Determination of Two Synthetic Pigments in Lifei Tablets by HPLC and LC-MS/MS Spectrometry

Zebing ZHU, Dewei ZHANG

Chongqing Key Laboratory of Development and Utilization of Dao-di Herbs in Three Gorges Reservoir Area, Chongqing, Chongqing Wanzhou Food and Drug Inspection Institute, Chongqing 404100, China

Abstract [Objectives] To identify stained Schisandrae Chinensis Fructus in Lifei Tablets with carmine and acid red 73 as target substances. [Methods] High performance liquid chromatography (HPLC) and liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used to analyze the 70% methanol extract of the drug. The HPLC was equipped with an Inertstain-C18 (250 mm×4.6 mm, 5 μm) chromatographic column. The mobile phase was acetonitrile-5 mmol/L ammonium acetate solution, gradient elution: volume flow rate of 1.0 mL/min, column temperature of 30 ℃, detection wavelength of 509 nm. LC-MS/MS was equipped with a Waters-C18 (2.1 mm×150 mm, 1.7 μm) chromatographic column, the mobile phase was methanol-0.1% formic acid solution (containing 5 mmol/L ammonium acetate), gradient elution; the volume flow rate was 0.3 mL/min; ESI ion source, positive ion mode, full scan of primary and secondary mass spectrometry. [Results] Carmine and acid red 73 showed good linear relationship in HPLC and LC-MS/MS, with r greater than 0.995. The sample recovery and RSD values of HPLC and LC-MS/MS met the requirements. The RSD of the results determined by HPLC and LC-MS/MS was not greater than 5.0%. Both methods had good compatibility and could be used for the examination of carmine and acid red 73 in Lifei Tablets. [Conclusions] The method is reliable and reproducible and can be used to identify stained Schisandrae Chinensis Fructus in Lifei Tablets.

Key words High performance liquid chromatography (HPLC), Liquid chromatography tandem-mass spectrometry (LC-MS/MS), Lifei Tablets, Carmine, Acid red 73, Schisandrae Chinensis Fructus, Synthetic pigments, Chinese patent medicine

1 Introduction

Lifei Tablets is a kind of preparation composed of nine flavors including Schisandrae Chinensis Fructus, Lilii Bulbus, Glycyrrhizae Radix Et Rhizoma,etc.This kind of preparation has the effects of expelling tuberculosis, nourishing the lung, relieving cough and resolving phlegm, and can be used for tuberculosis cough, expectoration, hemoptysis, asthma due to qi deficiency, chronic bronchitis,etc.[1-2]. In addition, modern clinical studies have shown that Lifei Tablets have a certain therapeutic effect on patients with chronic obstructive pulmonary disease (COPD) in both acute onset and stable stages[3]. Especially during the COVID-19 epidemic, Lifei Tablets has a strong adjuvant treatment effect, resulting in a shortage of Lifei Tablets in the market.

At present, there are 38 approval numbers issued by China National Medical Products Administration, with large output and high frequency of use, belonging to common commonly used drugs. Carmine and acid red 73 are prepared from chemical toxic substances such as benzene, toluene, naphthalene, anthracene,etc.as raw materials, and are synthesized through organic reactions such as nitration, acylation, and azo. Because of advantages of bright color, strong colorability and low cost, they are widely used. However, since synthetic pigments generally have toxic and side effects on the liver and kidneys on the human body, and can even cause teratogenicity, carcinogenicity, and mutagenesis, it brings great risks to the quality of subsequent Chinese patent medicines and clinical prescriptions[4]. For this consideration, Volume IV of theChinesePharmacopoeia(2020 Edition) stipulates that Chinese herbal medicines and Chinese herbal decoction pieces should not use pigments. In this prescription, Schisandrae Chinensis Fructus is often used as a variety that is easy to be added illegally because its color is similar to carmine and acid red 73. Illegal drug manufacturers often mix inferior medicinal materials into decoction pieces of high-quality medicinal materials through illegal staining. The Schisandrae Chinensis Fructus mixed by illegal dyeing results in a certain amount of residues in Lifei Tablets, which not only affects its clinical efficacy, but also causes harm to human health and seriously affects the safety of public medication[5]. Therefore, in this study, we used HPLC and LC-MS/MS to identify carmine and acid red 73 in Lifei Tablets.

2 Experiment

2.1 Instruments and drugsShimadzu-20A high performance liquid chromatograph; QE Focus ultra-high performance liquid chromatography-tandem mass spectrometer (Thermo Fisher Scientific, USA); Lifei Tablets (market available, purchased from different pharmacies in different provinces and cities, involving 35 manufacturers); carmine and acid red 73 reference substances (China National Institutes for Food and Drug Control, batch No.111771-201603 and 111773-201602, respectively); acetonitrile was chromatographically pure; water was ultrapure water; other reagents were guaranteed reagent (GR).

2.2 Methods and results

2.2.1Chromatographic conditions. HPLC used Inertstain-C18(250 mm×4.6 mm, 5 μm) column, acetonitrile as mobile phase A, 0.05 mol/L ammonium acetate solution as mobile phase B, gradient elution (0-30 min, 5%-45%A, 30-40 min, 5%A), the flow rate was 1.0 mL/min, the column temperature was 30 ℃; the injection volume was 10 μL; the detection wavelength was 509 nm. LC-MS/MS adopts Waters-C18(2.1 mm×100 mm, 1.7 μm) chromatographic column; acetonitrile as mobile phase A, 0.1% formic acid solution (containing 5 mmol/L ammonium acetate) as mobile phase B, gradient elution (0-10 min, 5%-90%A, 10-15 min, 5%A), flow rate 0.3 mL/min, column temperature 40 ℃, injection volume 5 μL: electrospray ionization (ESI), primary mass spectrometry full scan, and ion secondary scanning mass spectrometry, positive ion mode, the ionization voltage (IS) was 3.2 kV, the sheath gas was 35 mL/min, the auxiliary gas was 10 mL/min, and the ionization temperature (TEM) was 350 ℃.

2.2.2Preparation of solutions. Took an appropriate amount of carmine and acid red 73 reference substances, added 70% methanol to prepare 1 mL of mixed reference solution containing separately 20 μg of carmine red and acid red 73. The HPLC reference solution was diluted into a series of reference solutions with 10% methanol aqueous solution. Took an appropriate amount of sample, ground finely, precisely weighed 4 g, put it in a stoppered conical flask, and precisely added 20 mL of 70% methanol, weighed, ultrasonic treatment (power 400 W, frequency 40 kHz) for 20 min, cooled down, weighed again, made up for the weight loss with 70% methanol, shook well, filtered, took the filtrate as the HPLC sample solution. Precisely weighed 0.10 mL of liquid chromatography sample solution, placed it in a 100 mL volumetric flask, diluted it with 10% aqueous methanol solution and fixed the volume, shook well to obtain LC-MS/MS sample solution.

2.2.3Specificity test. Using Lifei Tablets as the research matrix, we studied the interference of other components and excipients in the preparation on the detection method after adding the pigment mixed with the reference substance. We prepared the test solution in accordance with the method in Section2.2.2, and prepared the blank matrix solution without adding the mixed reference solution, and compared the liquid chromatogram and mass spectrum (Fig.1-2). The results showed that the liquid chromatography in the blank matrix solution did not have the same chromatographic peaks as the mixed reference solution, while the test solution prepared by adding the mixed reference solution could detect the same parent ion peak and daughter ion peak as the mixed reference solution. Therefore, we judged that the traditional Chinese medicine components and excipients in the preparation did not interfere with the results.

Note: A. mixed control; B. test product+mixed control; C. test product+carmine; D. test product+acid red 73; E. matrix blank.

2.2.4Linearity, limit of detection (LOD) and limit of quantitation (LOQ) test. Precisely pipetted a certain amount of carmine and acid red 73 reference solution for linear relationship analysis, taking the mass concentration when the signal-to-noise ratio (S/N) was 3 as the limit of detection (LOD), and the mass concentration when the S/N was 10 as the limit of quantitation (LOQ). From Table 1, it can be seen that the linear relationship between carmine and acid red 73 determined by HPLC and LC-MS/MS is good.

Table 1 Linearity, LOD and LOQ determined by HPLC and LC-MS/MS (ng/mL)

2.2.5Precision, reproducibility, stability and sample recovery rate tests. Took the negative sample (Shanxi S1 pharmaceutical company), added the mixed reference solution, and prepared the test solution in accordance with the method in Section2.2.2, injected and measured the samples according to the chromatographic conditions in Section2.2.1, repeated the sample injection 6 times, measured the peak area, and calculated the precision. As indicated in Table, the precision is high. Took 6 pieces of negative samples (S1), added the mixed reference solution, prepared the test solution in accordance with the method in Section2.2.2, injected and measured the samples according to the chromatographic conditions in Section2.2.1, repeated the sample injection 6 times, as shown in Table 2, the reproducibility is high. Took the negative sample (S1), added the mixed reference solution, and prepared the test solution in accordance with the method in Section2.2.2. At the interval of 0, 3, 6, 9, 12, 24, 36 and 48 h after the preparation of test solution, injected the samples and measured according to the chromatographic conditions Section2.2.1. As shown in Table 2, the test solution is stable at room temperature within 48 h. Took negative samples (S1), added the mixed reference solution, prepared the test solution in accordance with the method in Section2.2.2, injected and measured the samples according to the chromatographic conditions in Section2.2.1, then calculated the recovery rate. As shown in Table 2, the recovery rate of carmine and acid red 73 is good in the samples determined by HPLC and LC-MS/MS.

Note: A. carmine; B. acid red 73; A1, B1. primary; A2, B2. Secondary.

Table 2 Methodological verification %

2.2.6Determination of samples. Took 122 batches of Lifei Tablets samples, prepared the test solution in accordance with the method in Section2.2.2, injected and measured the samples according to the chromatographic conditions in Section2.2.1(Table 3). TheRSDof the results determined by HPLC and LC-MS/MS was ≤5.0%, indicating that the two methods have good compatibility and can be used for the determination of carmine and acid red 73 in Lifei Tablets.

Table 3 Results of determination

3 Discussion

The results of methodological verification indicate that HPLC and LC-MS/MS have good linearity, precision, repeatability, stability and recovery rate in their respective ranges. Besides, HPLC and LC-MS/MS can also show good performance in the detection of samples. This means that the results of HPLC and LC-MS/MS determination of carmine and acid red 73 in Lifei Tablets are reliable and reproducible, and can be used for the identification of Schisandrae Chinensis Fructus in Lifei Tablets. By comparison, LC-MS/MS is 500 times more sensitive than HPLC. According to the above methods, the results of 122 batches of samples show that some enterprises still use stained Schisandrae Chinensis Fructus to take the place of Schisandrae Chinensis Fructus to produce Lifei Tablets. At present, there are extensive reports about the detection methods of synthetic pigments in medicinal materials and food, mainly including HPLC, HPLC-MS/MS,etc., while there are relatively few reports on pigments in Chinese patent medicines[6]. In view of these problems, it is very necessary to improve the identification technology of synthetic pigments in Chinese patent medicines and strengthen the supervision of illegally adding stained Chinese medicinal materials in Chinese patent medicines.