good, analysis time is short, can complete the detection of determinand in 3 minutes, need sample amount few (only need 1 μ l), solvent load is few, and cost is low, and assay result is accurate, repeatability, good stability.
3, Arecoline hydrobromide is the Effective Anti parasite veterinary drug of effectively preventing and treating infecting both domestic animals and human taeniasis and animal fluke disease, use method of the present invention, content that can be convenient and swift, detect accurately arecaline in animal blood plasma, for Arecoline hydrobromide pharmacokinetics and bioequivalence Journal of Sex Research in animal body established methodology basis.
Brief description of the drawings
Fig. 1 is blank plasma sample total ion current figure (TIC figure).
Fig. 2 is arecaline and internal standard compound nopinene total ion current figure (TIC figure).
Fig. 3 is arecaline one-level mass spectrogram.
Fig. 4 is arecaline second order ms figure.
Embodiment
The present invention is further illustrated to enumerate embodiment below, and this embodiment does not only limit the present invention for the present invention is described.
Embodiment:
(principal ingredient is Arecoline hydrobromide to the Arecoline hydrobromide tablet that the present invention uses, and auxiliary material is pregelatinized starch, sodium carboxymethyl starch, dolomol.Grown place: Lanzhou) be the novel anti-animal taeniasis veterinary drug of Lanzhou herding and veterinary drug research institute independent development, on veterinary clinic, be mainly used in treating Taenia elliptica disease, anthelminthic effect can arrive 100%, evident in efficacy, and nopinene is internal standard compound matter.
1 materials and methods
1.1 instrument
Aglient 1200-6410 type fast liquid chromatography-triple quadrupole bar GC-MS, (Aglient company of the U.S.), electric spray ion source (ESI); Hydro-extractor; AEL-1600 type electronic balance (Japanese Shimadzu); ZH-II type automatic whirlpool mixer, (Tianjin Pharmacopoeia Standard Instrument Factory).
1.2 medicines and reagent
Arecoline hydrobromide standard items (Nat'l Pharmaceutical & Biological Products Control Institute); Arecoline hydrobromide tablet ((Lanzhou herding and veterinary drug research institute independent development); Beta pinene (Japanese TCI company buy); Methyl alcohol is chromatographically pure, and acetic acid is pure for analyzing, and water is ultrapure water.
1.3 chromatographic condition
Chromatographic column is Aglient C18 post (2.1 × 30mm, 3.5um); Mobile phase is acetonitrile: water (ammonium formate is adjusted pH to 4)=8:92; Flow velocity 0.4mLmin
-1; Column temperature room temperature; Sampling volume is 1uL.
1.4 mass spectrum conditions
Electron spray ESI source; Spray voltage Is is 4kv; Atomization temperature: 350 DEG C; Atomization gas flow: l1 L/min; Collision airshed: 10 Lmin
-1; Capillary outlet terminal voltage: 50v; Collision energy: 20v; Deta EMV+:180v; Detection mode is positive ion multiple-reaction monitoring (MRM), for the arecaline mother/daughter ion of quantitative test to being m/z 156.2-53.0.
The preparation of 1.5 standard solutions and interior mark liquid
Precision takes Arecoline hydrobromide standard items 0.1mg, and water dissolves and constant volume obtains 1.0 μ gmL
-1arecoline hydrobromide solution; Precision takes nopinene 50 μ L, obtains 10 μ lmL with anhydrous alcohol solution constant volume
-1inner mark solution.
1.6 blood specimen collections and processing
1.6.1 animals administer
Get 4 of healthy beasle dogs, at administration prosodetic food 12h, weigh respectively, numbering.Before administration, first feed to the tincture of iodine 5 ~ 10mL of dilution and prevent vomiting, through 10 ~ 20min, by 3mg/kg body weight dosage, the oral Arecoline hydrobromide tablet that gives respectively.
1.6.1 the collection of blank plasma
Use before administration disposable EDTA vacuum blood collection venae vasorum to gather blood, blood sampling volume is 5 ml, centrifugal 10 min, separated plasma, draws supernatant, obtains blank plasma, be put in centrifuge tube be kept at-20 DEG C to be analyzed.
1.6.2 the collection of plasma sample
After administration, 40min uses disposable EDTA vacuum blood collection venae vasorum to gather blood, and blood sampling volume is 5 ml, centrifugal 10 min, separated plasma, draws supernatant, obtains plasma sample, be put in another centrifuge tube be kept at-20 DEG C to be analyzed.
The processing of 1.7 standard plasma solutions
Get " 1.6.1 " blank plasma 1ml in 10mL centrifuge tube, add the each 10 μ L of standard solution and inner mark solution, after fully mixing, add the AgNO of 0.1M
3100 μ L, vortex 30s, adds 0.4g NaCl, vortex 2min, add sherwood oil 3mL, vortex 2min, centrifugal 30min, separates organic layer and is placed in another 5mL centrifuge tube, in 40 DEG C of water-baths, nitrogen dries up, and adds 200 μ L ultrapure water dissolved residues, obtains standard plasma solutions, for subsequent use.
The processing of 1.8 need testing solutions
Get " 1.6.2 " plasma sample 1ml in 10mL centrifuge tube, add inner mark solution 10 μ L, after fully mixing, add the AgNO of 0.1M
3100 μ L, vortex 30s, adds 0.4g NaCl, vortex 2min, add sherwood oil 3mL, vortex 2min, centrifugal 30min, separates organic layer and is placed in another 5mL centrifuge tube, in 40 DEG C of water-baths, nitrogen dries up, and adds 200 μ L ultrapure water dissolved residues, obtains need testing solution, for subsequent use.
1.9 measure
Draw respectively the each 5 μ l of standard plasma solutions and need testing solution, with 0.22 μ m filtering with microporous membrane, laggard HPLC-MS/MS analyzes.
2 results
2.1 method specificities
Get dog blank plasma 1 mL, except not adding interior mark, other operate by " 1.7 ", and sample introduction 5 μ L, obtain total ion current figure, see Fig. 1; Certain density standard solution and inner mark solution are added in blank plasma, according to operating under " 1.7 " item, as can be seen from the figure: the endogenous material in blank plasma is not disturbed the mensuration of arecaline and nopinene.Total ion current figure and mass spectrogram are shown in Fig. 2-4.Fig. 2 is arecaline and internal standard compound nopinene total ion current figure (TIC figure).As can be seen from the figure two compound peak shapes are good, and peak degree of separation is good, analysis time the short 3min that only needs; Fig. 3 is arecaline one-level mass spectrogram.The arecaline parent ion obtaining is [M+H]
+;
Fig. 4 is arecaline second order ms figure, is m/z 53.0 for quantitative daughter ion, auxiliary quantitative daughter ion m/z 81.0
2.2 linear relationships are investigated
Get dog blank plasma 1mL, totally 6 parts, configure respectively 5.0,20,80,320ngmL
-1, 1.3 μ gmL
-1, 5.2 μ gmL
-1standard plasma solutions, process by under step " 1.7 " operation, by " 1.3,1.4 " lower chromatogram mass spectrum condition sample introduction, measure peak area.With arecaline and interior target peak area ratio (Y), arecaline concentration (X) in blood plasma is carried out to linear regression, obtaining regression equation is y=0.0075x+0.0584, R
2=0.9994, the range of linearity is 5-200 ngmL
-1.
The accurate plasma solutions of label taking, is configured to respectively concentration and is respectively 10,5,2.5,2,1 ngmL
-1solution, by " 1.3,1.4 " lower chromatogram mass spectrum condition sample introduction, measure lowest detectable limit (S/N=10) and the minimum quantitative limit (S/N=3) of arecaline.Record to detect and be limited to 1 ngmL
-1, be quantitatively limited to 2ngmL
-1.
Detectability and quantitative limit are measured and are obtained by standard plasma solutions, and assay method is first bioassay standard plasma solutions, obtains typical curve and regression equation; Then measure need testing solution, the peak area substitution regression equation calculation obtaining is obtained to ultimate density.
2.3 precision and accuracy
Prepare the standard plasma solutions of high, medium and low three kinds of concentration, add respectively the inner mark solution of 10 μ l, compound method is with " 1.7 ", and plasma sample obtains concentration and is respectively 5,40,100 ngmL after processing
-1standard plasma sample solution, by " 1.3,1.4 " lower chromatogram mass spectrum condition sample introduction, three kinds of concentration samples continuous sample introduction 5 times in 1 day, continuous 5 days sample introductions, carry out concentration determination, calculate a day within variance coefficient, the coefficient of variation in the daytime.Result shows, under this test condition the method accurately, reliable, favorable reproducibility.The results are shown in Table 1.
The precision that table 1 Arecoline hydrobromide detects in plasma sample
2.4 the recovery
Get 1ml plasma sample (n=6), to add high, medium and low three kinds of concentration be 5,40,100 ngmL to precision respectively
-1the each 1ml of standard solution, add the inner mark solution of 10 μ l, by " processing of 1.7 plasma samples " operation, by " 1.3,1.4 " lower chromatogram mass spectrum condition sample introduction, calculate respectively the recovery of basic, normal, high 3 kinds of concentration.The average recovery rate of the basic, normal, high 3 kinds of concentration of result arecaline is (92.41 ± 3.35) % respectively, (95.26 ± 3.24) %,
(97.64 ± 5.12) %, average RSD is 2.24%.Result shows, the satisfied mensuration of the recovery of the method requirement under this test condition.
2.5 assay
Get the Arecoline hydrobromide tablet of 20110305,20110306,20,110,307 3 different batches, respectively dog is passed through to oral administration, after medication, 40min gets blood separated plasma, by " processing of 1.8 need testing solutions " operation, by " 1.3, a 1.4 " lower chromatogram mass spectrum condition sample introduction, measure the content of arecaline in dog plasma.Assay result RSD (%) is all less than 3%, and there was no significant difference between 4 dogs, show the inventive method can Accurate Determining dog plasma in the content of arecaline, and stability is better, method is reliable.The results are shown in Table 2 and Fig. 2.
Arecaline assay result in table 2 dog plasma
Claims (3)
1. an assay method for Arecoline hydrobromide content in animal blood plasma, the steps include:
A, get on an empty stomach vein blank plasma of animal, add Arecoline hydrobromide standard solution, inner mark solution, vortex mixed, adds the AgNO of 0.1M
3, vortex 30-40s, adds NaCl, vortex 2-3min, adds sherwood oil, vortex 2-3min, centrifugal 30-40min, separate organic layer and be placed in another centrifuge tube, in 40 DEG C of water-baths, nitrogen dries up, and adds ultrapure water dissolved residue, obtain standard plasma solutions, cross 0.45 μ m or 0.22 μ m miillpore filter, obtain standard plasma solutions, to be measured;
B, get the oral blood plasma giving after Arecoline hydrobromide of animal, add inner mark solution, vortex mixed, adds the AgNO of 0.1M
3, vortex 30-40s, adds NaCl, vortex 2-2.5min, add sherwood oil, vortex 2-2.5min, centrifugal 30-40min, separate organic layer and be placed in another centrifuge tube, in 40-50 DEG C of water-bath, nitrogen dries up, and adds ultrapure water dissolved residue, crosses 0.45 μ m or 0.22 μ m miillpore filter, obtain need testing solution, to be measured;
Standard plasma solutions and the need testing solution that a, b step obtain measured respectively in c, high performance liquid chromatography-tandem mass coupling, the condition of measuring is that the ammonium formate aqueous solution ratio that mobile phase acetonitrile and pH are 4 is 8: 92 by the fixing phase chromatographic column of carbon octadecyl silane; Flow velocity 0.4mLmin
-1; Electron spray ESI source; Spray voltage Is is 4kv; Atomization temperature: 350 DEG C; Atomization gas flow: l1 L/min; Collision airshed: 10 Lmin
-1; Capillary outlet terminal voltage: 50v; Collision energy: 20v; Deta EMV+:180v; Detection mode is positive ion multiple-reaction monitoring (MRM), for the arecaline mother/daughter ion of quantitative test to being m/z 156.2-53.0;
D. the standard plasma solutions and the need testing solution that a, b step are obtained, injecting respectively high performance liquid chromatography-tandem mass instrument detects, obtain total ion current figure and mass spectrogram, measure peak area, with arecaline and interior target peak area ratio (Y), arecaline concentration (X) in blood plasma is carried out to linear regression, obtain linear relationship and regression equation, calculate testing concentration.
2. the assay method of Arecoline hydrobromide content in a kind of animal blood plasma according to claim 1, it is characterized in that, described Arecoline hydrobromide standard solution, inner mark solution are respectively precision to take Arecoline hydrobromide standard items appropriate, and water dissolves and constant volume obtains Arecoline hydrobromide standard solution; It is appropriate that precision takes nopinene, obtains inner mark solution with anhydrous alcohol solution constant volume.
3. the assay method of Arecoline hydrobromide content in a kind of animal blood plasma according to claim 1, is characterized in that, described animal can be dog, ox, pig.
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Cited By (3)
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| CN105738551A (en) * | 2016-04-22 | 2016-07-06 | 云南中烟工业有限责任公司 | Detection method of arecoline |
| CN107290468A (en) * | 2017-06-06 | 2017-10-24 | 山东省药学科学院 | A kind of method that LC-MS quantitatively detects betel nut alkali content in children's indigestion tablet |
| CN108535382A (en) * | 2018-05-07 | 2018-09-14 | 中国热带农业科学院农产品加工研究所 | A kind of rapid detection method of arecaline |
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| WO2008011713A1 (en) * | 2006-07-26 | 2008-01-31 | Diamedica Inc. | Methods of diagnosis and treatment for metabolic disorders |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738551A (en) * | 2016-04-22 | 2016-07-06 | 云南中烟工业有限责任公司 | Detection method of arecoline |
| CN105738551B (en) * | 2016-04-22 | 2017-09-22 | 云南中烟工业有限责任公司 | A kind of detection method of arecaline |
| CN107290468A (en) * | 2017-06-06 | 2017-10-24 | 山东省药学科学院 | A kind of method that LC-MS quantitatively detects betel nut alkali content in children's indigestion tablet |
| CN108535382A (en) * | 2018-05-07 | 2018-09-14 | 中国热带农业科学院农产品加工研究所 | A kind of rapid detection method of arecaline |
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Application publication date: 20140723 |