Determination of Adulteration Dye Auramine O in Gegen Qinlian Tablets by HPLC-MS

*

1. Guangxi Institute for Food and Drug Control, Nanning 530021, China; 2. Guangxi Institute of Traditional Chinese Medicine & Pharmaceutical Science, Nanning 530021, China

Abstract [Objectives] This study aimed to establish a method for determination of adulteration dye auramine O in Gegen Qinlian tablets by HPLC-MS/MS. [Methods] The instrument conditions were as follows: column, Agilent Eclipse plus C18 (2.1 mm × 100 mm, 1.8 μm); mobile phase, acetonitrile-0.01 mol/L ammonium acetate; gradient elution; flow rate, 0.3 mL/min; column temperature, 35 ℃; injection volume, 2 μL; ion source, electrospray ionization source (ESI); and scanning mode, multiple reaction monitoring (MRM). [Results] Methodological validation shows that auramine O had a good linear relationship in the range of 0.093 4-1.869 pg (r=0.999 9), and the average recovery rate was 94.38% (RSD=3.60%, n=9). [Conclusions] The method established is accurate, convenient, precise, reproducible, and stable, with good linear relationship, and it can be used determine the content of adulteration dye auramine O in Gegen Qinlian tablets.

Key words Gegen Qinlian tablet, HPLC-MS/MS, Adulteration dye, Auramine O

1 Introduction

Gegen Qinlian tablets are composed of Radix Puerariae, Radix Scutellariae, Rhizoma Coptis Chinensis and Radix Glycyrrhizae Preparata, recorded in the 2015 edition of theChinesePharmacopoeia, with effects of resolving the flesh, clearing away heat, and relieving diarrhea. At present, illegal dyeing and adulteration of Chinese medicinal materials and decoction pieces of Chinese medicinal products is widespread, and reports on the inclusion of pigments such as auramine O in Radix Scutellariae and Rhizoma Coptis Chinensis have also emerged. Jiang Lingetal.[1]conducted qualitative and quantitative analysis on commercially available Rhizoma Coptis Chinensis samples, and chemical dye auramine O was detected in some of them. Radix Scutellariae and Rhizoma Coptis Chinensis are used as raw materials for Gegen Qinlian tablets, so the dyes in Radix Scutellariae and Rhizoma Coptis Chinensis may be brought into Gegen Qinlian tablets[2-3], and so, it is necessary to carry out research on the detection of auramine O. To further control the quality of this product, LC-MS/MS technology[4-5]was used in this study to detect the adulteration dye auramine O in Gegen Qinlian tablets. The method is simple, stable and reproducible. This study provides a sensitive and reliable inspection basis for the quality and safety control of Gegen Qinlian tablets.

2 Instruments and reagents

The used instruments mainly included Agilent 6490 liquid chromatography-mass spectrometry and MILLI-PROA pure water processor. The reference substance of auramine O (batch No.111769-200701; purity: 89.92%) was provided by China Food and Drug Inspection Institute. Acetonitrile was chromatographically pure. Water used was ultrapure water. Other reagents were of analytical grade. Methanol was chromatographically pure. Gegen Qinlian tablets were purchased from a local market.

3 Methods and results

3.1 Instrument conditions

3.1.1Chromatographic conditions. The chromatographic conditions were as follows: column, octadecyl-bonded silica gel column; column temperature, 35 ℃; flow rate, 0.3 mL/min; mobile phase A, 0.01 mol/L ammonium acetate solution; mobile phase B, acetonitrile; gradient elution (Table 1); and injection volume, 2 μL.

Table 1 Gradient elution

Time∥minMobile phase A∥%Mobile phase B∥%0-490→1010→904-810908-8.510→9090→108.5-139010

3.1.2Mass spectral conditions. The mass spectral conditions were as follows: ion source, ESI; detection mode, positive ion; dry gas temperature, 200 ℃; dry gas flow rate, 12 L/min; nebulizer pressure, 20 Psi; sheath gas temperature, 350 ℃; sheath gas flow rate, 11 L/min; capillary voltage, 3 500 V; nozzle voltage, 500 V; dwell time, 20 ms; and scanning mode, multiple reaction monitoring (MRM). The molecular formula of auramine O is C17H21N3, with an exact mass of 267.17, and its precursor, product ion, collision energy (CE) and fragmentation voltage are shown in Table 2.

3.2 Preparation of reference solutionsAccurate amounts of the reference substance of auramine O were dissolved in certain volumes of methanol to prepare into reference solutions of 0.05, 0.1, 0.2, 0.4, 0.8 and 1.0 ng/mL.

Table 2 Optimized conditions for mass spectrometry

PrecursorProductionCollisionenergy (CE)∥VFragmentationvoltage∥V268147293802681073338026813135380

3.3 Test solutionGegen Qinlian tablets were ground into powder. An accurate amount (around 0.5 g) of the powder was placed in a conical flask, added with 10 mL of methanol, sealed, weighed, sonicated (500 W, 80 kHz) for 30 min, cooled, and weighed again. The lost weight was supplemented with methanol. The new solution was filtered. An accurate volume (1 mL) of the subsequent filtrate was diluted with methanol to 10 mL, and the dilution obtained was the test solution for analysis.

3.4 Investigation on linear rangeThe test solutions prepared in Section3.2were detected by LC-MS/MS, respectively. Taking the integral value of the peak area as the ordinate (Y) and the injection volume as the abscissa (X), linear regression was performed, and the obtained regression equation wasY=22 188X+174.89 (r=0.999 9). The results show that there is a good linear relationship between injection volume and peak area of auramine O within the range of 0.093 4-1.869 pg.

3.5 Accuracy testAccurate volumes (1, 2 and 5 mL) of the reference solution of auramine O (9.343 ng/mL), three portions for each volume, were placed in conical flasks, respectively. After flushed with nitrogen gas, the reference solutions were added with accurate amount (around 0.5 g) of the test sample (batch No. 20130302, content 0), respectively. Then, they were detected under the conditions described in Section3.3to calculate the average recovery rate. The results show that the average recovery rate was 94.38% (RSD=3.60%,n=9), meeting the requirements of quantitative analysis.

3.6 Precision testThe test solution used in Section3.5was detected by LC-MS/MS six times repeatedly under the conditions described in Section3.1. TheRSDvalue of the peak areas of auramine O was 2.96% (n=6), indicating that the precision of this method is good.

3.7 Stability testThe test solution used in Section3.5was detected by LC-MS/MS once at a certain interval, for a total of 6 times. TheRSDvalue of the peak areas of auramine O was 1.46% (n=6), indicating that the test solution is stable within 24 h.

3.8 Detection limitThe reference solution of auramine O (9.343 ng/mL) was diluted and detected under the instrument conditions described in Section3.1. The results show that the detection limit (S/N=3) was 3 ng/g.

3.9 Determination of test samplesAccording to the method described in Section3.3, 85 batches of Gegen Qinlian tablets from 11 manufacturers were prepared into test solutions, and the contents of the adulteration dye auramine O in the test solutions were determined according to the method drawn up in the text. The results show that the contents of auramine O in 15 bathes of Gegen Qinlian tablets from 5 manufacturers ranged from 0.013 to 83.51 μg/g, indicating that the raw materials of some of the Gegen Qinlian tablet samples were ever added with the adulteration dye auramine O. The LC-MS/MS chromatograms of the reference solutions and test solutions are shown in Fig.1 and Fig.2.

Fig.1 LC-MS/MS chromatograms of reference substances of auramine O

Fig.2 LC-MS/MS chromatograms of Gegen Qinlian tablets

4 Discussion

The 85 batches of Gegen Qinlian tablets from 11 manufacturers were detected by LC-MS/MS, respectively. The results show that the adulteration dye auramine O was detected in some batches of Gegen Qinlian tablets from some of the manufacturers, indicating that some Chinese medicine preparations invested by pharmaceutical enterprises have dyeing phenomenon, and the source and quality of proprietary Chinese medicines need attention. In this study, the adulteration dye auramine O was detected in some of the Gegen Qinlian tablet samples, consistent with the reports of the addition of auramine O to Radix Scutellariae and Rhizoma Coptis Chinensis. Based on the research results, it could be speculated that dyed medicinal materials have entered proprietary Chinese medicine manufacturing enterprises, and produced a serious impact on the safety of proprietary Chinese medicine.

The production of Chinese herbal medicines and traditional

Chinese medicine decoction pieces is the source of the production and operation of proprietary Chinese medicine, and dyeing and adulteration in Chinese medicinal materials and Chinese medicine decoction pieces will bring serious harm to the subsequent production and clinical prescriptions of Chinese medicine. At present, China has specified the test methods for auramine O in Chinese herbal decoction pieces such as Cortex Phellodendri Chinensis and Rhizoma Coptis Chinensis, but there are few reports on the detection of auramine O in proprietary Chinese medicines. In this study, the content of the adulteration dye auramine O in Gegen Qinlian tablets was determined by LC-MS/MS, in order to achieve the purpose of effective quality control of the prescription.