Introduction

Aconite and aconitine in Aconitum and Aconitum carmichaelii, as well as strychnine and brucine in Semen Strychni, are highly toxic alkaloids that can cause severe poisoning or even death in trace amounts. In the 2020 edition of the Chinese Pharmacopoeia, there are still nearly 60 traditional Chinese patent medicines and simple preparations and varieties of Chinese medicinal materials that use aconitine and strychnine as reference substances. As highly toxic substances under strict control, the Ministry of Public Security requires that no corresponding reference substances be issued [1]. To ensure the scientificity, rationality, and applicability of pharmacopoeia standards, it is urgent to replace the reference standards of aconitine and strychnine with reference extracts of aconitine diester alkaloids and total alkaloids of Fructus Strychni. As early as 2016, the China National Institute for Food and Drug Control began relevant research work [2] [3]; Recently, the National Pharmacopoeia Committee has completed the addition and revision of 57 related varieties, which have been included in the 2025 edition of the Chinese Pharmacopoeia.

Aconitum diester alkaloids

After processing (soaking, steaming, sanding, etc.), the C-8 acetyl group hydrolyzes to form aconite monoester alkaloids (such as benzoyl aconitine, benzoyl neoaconitine, benzoyl hypaconitine, etc., low toxicity), while the C-14 benzoyl group further hydrolyzes to form aconitine amine alkaloids (such as aconitine, neoaconitine, hypaconitine, etc., slightly toxic)[4]As shown in Figure 1.

The aconitine diester alkaloids contained in Chuanwu (processed from Chuanwu), Caowu (processed from Caowu), and Fuzi are mainly aconitine, hypaconitine, and neoaconitine, while the aconitine diester alkaloids contained in Huangcao Wu (processed from Huangcao Wu) are mainly aconitine and aconitine[5]At present, the content of control extract of aconitine diester alkaloids sold in the market is calculated as neoaconitine, hypaconitine and aconitine, and the determination of aconitine diester alkaloids in some traditional Chinese patent medicines and simple preparations varieties is seriously disturbed by the sample matrix. Thermo Fisher Laboratories has established a novel method for the determination of five types of aconite diester alkaloids and three types of aconite monoester alkaloids, in order to enable pharmaceutical companies to more comprehensively control the quality of related formulations.

Slide down to view all content

Figure 1: Eight structural formulas of aconitine

The complete application solution of Thermo Fisher Scientific for the feature map of raw materials

01

Preparation of reference solution and test solution

8 types of aconitine reference solutions were purchased from standard suppliers, and 2 batches of Jiangyou Shengfu tablets test solutions were provided by customers.

Preparation of reference solution:Take appropriate amounts of benzoyl aconitine reference substance, benzoyl aconitine reference substance, benzoyl aconitine reference substance, new aconitine reference substance, aconitine reference substance, aconitine reference substance, aconitine reference substance, Dianaconitine reference substance, and crude stem aconitine A (Caowusu) reference substance solution, weigh accurately, and add acetonitrile to prepare a mixed solution containing 10 μ g per 1 mL.

Preparation of test solution:Take about 1.0 g of the powder of this product, weigh it accurately, and place it in a 25 mL stoppered glass test tube. Accurately add 10 mL of isopropanol ethyl acetate (1:1) mixed solution (containing 0.6 mL of ammonia test solution), seal it tightly, extract it with ice water bath ultrasound for 30 minutes, and let it settle at room temperature. Transfer 5 mL of supernatant to a 15 mL centrifuge tube, centrifuge at 8000 rpm at 20 ℃ for 5 minutes, accurately take 4.00 mL and blow it to a clean nitrogen blowing tube in a 30 ℃ water bath until almost dry. Accurately add 2.00 mL of acetonitrile for reconstitution (vortex/ultrasound/vortex), centrifuge at 5000 rpm at 20 ℃ for 3 minutes, pass the reconstitution solution through a 0.2 μ m hydrophilic PTFE filter membrane (P/N: 42213-NPL), and test the filtrate on the machine.

Measurement method:Accurately aspirate 5 μ L of each of the reference solution and the test solution, inject them into the liquid chromatograph, and measure to obtain the results.

Slide down to view all content

02

Instrument configuration and chromatographic conditions

Thermo Scientific ™ Vanquish Core ™ High performance liquid chromatography system, configuration: pump Quaternary Pump C (VC-P20-A), automatic sampler Split Sampler CT (VC-A12-A), column temperature box Column Comparison C (VC-C10-A), detector Diode Array Detector CG (VC-D11-A), data processing Chromeleon ™ seven point three

色谱柱： Acclaim Mixed-Mode WCX-1, 3 μm, 3.0×150 mm (P/N: 070092)

Mobile phase: A: Ultra pure water B: 100 mM (NH)4)H2PO4(Adjust pH to 3.0 with phosphoric acid) C: Acetonitrile

Flow rate: 0.5 mL/min

Gradient elution program: see attached application solution for details

Injection volume: 5 µ L

Column temperature: 40 ℃

Detector: DAD, acquisition wavelength: 230/260 nm; acquisition frequency: 5 Hz; spectral acquisition: 190-400 nm

Slide down to view all content

03

Spectrum and data

3.1

Reference solution spectrum

Figure 2 Separation spectra and data of 8 mixed reference solutions of aconitine (10.0 μ g/mL) (click to view larger image)

Figure 3 Localization spectra of 8 types of aconitine single reference solution (10.0 μ g/mL) (click to view larger image)

Swipe to view more

Eight types of aconitine in the test solution can be effectively separated, among which five types of aconitine diester alkaloids have good peak shapes, excellent column efficiency, and can be accurately determined and quantified; Three types of aconitine monoester alkaloids, benzoyl aconitine and benzoyl aconitine, could not be separated at baseline and can be used for preliminary qualitative analysis.

3.2

Spectral diagram of the test sample solution

Figure 4 Separation spectrum and data of the test sample solution for raw tablets (S1)

(Click to view large image)

Figure 5 Separation spectrum and data of the test sample solution for raw tablets (S2)

(Click to view large image)

Swipe to view more

Aconitine, aconitine, and neoaconitine were all detected in two batches of secondary aconite tablets, and the target peaks were able to achieve baseline separation and quasi deterministic quantification, while Dianaconitine and Aconitum carmichaelii were not detected.

3.3

Characteristic map of Shengfu tablets

Figure 6 Spectral diagram of adjacent peaks of aconitine in the test solution of Shengfu tablets

(Click to view large image)

At 230 nm, two unknown peaks can be observed before and after the target peak of aconitine in the test solution of Shengfu tablets. Through spectral identification, they are suspected to be other types of aconitine diester alkaloids, and corresponding research can be carried out in the future.

Figure 7: Overlapping spectra of six reference solutions of aconitine and the test solution of Shengfu tablets (click to view larger image)

In the continuous injection superimposed spectrum of the test solution of Shengfu tablets, the retention time and peak area of each target peak are stable, with RSD less than 1%. The method has excellent reproducibility and stability, and can be used to establish the characteristic spectrum of aconite diester alkaloids in Shengfu tablets.

04

Results and Discussion

1)This experiment used the Acclaim Mixed Mode WCX-1 mixed mode chromatography column (reverse phase+weak cation exchange) and a mobile phase without ion pair reagents or tetrahydrofuran, combined with the Vanquish Core high-performance liquid chromatography system, to effectively separate 8 types of aconitines. Among them, 5 types of aconitine diester alkaloids had good peak shapes, excellent column efficiency, and can be accurately determined and quantified. The 3 types of aconitine monoester alkaloids can be used for preliminary qualitative analysis.

2)Optimization of chromatographic conditions was carried out for the separation of five aconite diester alkaloids in the test solution. Two unknown peaks were separated before and after the peak of aconitine, and suspected other aconite diester alkaloids were identified through spectral analysis. However, neither aconitine nor aconitine were detected.

3)This method is suitable for monitoring the hydrolysis degree of aconitine during the processing of raw aconite, and can also be used for tracing the origin of authentic medicinal materials such as Sichuan aconite and Yunnan aconite.

5. References

[1] Li Hao, Shen Mingrui, Zhang Peng, et al. [J]. Introduction to the main additions and revisions of the 2025 edition of the Chinese Pharmacopoeia [J]. Chinese Pharmaceutical Standards, 2025, 26 (01): 17-22. DOI: 10.19778/j.cp.2025.01.003

[2] Zhu Yue, Wang Ruizhong, Zhang Yumei, etc Preparation and Application Study of Dual ester Aconitum Alkaloids Reference Extract [J]. Chinese Pharmaceutical Standards, 2016,17 (04): 252-257. DOI: 10.19778/j.cp.2016.03.003

[3] Wang Feifei, Wu Shouhai, Zhang Yumei, etc Research on the Comparative Extract of Malvaceae and Its Application in Quality Control of Malvaceae Medicinal Materials and Compound Preparations [J]. Journal of Pharmaceutical Analysis, 2018, 38 (07): 1226-1230. DOI: 10.16155/j.0254-1793.2018.07.18

[4] Hu Jiahao, Li Wenru, Shi Qingxin, etc Research progress on chemical composition and processing technology of Aconitum carmichaelii [J]. Chinese Journal of Traditional Chinese Medicine, 2025, 50 (06): 1458-1470. DOI: 10.19540/j.cnki. cjcmm. 2024, 1214.201

[5] Xu Yi, Qin Bo, Cao Hongyun, etc Simultaneous determination of Dianwualkaloid and Caowujia in Huangcao Wu by HPLC method [J]. Yunnan Journal of Traditional Chinese Medicine, 2017, 38 (09): 70-72. DOI: 10.16254/j.cnki. 53-1120/r. 2017.09.033

Want to learn more about application details

Please scan the QR code to download

If you need to collaborate in reprinting this article, please leave a message at the end of the article.