Abstract: Thin layer chromatography (TLC,) high performance liquid chromatography-photoelectric two-tube array detector (HPLC / DAD), animal experiments and other methods were used to compare aconite alkaloids in biological samples, and these methods were compared. It was found that the TLC method is the preferred method for the screening of such drugs, and the minimum detection amount is 0.3 μg. When performing HPLC detection, the characteristic ultraviolet absorption spectrum of aconite alkaloids and animal experiment results are important qualitative means, and the characteristic absorption wavelengths are (228 ± 2) nm and 275 ± 2) nm. Aconitum alkaloids have a good linear shape of peak area and mass concentration at 2g / L ~ 50MG / l, and the country coefficient is 0.9996. The actual case proves that the method is accurate and sensitive, and can be used for the detection of aconitoid alkaloids in biological samples.
Key words: thin layer chromatography; high performance liquid chromatography 'Aconitum alkaloids; animal experiments
CLC number: O658 Document identification code: A Article number: 1000-8713 (2002) 01-03
Aconitum alkaloids are a common class of highly toxic poisons. It has many types, complex structure, and poor stability. Its effective ingredients and toxicity vary greatly with the origin, season, and processing method. In our country, cases of aconite alkaloid poisoning have occurred frequently, especially in cases of overdose and misuse during the processing of Chinese medicine. In the actual inspection of such cases, the inspection of poisonous plants and suspicious drugs is more, and the inspection of aconite alkaloids in biological samples is rarely reported. Inspection by high-performance liquid phase-photodiode array detector (HPLC / DAD) method and animal experiment method is even rarer. In several cases in which the death was suspected to be aconitine poisoning for unknown reasons, the authors tested the drugs and the stomach contents and liver of the deceased separately, and achieved good results.
1 Experimental part
1.1 Instruments and reagents
High-efficiency thin-layer plate (GF254 (Qingdao Ocean Chemical Plant); centrifuge, rotating speed is 4000r / min (Shanghai Surgical Instruments Factory); high-performance liquid chromatograph HP-1100 HPLC with DAD (US Hewlett-Packard Company).
A blank liver (taken from the deceased in the case of ethanol poisoning); the experimental mice were 15-20g mice, two weeks old.
Reagents: methanol and acetonitrile are chromatographically pure; chloroform, absolute ethanol, anhydrous sodium sulfate, glacial acetic acid, and hydrochloric acid are all analytically pure; deionized water. Aconitum alkaloid reference substance (purchased from China National Institute for the Control of Pharmaceutical and Biological Products).
Aconitum alkaloid standard solution (100mg / L): Accurately weigh 10mg of aconite alkaloid reference substance and dissolve it in 100ml of anhydrous methanol. Potassium bismuth iodide: 200mg bismuth subnitrate, 5g potassium iodide, 2g iodine, 1ml concentrated hydrochloric acid and 1ml glacial acetic acid, dissolved in water, diluted to 250ml, and stored in the refrigerator.
1.2 Inspection material extraction
Take 5ml of the stomach contents of the dead and centrifuge for 5min. The supernatant was taken to adjust the pH to 8-9, extracted with chloroform, dehydrated with anhydrous sodium sulfate, and dried in a KD concentrator at 60 ° C for inspection.
Take 10g of crushed dead liver, add 20ml of 2mol / L Hcl, hydrolyze at 80 ℃ for 2h, cool and centrifuge. Take the supernatant and adjust the pH to 8 ~ 9, extract with chloroform, dehydrate with anhydrous sodium sulfate, evaporate at 80 ℃, and prepare for inspection. The extraction of blank liver is performed in parallel with the same amount as above.
Take 5g of the suspicious drug, soak it in 15ml of absolute ethanol for 24h, centrifuge, take the supernatant and dry it for inspection.
1.3 Inspection
1.3.1 Thin layer chromatography (TLC) inspection
Adsorbent: silica gel G; developing agent: A is cyclohexane-diethylamine (volume ratio 9: 1) solution, B is cyclohexane-ethyl acetate (volume ratio 1: 1) solution, C is benzene -Dioxane-diethylamine (volume ratio is 7: 2: 1) solution; developer: potassium bismuth iodide;
The above test substance extracts were dissolved in 200 μl of methanol, respectively, and subjected to TLC analysis. After unfolding, the developing agent was dried, placed in bismuth potassium iodide for color development for 3 seconds, and taken out for observation.
1.3.2 HPLC analysis
Column: 1 Phenosphere CN 5μm (250mmx4.6mm ID); 2 Pypersil 5μm C18 BDB (250mmx4.6mm ID). Mobile phase A: 0.001mol / L KH2PO4 + 0.001mol / L sodium dodecyl sulfonate; B: 5% acetonitrile; C: methanol. Mobile phase gradient 0min ~ 10min, V (A): V (B): V (C) = 65: 5: 30; 10min ~ 20min, V (A): V (B): V (C) = 40: 14 : 46; 20min ~ 25min. V (A): V (C) = 15: 85; 25min, V (A): V (B): V (C) = 44: 10: 46.
The above test material extracts were respectively dissolved in 200 μL of methanol, and 20 μL were taken for HPLC distraction.
1.3.3 Standard working curve
Prepare aconite standard solution 2mg / L, 5mg / L, 10mg / L, 30mg / L, 50mg / L, 80mg / L, 100mg / L for HPLC analysis, and compare the peak area Y to the mass concentration X (mg / L) As a standard working curve, the linear equation Y = -0.2805908 + 14.11653XM, r = 0.9996 linear range 2mg / L ~ 50mg / L.
1.3.4 Animal experiment
Preparation of poison bait (divided into 3 groups according to different extraction methods):
The first group took 5g of suspicious drug, soaked it in 10ml of water for 24, waved it to 3, and set aside.
The second group took 5g of the drug, placed it in 10ml of 1% acetate red, heated and soaked it for 24h, and waved it to 3mL for use.
The third group took 5g of the suspicious drug, placed it in 15ml of absolute ethanol, heated and soaked it for 24h, evaporated it, then dissolved it with 5ml of 1% hydrochloric acid, added alkali to neutrality, and concentrated to 3ml for use.
Nine white mice were taken and divided into three groups on average. After 12 hours of fasting, the rats in each group were injected with 1ml / suspect of the suspicious drug extract of the corresponding group, and the rats in groups 1 and 3 died of poisoning.
(1) In several cases, we first used TLC to test aconitum alkaloids. The results show that the TLC method is simple and easy to perform, with high sensitivity and a minimum detection amount of 0.3μg, which can be used as the preferred method for aconite alkaloid test screening. If the color development result is negative, it can be used as a basis for denying the existence of such drugs.
(2) Due to the wide variety of aconite alkaloids, complex structure, and poor chemical stability, the results obtained by different extraction processing methods for the same test materials are quite different. This is very obvious in TLC and HPLC inspection. Not only is the Rf value and tR value of the test material difficult to match the Rf value and tR value of the standard aconitum alkaloids, even the standard aconitum alkaloids have different Rf and tR values ​​due to different origins and different storage times. Obviously, aconite alkaloids cannot be qualitatively based on Rf and tR values. At the same time, we also found that although the tR value of aconitoid alkaloids in HPLC test is complex and difficult to control and master, the absorption wavelength of its specific structural group in UV detection is relatively stable, the peak shape characteristic is obvious, and the characteristic absorption wavelength is 228 ± 2) nm, (275 ± 2) nm, and does not change with its tR change. The control test of normal liver, which is equivalent to the liver corruption of the test material, confirmed that the endogenous substances in the liver tissue did not interfere with the test, so it can be used as an important basis for the identification of aconite alkaloids. In our experiments, aconitoid alkaloids were detected from the liver and stomach contents of the poisoned dead, with good separation, stable absorption wavelength, and obvious ultraviolet absorption spectrum characteristics. In particular, the detection of aconitine in the liver is of great significance.
(3) According to the literature, the most toxic of aconitine alkaloids are aconitine, medium aconitine, hypoaconitine and other diester alkaloids. This kind of medicine has strong ester affinity and high toxicity, and people can die by oral administration of 4mg. However, during the processing and processing of drugs, these drugs are easily hydrolyzed, losing benzoyl groups, and generating acetyl aconitine, which has a significantly reduced toxicity; further hydrolysis, generating acetamides, the toxicity is further reduced. This view has been reflected in animal experiments. White mice were immersed and fed with suspicious drug color dilute acetic acid, but no death was observed; while the mice were fed with suspicious water and ethanol extract of the same weight, all the tested mice died. It can be seen that the drug extraction method is different, that is, the degree of hydrolysis is different, its toxicity shows a significant difference, this feature is significantly different from other drugs.
(4) According to the literature, the toxic effects of aconite alkaloids mainly act on the heart and nervous system of humans and animals, so that their heart rate is frequent, and the peripheral nerves are excited first and then suppressed, or even paralyzed. Our animal experiments reproduced this phenomenon intuitively. The poisoned rats began to show excitement and restlessness; they immediately developed numbness, difficulty walking, weak limbs, and difficulty supporting the body; finally, the limbs were paralyzed and died prone. The symptoms of this poisoning symptom are obvious and intuitive, and there are obvious differences from other poisoning symptoms. The results of this animal experiment can be used as an important basis and side evidence for the testing of aconite alkaloids.
(5) Due to the wide variety of aconitum alkaloids, the toxic components have poor thermal stability and do not vaporize, so it is difficult to analyze by conventional GC and GC / MS methods, and qualitativeness is difficult under lack of detection conditions such as LC / MS. Since we have not yet clarified the changes in the effective components of alkaloids in the body, no specific quantitative analysis was carried out in the test. Knowledge used two chromatographic columns (C18 column and CN column) combined with DAD spectrogram for qualitative analysis .
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