CN1430059A - Analytical method for concentration of xipulin and huperzine A in blood - Google Patents
Analytical method for concentration of xipulin and huperzine A in blood Download PDFInfo
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- CN1430059A CN1430059A CN 01145584 CN01145584A CN1430059A CN 1430059 A CN1430059 A CN 1430059A CN 01145584 CN01145584 CN 01145584 CN 01145584 A CN01145584 A CN 01145584A CN 1430059 A CN1430059 A CN 1430059A
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Abstract
A method for measuring the concentrations of Xipulin and its metabolin Transcutaneous huperzine A in blood at same time features the double bond of Xipulin molecular is reduced by sodium tetrahydroborate to prevent it from being converted to Tan scutaneous huperzine A, and an LC/MS/MS system is used for analysis.
Description
Technical field
The present invention relates to biological substance blood concentration analytical approach and more particularly be Xi Pulin and huperzine (Huperzine A) blood concentration analytical approach.
Background technology
Degenerative brain disorder (A1zheimer ' s Disease, AD) be the damaged disease of class intelligence that betides in the elderly population, mainly show as carrying out property cognitive decrease clinically, and with behavior disorder and mood impermanence etc., until can't take care of oneself, bring serious burden with society to patient and family.Along with increasing sharply of the world aged, patient's AD is increasing, and AD has become the 3rd disease that threatens the elderly's life after cardiovascular disease, neoplastic disease.
In the world the research of disease of old people is attached great importance at present, the U.S. drops into half that accounts for the pharmaceuticals industry research and development to this research.Seeking effective chemoprophylaxis and treatment AD has become medicine scholar and clinical medicine worker's urgent task.
Huperzine is the acetylcholinesteraseinhibitors inhibitors of the new high selectivity of China's discovery, because its unique pharmacological property and definite curative effect, and cause people's extensive attention.At present, be widely used in clinical as the new drug for the treatment of AD at home.Sell on the internet as food additives or nutrient and healthcare products at U.S.'s huperzine, be used to improve memory function.Given this, the countries in the world scientist has carried out a large amount of research to the structure structure-activity relationship and the structure of modification of huperzine, synthetic and screened thousands of huperzine analogs, it is better to attempt to search out the huperzine that curative effect compares, the compound that toxicity is lower, but the research of this respect does not have breakthrough so far.Utilize the resources advantage of China's huperzine, we have carried out structure optimization and structural modification to the structure of huperzine, from semisynthetic up to a hundred compounds, found noval chemical compound Xi Pulin, its acetylcholine esterase inhibition activity and huperzine are close, but the inhibiting effect to the periphery butyrylcholine esterase far is weaker than huperzine, therefore it much larger than huperzine, toxicity is 1/12～1/11 of huperzine only to the selectivity of acetylcholinesterase, therapeutic index is apparently higher than huperzine.E2020 (domepezil hydrochloride, trade name Acricept) is the common acetylcholinesteraseinhibitors inhibitors of developing of new generation of Pfizer Inc. and Japanese Wei Cai company, and in 1997 in states such as American and Britain listings, it is reported that its curative effect and toxicity obviously are better than Tacrine.For this reason, we improve aspect such as memory disorders and toxic and side effect and huperzine, Tacrine and E2020 and contrast in the anti-AChE effect of test, assessment Xi Pulin.External anti-AChE effect comparative experiments result shows that Xi Pulin is the strongest to the selectivity of AChE, is better than huperzine, E2020 and Tacrine; Under the rat oral administration condition, Xi Pulin and huperzine all have the obvious suppression effect to cortex and hippocampus AChE, with grammol dosage relatively, the anti-AChE effect of Xi Pulin is stronger more than 8 times than E2020, and the bioavilability of Xi Pulin reaches and is better than E2020 and Tacrine by the blood-brain barrier ability; Experiment confirms that also effect is weaker than huperzine, Tacrine and E2020 to Xi Pulin to the periphery cholinergic system.
Be used to measure the analytical approach of Xi Pulin and metabolin huperzine blood concentration thereof in the past, used the PHLC technology, analyzed the plasma sample after liquid-liquid extracts.The problem that this analytical approach exists is difficult to measure exactly the concentration of Xi Pulin in the blood when being blood sampling, and sensitivity for analysis is low.Its reason is that Xi Pulin is extremely unstable in water under the normal temperature, easily changes into huperzine.In the method, this conversion mainly occurs in and prepares in the blood plasma process under (1) normal temperature; (2) in the plasma sample pre-treatment process; In the detachment process in (3) 30 ℃ the HPLC pillar.Simultaneously, the sensitivity of UV detecting device is lower.
Need carry out relevant animal and human pharmacokinetics research work for the features relevant that can be directly acquainted with Xi Pulin absorption in vivo, distribution, metabolism and drainage, set up the analytical approach that to measure Xi Pulin and metabolin huperzine blood concentration thereof simultaneously.
Summary of the invention
The present invention seeks to seek and a kind ofly can overcome Xi Pulin easily changes into 5-Cl-O-Vanillin and huperzine (reaction equation as follows) in blood or water method, set up the analytical approach that to measure Xi Pulin in the blood and metabolic product huperzine blood concentration thereof simultaneously.
Xi Pulin huperzine 5-Cl-O-Vanillin the present invention adopts:
The two hydrogen Xi Pulin of Xi Pulin
The two keys of Xi Pulin are by NaHB
4Obtain two hydrogen Xi Pulin after the reduction, its molecular structure stabilized, hydrogenation is incomplete, therefore records the concentration that two hydrogen Xi Pulin concentration are equal to the former Xi Pulin of sample in sample.
Owing to after Xi Pulin is water-soluble or in blood, easily change into huperzine, the present invention adopts two keys of the molecule of tetrahydro boron sodium reduction Xi Pulin, is converted into huperzine to prevent it in aqueous solution or in blood.
The present invention adopts poly-enforcement of following step: for avoiding Xi Pulin to be dissolved in behind the water or change into huperzine in blood, should adopt each animal used as test to prepare the Xi Pulin soup respectively, add the Xi Pulin soup of pvp with 4 ℃ of fresh preparations of physiological saline, and within one minute, pass through to irritate stomach or intravenously administrable.During blood sampling, with animal blood (about 250～300 μ l), directly be collected in the test tube that fills tetrahydro boron sodium and heparin, jolting rapidly makes Xi Pulin reduction in the blood.Carry out the blood concentration analysis with whole blood.If the tetrahydro boron sodium solid directly is added in the blood, can produce too much hydrogen because of its consumption is excessive influences quantitative test work with a large amount of foams.The present invention adopts tetrahydro boron sodium to be dissolved in the absolute ethyl alcohol, makes saturated solution, saturated solution is added in the test tube again, volatilizes alcohol solvent and makes the test tube of tetrahydro boron sodium and be used for blood sample collection.Its characteristics are: 1) add tetrahydro boron sodium in right amount in every part of blood sample.2) adopt this method that the surface area of tetrahydro boron sodium is increased greatly, thereby the hydrogenation that makes Xi Pulin more rapidly and one complete.
Isopropyl alcohol=1: 0.05～0.2) or methylene chloride, the extraction of chloroform equal solvent the Xi Pulin of reduced form and huperzine adopt the mixed solvent (ethyl acetate: of ethyl acetate and isopropyl alcohol in the blood after above-mentioned processing, extract evaporates into dried, and residue injects the LC/MS/MS system with moving phase dissolving back and analyzes.Moving phase comprises solvent orange 2 A and B.Wherein solvent orange 2 A is 2%-5% methanol aqueous solution (including 0.05% ammonium formate), and solvent B is 80%-90% methanol aqueous solution (including 0.05% ammonium formate), to carry out gradient elution.Mass detector in the LC/MS/MS system is the ion trap type.Comprise QC standard items, typical curve in the sample analysis catalogue, standard items and unknown blood sample.During the production standard curve, Xi Pulin storing solution (20 μ g/ml) is diluted to the solution of variable concentrations with absolute ethyl alcohol, respectively getting 10 μ l adds in the tetrahydro boron sodium pipe, fling to solvent, add the blank blood of 200 μ l fresh rat, the mixing reaction is after adding internal standard compound Huperzine B (huperzine B) and the damping fluid (PH 10), with the ethyl acetate extraction that contains 5% isopropyl alcohol, make the Xi Pulin typical curve.
The standard curve making and the Xi Pulin of huperzine are basic identical, but use common test tube without the tetrahydro boron sodium test tube.In sample pretreatment process, need careful operation, be brought in the extract to reduce organic impurities.
Advantage of the present invention:
1. the method for a kind of quantitative test Xi Pulin simultaneously and metabolin huperzine blood concentration thereof is provided, and it is more accurate to make Xi Pulin and huperzine pharmacokinetics The Characteristics in animal body.
2. the present invention adopts tetrahydro boron sodium earlier two keys of Xi Pulin to be reduced, to prevent continuing to change into huperzine at Xi Pulin after the blood sampling in blood.Therefore the present invention can accurately measure Xi Pulin and metabolin huperzine thereof in the blood.
Description of drawings
After Fig. 1 was dosage Xi Pulin of male rat 1 oral administration gavage (0.5mg/kg body weight), male SD rat body Nei Xipulin and metabolin huperzine blood concentration thereof be change curve in time.Among the figure
Expression Xi Pulin;-O-is a huperzine.
After Fig. 2 was dosage Xi Pulin of male rat 2 oral administration gavages (0.5mg/kg body weight), SD male rat body Nei Xipulin and metabolin huperzine blood concentration thereof be change curve in time.Among the figure
Expression Xi Pulin;-O-is a huperzine.
After Fig. 3 is dosage Xi Pulin of oral administration gavage (0.5mg/kg body weight), 1 hour (on) and 2 hours (descending) sampled point " male rat 2 " whole bloods in Xi Pulin and metabolin huperzine LC/MS/MS thereof analyze.
Embodiment
The making of tetrahydro boron sodium pipe: tetrahydro boron sodium is dissolved in the absolute ethyl alcohol, makes saturated solution, again this saturated solution (200 μ l) is added in the test tube, volatilize alcohol solvent and make " tetrahydro boron sodium pipe " and be used for blood sample collection.
Zoopery: male SD rat (about 300g) carries out Xi Pulin administration experiment after a laundering period in week.In PVP-Xi Pulin, the content of Xi Pulin is 2%.With 4 ℃ of physiological saline is the Xi Pulin soup of every fresh preparation of rat with the PVP hydrotropy, and squeezes in the animal body by irritating stomach (i.g.) or intravenous injection (i.v.) within 1min.Before administration and after the administration 5,15,30min, 1,2,4,6,8,12,20h time blood sampling (250～300 μ L).
Sample pre-treatments: blood sampling back blood sample is contained rapidly in the tetrahydro boron sodium pipe that contains heparin and is reacted, and Xi Pulin is wherein changed into the Xi Pulin of reduction.Reacted blood sample (200 μ L whole blood) adds 10 μ L internal standard compounds earlier, and (Huperzine B is 100ng/mL) with 100 μ L buffer solution (0.1M NaOH+0.05 Na
2HPO
4, pH12), again with the ethyl acetate extraction that contains 5% isopropyl alcohol.After extract (1350mL) volatilized, residue dissolved with moving phase, and injected the LC/MS/MS system and analyze.
LC/MS/MS analyzes: chromatographic resolution is carried out on one 0.5 μ mODS reversed-phase column (10 * 3i.d.mm, 30 ℃), and moving phase comprises: solvent orange 2 A and B, and to carry out gradient elution.Solvent orange 2 A is that 2% methyl alcohol (containing 0.05% ammonium formate), solvent B are 90% methyl alcohol (containing 0.05% ammonium formate).Mass detector in the LC/MS/MS system is the ion trap type.Respectively with Xi Pulin and huperzine to the ionogenic work of ESI with reference to being optimized, to improve sensitivity for analysis.Comprise QC standard items, typical curve standard items and unknown blood sample in the analytic sample catalogue.
The making of typical curve: Xi Pulin storing solution (25 μ g/mL) is diluted to the solution of variable concentrations with absolute ethyl alcohol, respectively gets 10 μ L and add in the tetrahydro boron sodium pipe and fling to solvent rapidly.Add the blank blood of the fresh rat of 200 μ L, the mixing reaction.(Huperzine B is 100ng/mL) and behind the 100 μ L buffer solution (pH 12), with the ethyl acetate extraction that contains 5% isopropyl alcohol adding 10 μ L internal standard compounds.Under the same condition, carry out LC/MS/MS and analyze, to make the Xi Pulin typical curve.The making of huperzine typical curve and Xi Pulin's is basic identical, just without the tetrahydro boron sodium pipe, and uses common tube.
Claims (7)
1. the energy method of quantitative measurement Xi Pulin and metabolic product huperzine blood concentration thereof simultaneously, the step that it is characterized in that this method is as follows: be the Xi Pulin soup of every fresh preparation of animal used as test with the pvp hydrotropy with 4 ℃ of physiological saline 1), and passed through to irritate stomach or intravenous administration in one minute; 2) before administration and after the administration 5,15,30min, 1,2,4,6,8,12,24h blood sampling.Animal blood 250～300 μ l directly are collected in the test tube that fills tetrahydro boron sodium and heparin for preparing in advance, jolting rapidly, the two keys in the reduced blood in uncommon general standing forest; 3) through the Xi Pulin whole blood blood sample 200 μ l of reduction, add the damping fluid of 10 μ l internal standard compound Huperzine Bs and 100 μ l, pH12 earlier, volatilize with organic solvent extraction and with extract again; 4) above-mentioned waving carried out the LC/MS/MS analysis after the thyraden residue dissolves with moving phase.
2. it is characterized in that according to the described method of measuring Xi Pulin and metabolic product huperzine blood concentration thereof simultaneously of claim 1 that the blood sample after the administration is handled with tetrahydro boron sodium earlier before mensuration, with reduction Xi Pulin wherein.
3. according to the described method of measuring Xi Pulin and metabolic product huperzine blood concentration thereof simultaneously of claim 1, it is characterized in that tetrahydro boron sodium is dissolved in the absolute ethyl alcohol, make saturated solution, this saturated solution 20 μ l are moved in the test tube again, the volatilization alcohol solvent gets the test tube of tetrahydro boron sodium.
4. according to the described method of measuring Xi Pulin and metabolic product huperzine blood concentration thereof simultaneously of claim 1, it is characterized in that Huperzine B is as the quantitative test internal standard compound.
5. according to the described method of measuring Xi Pulin and metabolic product huperzine blood concentration thereof simultaneously of claim 1, it is characterized in that in the blood sample that Xi Pulin is reduced, adding the internal standard compound Huperzine B and add damping fluid 0.1MNaOH+0.05NaHPO
4, the pH value is 12.
6. according to the described method of measuring Xi Pulin and metabolic product huperzine blood concentration thereof simultaneously of claim 1, it is characterized in that with the ethyl acetate extraction Xi Pulin, huperzine and the internal standard compound Huperzine B that include 5% isopropyl alcohol.
7. according to the method for claim 1 described while quantitative measurement Xi Pulin and metabolic product huperzine blood concentration thereof, it is characterized in that when carrying out the LC/MS/MS analysis, carrying out the binary gradient elution with the 2%--5% aqueous methanol that includes 0.05% ammonium formate and the two kinds of solvents of 80%--90% aqueous methanol that include 0.05% ammonium formate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102097026B (en) * | 2009-12-14 | 2013-04-10 | 中国科学院上海药物研究所 | Device and method for simulating pharmacokinetics characteristics in vitro |
| CN108333018A (en) * | 2018-02-12 | 2018-07-27 | 李恩有 | In a kind of art in whole blood Etomidate and fentanyl drug extracting method |
-
2001
- 2001-12-29 CN CN 01145584 patent/CN1430059A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102097026B (en) * | 2009-12-14 | 2013-04-10 | 中国科学院上海药物研究所 | Device and method for simulating pharmacokinetics characteristics in vitro |
| CN108333018A (en) * | 2018-02-12 | 2018-07-27 | 李恩有 | In a kind of art in whole blood Etomidate and fentanyl drug extracting method |
| CN108333018B (en) * | 2018-02-12 | 2020-07-10 | 李恩有 | Method for extracting etomidate and fentanyl from whole blood during operation |
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