Liquid Chromatography – Mass Spectrometry Method for the Simultaneous Determination and Confirmation of Seven Active Components in Chinese Medicine Kumu Injection

Purpose: To develop and validate a simple and selective high performance liquid chromatography photo diode array mass spectrometry (HPLC-PDA-MS/MS) method for simultaneous determination and confirmation of seven major active alkaloids (6-Hydroxy-β-Carboline-1-carboxylic acid, β-Carboline-1-carboxylic acid, β-Carboline-1-propanoic acid, 3-Methylcanthin-5,6-dione, 4-Methoxy-3-methylcanthine-5,6-dione, 5-Hydroxy-4-methoxycanthin-6-one, 4,5-Dimethoxycanthin-6-one) in Kumu injections (KMIs) Methods: For the analysis of the preparation, the optimal chromatographic condition was achieved on a Phenomenex Gemini C 18 column with gradient elution of 25 mM aqueous ammonium acetate (pH = 4.0 adjusted by glacial acetate acid) and acetonitrile with ﬂow rate at 1.0 mL/min, column temperature at 35 o C and detection wavelengths at 245, 260 and 271 nm. Results: Excellent linear behavior over the investigated concentration ranges was observed with regression coeffcient (R 2 ) > 0.9997 for all analytes. Intra- and inter-day precisions for all studied constituents ranged from 0.20 to 1.80 %. Recoveries of the assayed constituents were in the range of 98.73 to 100.34 %. The results showed the contents of these seven marker compounds differed significantly among different batches of KMIs both from the same and different manufacturers. Conclusion: The validated method was reliable, accurate, repeatable and can be applied to routine quality assessment of these active components in KMIs.


INTRODUCTION
It was about seventy years ago that injections were introduced into traditional Chinese medicine (TCM) domain as a new and important dosage form, which has since substantially changed the impression of TCM from being perceived as slow and weak acting into one with rapid onset and higher efficacy [1]. TCM injections have played an indispensable role in emergency medicine today owing to its ability to bypass the first pass metabolism and the active ingredients can be directly distributed into blood circulation to exert rapid therapeutic effect. However, the use of TCM injections is often associated with adverse drug reactions (ADR). The ADR of TCM injections can manifest as drug fever, disorders of skin and appendages, circulatory system allergy reaction, and even anaphylactic shock in severe case. TCM injection-associated ADR could occur when changing the injection products to that of different manufacturers or to different batches of the same manufacturer. This observation underscores the generally undesirable quality of TCM injection products in the market and the huge challenge facing quality control of injection form of Chinese medicine [2]. Kumu injection (KMI), a widely used Chinese herbal preparation in China and officially recorded in the Drug Standard of Ministry of Public Health of the People's Republic of China, is made from a single Chinese herbal Picrasma quassioides (kumu in Chinese). Kumu has the functions of heat-clearing, detoxification, and anti-inflammation in Chinese medicine practice. Because of the above therapeutic functions, KMI is extensively used for the treatment of cold, upper respiratory tract inflection, acute tonsillitis, enteritis, and bacillary dysentery [3]. Pharmacological and phytochemical studies on P. quassioides and KMI have shown that alkaloids are the main active ingredients responsible for the overall therapeutic effects of KMI. The P. quassioides-derived alkaloids, which can be broadly divided into β-carboline and canthinones types, have shown potent activities against infection and abscess of respiratory, digestive and urinary systems [4][5][6][7][8]. Among these alkaloids, 4,5-dimethoxycanthin-6-one possesses therapeutic action against ulcerative colitis [9], 5-hydroxy-4-methoxycanthin-6-one exhibits inhibitory effect against tobacco mosaic virus [6], while 3-methylcanthin-5,6-dione shows significant anti-inflammatory and antioxidant activities [10]. Due to the biological activities of these alkaloids, their quantitative measurement in Kumu product is of great importance for its quality control.
Several qualitative and quantitative analytical methods such as thin layer chromatography [11], gravimetry [3] and HPLC [12] have been developed for quality assessment of KMI. However, all these methods suffered from either low resolution, low sensitivity or identification of few marker constituents (less than three analytes) and are inadequate for revealing the synergistic effects and complex constituents of KMI. Therefore, an analytical method with capability for multi-targets determination is urgently needed to establish the quality control and enhance the clinical safety and efficacy of KMI.

EXPERIMENTAL Chemicals and materials
Acetonitrile, methanol, ammonium acetate and glacial acetate acid were of HPLC-grade (Merck, Darmstadt, Germany). LC-MS grade water was purchased from Fisher Scientific (Massachusetts, USA). Redistilled water was used for the preparation of two-phase mobile solvent system. All other reagents used in this study were of analytical grade from Guanghua Chemicals Co., Ltd (Guangzhou, Guangdong Province, China).
Negative control preparation (NC) and a total of 20 batches of KMIs were provided by Qingfeng Pharmaceutical Company (Ganzhou, Jiangxi Province, China) and Wannianqing Pharmaceutical Company (Shantou, Guangdong Province, China).

HPLC system and conditions
Shimadzu LC-20A HPLC system (Shimadzu, Kyoto, Japan) comprising of a SPD-M20A PDA detector, a LC-20AT pump, a SIL-20AC automatic sampler, and a CTO-20A thermostatic column compartment was applied for chromatographic analysis. The separation was performed on Gemini C 18 column (4.6 x 250 mm, 5 μm, Phenomenex Inc., CA, USA) protected by a Security Guard C 18 guard column (

LC-MS system and conditions
Agilent G6410 Triple Quad LC/MS (Agilent Technologies, MA, USA) was used for mass spectrometric measurements. The same separation conditions as described in HPLC-PDA analysis were used. By solvent splitting, 0.5 mL/min portion of the column effluent was delivered into the ion source of the mass spectrometer. Data acquisition was performed on a MassHunter software system. The conditions of MS analysis were as follows: dry gas (N 2 ) flow rate, 10 L/min; dry gas temperature, 325 o C; nebulizer pressure, 35 psi; source voltage, 4000 V. The mass spectrometric data was acquired from m/z 100 to 1000 in positive ion mode.

Statistical analysis
All data analyses were carried out using GraphPad Prism 5 statistical software. The datasets were analyzed using the unpaired t-test. A p-value of < 0.05 was considered statistically significant.

Optimization of chromatographic conditions
In order to obtain chromatograms with a good resolution of the targeted analyte peaks, various chromatographic parameters were optimized. Five different analytical columns, i.e., Phenomenex Luna C 18 , Phenomenex Gemini C 18 , YMC Pack ODS, Agilent Zorbax SB-C 18 and Waters Symmetry Shield RP 18 were initially screened and the best resolution was achieved with Gemini C 18 from Phenomenex. Suitable mobile phase compositions (acetonitrile-aqueous ammonium acetate, acetonitrile-phosphoric buffer, acetonitrile-aqueous sodium dodecyl benzene sulfonate, acetonitrile-ammonia) were also investigated and the acetonitrile-aqueous ammonium acetate system showed more powerful separation ability than other systems. The retention behavior of the compounds on the reversed-phase HPLC column was significantly affected by the pH of the mobile phase, thus different mobile phase pH (pH 3.5, 4.0, 4.5 and 5.0) adjusted by glacial acid were compared and the peak tailing eliminated at pH 4.0. Besides, column temperatures (25, 30 and 35 o C) were also optimized. As a result, better peak shape was achieved at temperature of 35 o C. Furthermore, according to maximum absorption of the standards, the optimal wavelengths were determined to be 245 nm for compound c, d e, f and g, 260 nm for compound b and 271 nm for compound a, respectively. Typical HPLC-PDA chromatograms of reference compounds, KMI samples and negative control preparation are shown in Figure 2.

Calibration curves, LODs and LOQs
The calibration curves were performed with working solutions at 7 different concentrations mentioned above in triplicate. The regression equations were calculated using the formula y = ax + b, where y and x were peak area and concentration (μg/mL), respectively. The working solutions were further diluted to a series of concentrations with methanol to calculate the limits of detection (LODs) and limits of quantification (LOQs) when signal-to-noise ratio (S/N) amounted to 3 and 10, respectively. Good linearity (R 2 > 0.9997) was achieved within the investigated ranges for all the analytes. The LODs of seven alkaloids ranged from 0.022 to 0.345 μg/mL and LOQs were within 0.073-1.153 μg/mL (Table 1).

Precision, repeatability, stability and accuracy
Intra-day and inter-day variations were chosen to measure the precision of the developed method by analyzing 1, 1/3, 1/10 dilutions of the mixed standard solution. The intra-day and inter-day precisions were determined by assaying standard solutions at the three concentrations in six replicates within a single day and once a day for three sequential days, respectively. The RSDs of intra-day and inter-day precisions for all the components under investigation were less than 2% (Table 2).
Repeatability was investigated by analyzing six independently prepared solutions (No. 20110321) and each of them was injected into the apparatus at 0, 4, 8, 12, 24 and 48 h, respectively, to determine the stability of the solution. The RSDs of repeatability and stability of the seven compounds were all less than 3% ( Table 2). Accuracy was determined by recovery test. The known quantities of the marker compounds were spiked to the known aliquots of the sample solution (No. 20110321) that had previously been analyzed. Recovery was between 98.73% and 100.34% with RSDs less than 3% for all the seven marker compounds ( Table 2).
The validation results strongly indicated that the HPLC-PDA method was reproducible and suitable for simultaneous quantitation of seven alkaloids in KMI.

Sample analysis
The proposed method was subsequently applied to simultaneously determine the seven alkaloids in 20 batches of commercially-available KMI from two manufacturers, and the contents of the investigated constituents are tabulated in Table  3, in which 6-hydroxy-β-carboline-1-carboxylic acid (a), β-carboline-1-carboxylic acid (b) and βcarboline-1-propanoic acid (c) were observed to be more abundant than the rest four marker constituents among all the 20 batches of KMI samples. However, there were large differences between the samples of the two origins, which not only manifested in that β-carboline-1carboxylic acid (b) was highest in content, accounting for about 60% of the total seven alkaloids among the samples from Qingfeng while β-carboline-1-propanoic acid (c) was the most abundant accounting for approximately 40% of the seven analytes in samples from Wannianqing. Also the total contents of the seven target compounds showed significant variations between samples from the different manufacturers according to statistical results. Furthermore, the average contents of compounds a-e were much higher among samples from Qingfeng than those from Wannianqing with only exception of β-carboline-1-propanoic acid (c). Also, large quality fluctuations obviously existed among the samples from the same manufacturer.

DISCUSSION
Based on the results of these analyses of the contents of the seven bioactive compounds of KMI, large quality variations among all these products obviously existed among the samples both from the same and different manufacturers, which may arise from the different sources of the raw herbal materials, material collection if plant were at different times of the year, disparity in preparation technology in different factories, and lack of effective quality control method to maintain the quality consistency of the preparation. All these factors would definitely affect their therapeutic efficacies and even safety. It is also worth noting that compound d, e, f, g believed as important bioactive compounds [6,9,10], were prone to huge loss during the preparation due to their small polar and weak water-soluble properties. There is certainly room for proper care in plant collection, processing and technical improvement in the preparation of the KMI to reduce the loss of these constituents.

CONCLUSION
Efficient and reliable analytical protocols to evaluate and control the quality of TCM injections are urgently needed to minimize the ADR associated with the use of this dosage form of Chinese herbal products. We have successfully developed a powerful and reliable analytical method for quality evaluation of KMI through identification and simultaneous quantitation of seven major alkaloids of KMI, namely 6-hydroxyβ-carboline-1-carboxylic acid, β-Carboline-1carboxylic acid, β-carboline-1-propanoic acid, 3methylcanthin-5,6-dione, 4-methoxy-3-methylcanthine-5,6-dione, 5-hydroxy-4-methoxycanthin-6-one, 4,5-dimethoxycanthin-6-one by HPLC-PDA-MS-MS. This method has been proven to be sensitive, accurate and reproducible and could provide valuable quantitative information for the quality assessment of KMI.