Rapid discrimination and quantification of alkaloids in Corydalis Tuber by near-infrared spectroscopy

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Abstract

With the application of near-infrared spectroscopy (NIRS), a convenient and rapid method for determination of alkaloids in Corydalis Tuber extract and classification for samples from different locations have been developed. Five different samples were collected according to their geographical origin, 2-Der with smoothing point of 17 was applied as the spectral pre-treatment, and the 1st to scaling range algorithm was adjusted to be optimal approach, classification model was constructed over the wavelength range of 4582–4270 cm−1, 5562–4976 cm−1 and 7000–7467 cm−1 with a great recognition rate. For prediction model, partial least squares (PLS) algorithm was utilized referring to HPLC-UV reference method, the optimum models were obtained after adjustment. Pre-processing methods of calibration models were COE for protopine and min–max normalization for palmatine and MSC for tetrahydropalmatine, respectively. The root mean square errors of cross-validation (RMSECV) for protopine, palmatine, tetrahydropalmatine were 0.884, 1.83, 3.23 mg/g. The correlation coefficients (R2) were 99.75, 98.41 and 97.34%. T test was applied, in the model of tetrahydropalmatine; there is no significant difference between NIR prediction and HPLC reference method at 95% confidence interval with t = 0.746 < t(0.05,20) = 2.086, therefore NIRS is a reliable analytical tool in establishing prediction models.

Highlights

► To compare the quality and efficacy and facilitate discrimination of Corydalis Tuberi originating from different geographic position and growing environment. ► A reliable analytical technique was developed to determine its main alkaloids. ► Near-infrared reflectance spectroscopy has been found an excellent tool for this purpose.

Introduction

Corydalis Tuber (named Yanhusuo in Chinese) of the family Papaveraceae is a perennial herb that is widely distributed in the Zhejiang, Hubei, Sichuan, Jiangsu province and Northeast of China. It is a traditional Chinese medicine (TCM) that shows therapeutic effects of promoting blood circulation, reinforcing vital energy and alleviating pain, for instance headache/chest pain, epigastric pain, abdominal pain, backache, arthralgia or trauma [1], [3], cardiac arrhythmia, gastric and duodenal ulcer and menorrhalgia [4], [5]. Tertiary and quaternary alkaloids are the main chemical ingredients of C. Tuber [2]. Nearly 20 alkaloids of these two types have been isolated. An HPLC method using unmodified silica with reversed-phase eluents was developed for the determination of dl-tetrahydropalmatine in Corydalis Yanhusuo W.T. Wang and its set prescription preparations [6], and a high-performance liquid chromatography (HPLC) method with MS and UV-detection was developed for the qualitative and quantitative determination of alkaloids in C. yanhusuo [7]. The content of most alkaloids varies with the geographic position and growing environment etc. Consequently, to compare the quality and efficacy and facilitate discrimination of C. Tuber, a reliable analytical technique should be developed. NIR qualitative model establishment had been applied to C. Tuber from two different origins in a previous study [8], but no literature has been reported to determine the contents of alkaloids in C. Tuber and various species have not been identified with the application of near-infrared reflectance spectroscopy.

Near-infrared spectroscopy analysis is suitable for different state of samples and a wide range of analytical applications. It is a rapid, highly sensitive, nondestructive method with minimal or no sample preparation, suitable for on-line analysis. Conventional analysis is time consuming while near infrared spectral scanning just needs a few seconds. With these advantageous features, NIRS has been extensively used as a qualitative and quantitative analysis tool in the chemical [9] pharmaceutical [10] and food [11], [12] industries.

In this work, NIR spectroscopy and chemometric techniques were applied for quantitative determination of protopine, palmatine and tetrahydropalmatine in C. Tuber, using PLS regression for the calibration and validation. The precision and accuracy of the new NIR method and the standard HPLC method were compared. On the basis of the NIR qualitative model the source of five C. Tuber samples of different origin was also identified.

Section snippets

Samples and reagents

For the classification model, samples of raw C. Tuber were collected from five main growing localities in China (Zhejiang, Sichuan, Northeast, Anhui, Jiangsu), respectively. For the quantitative analysis model 70 samples were used while 30 were used as calibration set, 20 as validation set, and the rest formed the prediction set.

All samples were first milled into powder with a high-speed medicinal herb grinder (AK-1000A, 1000 W, Aoli Medicine Manufactory, China) and then passed through a

HPLC studies of C. Tuber

Fig. 1 shows a typical HPLC-UV chromatogram of C. Tuber extract solution. Since the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, PR China) can only supply three reference standards: protopine, palmatine, tetrahydropalmatine, we dealt with the quantitative analysis of these three alkaloids. Criteria for assessing the suitability of the system are described in the chapter on chromatographic separation techniques. The results are listed in Table 1. The

Conclusion

A NIR spectroscopic method was developed to analyze a complex TCM, C. Tuber, and a qualitative and quantitative model was successfully constructed. Five samples of C. Tuber from different locations were collected and distinguished showing that NIR method can be used as an efficient analytical tool for classification of similar samples. The prediction results for PLSR method with different pre-processing were investigated to produce optical calibration model, while true content of three

Acknowledgments

We are particularly grateful to Mei li, Xin Xie, Qiang Gao from Pfizer Inc. who were generous enough to provide support in the whole study. We are also indebted to the valuable and detailed comments from Bohai Jiang from Dalian University of Technology, and we acknowledge the supply of standards from Lei Wang.

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