A series of strategies for solving the shortage of reference standards for multi-components determination of traditional Chinese medicine, Mahoniae Caulis as a case.

In order to solve the bottleneck of reference standards shortage for comprehensive quality control of traditional Chinese medicines (TCMs), a series of strategies, including one single reference standard to determine multi-compounds (SSDMC), quantitative analysis by standardized reference extract (QASRE), and quantitative nuclear magnetic resonance spectroscopy (qNMR) were proposed, and Mahoniae Caulis was selected as an example to develop and validate these methods for simultaneous determination of four alkaloids, columbamine, jatrorrhizine, palmatine, and berberine. Comprehensive comparisons among these methods and with the conventional external standard method (ESM) were carried out. The relative expanded uncertainty of measurement was firstly used to compare their credibility. The results showed that all these three new developed methods can accurately accomplish the quantification by using only one purified reference standard, but each of them has its own advantages and disadvantages as well as the specific application scope, which were also discussed in detail in this paper.

Application of quantitative 1H NMR for the calibration of protoberberine alkaloid reference standards.

Quantitative nuclear magnetic resonance spectroscopy (qNMR) has been developed into an important tool in the drug analysis, biomacromolecule detection, and metabolism study. Compared with mass balance method, qNMR method bears some advantages in the calibration of reference standard (RS): it determines the absolute amount of a sample; other chemical compound and its certified reference material (CRM) can be used as internal standard (IS) to obtain the purity of the sample. Protoberberine alkaloids have many biological activities and have been used as reference standards for the control of many herbal drugs. In present study, the qNMR methods were developed for the calibration of berberine hydrochloride, palmatine hydrochloride, tetrahydropalmatine, and phellodendrine hydrochloride with potassium hydrogen phthalate as IS. Method validation was carried out according to the guidelines for the method validation of Chinese Pharmacopoeia. The results of qNMR were compared with those of mass balance method and the differences between the results of two methods were acceptable based on the analysis of estimated measurement uncertainties. Therefore, qNMR is an effective and reliable analysis method for the calibration of RS and can be used as a good complementarity to the mass balance method.

Development of a rapid resolution liquid chromatographic method for simultaneous analysis of four alkaloids in Rhizoma coptidis under different cultivation conditions.

A sensitive and specific method using rapid resolution liquid chromatography coupled with UV-Vis detection was developed for fingerprint analysis of Rhizoma coptidis and simultaneous determination of 4 alkaloids: jatrorrhizine, coptisine, palmatine, and berberine. Samples of R. coptidis grown under different cultivation conditions and from different habitats were analyzed. The analysis was performed using a reversed-phase octylsilyl (C8) column and gradient elution. The mobile phase consisted of acetonitrile and 20 mmol/L KH2PO4. Each analysis was completed within 3.5 min. The method showed good linearity within test ranges of 4.75-47.50 microg/mL for jatrorrhizine, 20.60-164.80 microg/mL for coptisine, 18.07-180.73 microg/mL for palmatine, and 89.70-717.57 microg/mL for berberine. The method showed good precision, repeatability, and stability for quantification of the 4 alkaloids. The lower limit of detection was 0.19 ng for jatrorrhizine, 0.21 ng for coptisine, 0.15 ng for palmatine, and 0.14 ng for berberine. The lower limit of quantification was 0.57 ng for jatrorrhizine, 0.82 ng for coptisine, 0.55 ng for palmatine, and 0.27 ng for berberine. The overall recovery ranged from 96.30 to 104.10% for the 4 alkaloids. The method is accurate, rapid, and convenient, and it is suitable for routine quality control of R. coptidis.

Simultaneous determinations of paeonol and palmatine hydrochloride in Shangshi Aerosols by HPLC method.

A simple and rapid HPLC method was described for the simultaneous determination of paeonol and palmatine hydrochloride in Shangshi Aerosols. The optimum separation for these analytes was achieved using the mixture of 0.025 M sodium dihydrogen phosphate-acetonitrile-diethylamine (64:35:1, v/v/v) as the mobile phase and a Nova-Pak((R)) C8 column. The linear ranges of paeonol and palmatine hydrochloride were 0.2-80 and 0.06-60 microg/ml with the regression equations being Y=11716.4+2.96 x 10(6)X (R=0.99969), Y=-6388.8+1.89 x 10(5)X (R=0.99976), and limit of quantifications (LOQ) for paeonol and palmatine hydrochloride were 0.2 and 0.06 microg/ml, respectively (n=6). Other validation parameters: intra-day precision (R.S.D.: 0.71-1.65%) and inter-day precision (R.S.D.: 0.89-2.11%), and reproducibility (recoveries values: 94.6-98.2% for paeonol, 94.85-97.58% for palmatine hydrochloride) were found to be satisfactory. The proposed HPLC method had been applied for the determination of paeonol and palmatine hydrochloride in Shangshi Aerosols; R.S.D. values were 1.45 and 1.13%, respectively. In short, this method was rapid and convenient, which could be used for the routine control of paeonol and palmatine hydrochloride in Shangshi Aerosols.