Quality Assessment of Insamyangpye Decoction by Liquid Chromatography Tandem Mass Spectrometry Multiple Reaction Monitoring

: Insamyangpye decoction (ISYPD) is an oriental herbal prescription used in Korea to treat lung-related diseases such as chronic obstructive pulmonary disease. ISYPD is a complex prescription consisting of 13 herbal medicines, and ISYPD sample was obtained by adding 50 L of distilled water to a mixture (5 kg) of 13 herbal medicines, extracting at 100 ◦ C for 2 h using an electric extractor, and freeze-drying. In this study, an accurate and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method based on multiple reaction monitoring (MRM) was developed and veriﬁed for quality assessment of ISYPD using 10 marker components: mulberroside A ( 1 ), amygdalin ( 2 ), liquiritin apioside ( 3 ), naringin ( 4 ), poncirin ( 5 ), platycodin D ( 6 ), ginsenoside Rb1 ( 7 ), glycyrrhizin ( 8 ), saikosaponin A ( 9 ), and schizandrin ( 10 ). These marker compounds were separated using an Acquity UPLC BEH C 18 column (2.1 mm × 50 mm, 1.7 µ m) maintained at 30 ◦ C with a mobile phase elution gradient of acetonitrile in distilled water, both containing 0.1% ( v / v ) triﬂuoroacetic acid. Marker components were quantiﬁed using the LC–MS/MS MRM method developed and validated, and found at 0.09–7.47 mg/g.


Introduction
Standardization is essential for quality consistency of traditional Korean medicines (TKM), traditional Chinese medicines, and Kampo medicines. These medicines are difficult to standardize because their formulas are composed of various kinds of herbal medicines and numerous components. For their standardization, equipment for analysis such as high-performance liquid chromatography combined with a photo-diode array (HPLC-PDA) detector, liquid chromatography tandem mass spectrometry (LC-MS/MS), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS) have been widely used [1][2][3][4][5]. In particular, LC-MS/MS, which can produce a sensitive, accurate, and quick analysis, is currently one of the most widely used type of analytical system.

Plant Materials
In March 2018, the 13 types of medicinal herbs that make up ISYPD were obtained from a specialized supplier of herbal medicines, Kwangmyungdang Medicinal Herbs (KMH; Ulsan, Korea). Each raw herb was identified morphologically by Dr. Seung-Yeol Oh, pharmacognosist, (CEO of KMH) according to the guidelines of the Korean Ministry of Food and Drug Safety [19]. Specimens of each (2018CA03-1 to 2018CA03-13) have been deposited in the herbarium Korea Institute of Oriental Medicine.

Chemicals and Reagents
Authentic reference standards ( Figure S1) for LC-MS/MS quantitative analysis of ISYPD samples were supplied by manufacturers of phytochemical standards: compound 1 (C 26

Preparation of Samples and Standard Solutions for the Quantitative Analysis
To analyze the main marker components in the ISYPD sample using LC-MS/MS, 10.0 mg of the powdered ISYPD sample was added to a 100-mL volumetric flask filled with 50% methanol. The mixed solution was continuously subjected to ultrasonic extraction and vortexing for 10.0 min each. The extract was filtered through a 0.2 µm membrane filter and used for the analysis.
A solution of an authentic standard for each marker component used in this study was prepared at a concentration of 100.00 µg/mL using methanol, and then stored in a refrigerator (approximately 4 • C) and used for further analysis.

Instrument and Operating Conditions for Simultaneous Analysis of Compounds 1-10 in the ISYPD Sample
The instruments and methods for LC-MS/MS simultaneous quantification of compounds 1-10 in the ISYPD sample were applied to this study by modifying a previously published protocol [5]. Briefly, a simultaneous analysis of major markers was conducted using an Acquity UPLC H-Class system (Waters, Milford, MA, USA), consisting of a quaternary solvent manager and sample manager, with a Xevo TQ-S micro triple quadrupole MS system (Waters, Milford, MA, USA). Detailed operating conditions for UPLC and MS are presented in Table 1. The ionization mode, MRM transition (precursor ion to product ion), cone voltage, and collision energy for LC-MS/MS MRM analysis of compounds 1-10 are summarized in Table 2.

Validation of the LC-MS/MS MRM Method
The LC-MS/MS MRM method was validated for specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), and precision according to the International Conference on Harmonization guidelines and previous methods [2,20].
Briefly, the linearity was determined by the coefficient of determination (r 2 ) of the calibration curve prepared for various concentrations, and the LOD and LOQ were calculated from the following Equations (1) and (2) using the calibration curve data.
where σ and S are the standard deviation of the y-intercept and the slope of the calibration curve, respectively. Accuracy was determined using a recovery test by adding authentic standards to various concentrations. Briefly, known standard concentrations (low, medium, and high) were added to 10 mg of each ISYPD sample, adjusted to 100 mL with 50% methanol, and extracted according to the extraction protocol in Section 2.4. Precision was determined using the relative standard deviation (RSD) for repeatability (intraday, within one day), intermediate precision (interday, 3 consecutive days), and reproducibility.

Development of the LC-MS/MS MRM Method
In the development of the LC-MS/MS MRM method for ISYPD samples, the UPLC conditions for simultaneous analysis of compounds 1-10 were established as follows: an Acquity UPLC BEH C 18 column (2.1 mm × 50 mm, 1.7 µm), a column temperature of 30 • C, and a mobile phase gradient of acetonitrile in distilled water, both 0.1% (v/v) TFA (Table 1). In addition, the MS system was optimized for various parameters as shown in Table 2

MRM Transition of Each Marker Compound in the LC-MS/MS Simultaneous Analysis
In the MS spectra of the 10 marker analytes measured using the positive/negative ESI sources, compounds 1, 2, and 4-10 were detected in the positive ion mode and compound 3 was detected in negative ion mode, respectively ( Figure 1, Table 2). For LC-MS/MS simultaneous analysis, MRM transition conditions for each marker analyte were determined. In the optimized LC-MS/MS system, the precursor ion (Q1) peak of compound 1 was detected at m/z 569.21, and a product ion (Q3) peak was detected at m/z 244.99 in the form of [M + H − 2Glc] + in which two glucopyranosyl groups were released from the Q1 peak [21]. Compound 2 was observed in the form of [M + Na − aglycone] + at m/z 347.01 (Q3) in which the aglycone was eliminated from the Q1 peak [22]. Compounds 3 and 4 produced Q3 peaks formed by a retro-Diels-Alder reaction of the aglycone of each compound at m/z 118.94 and m/z 152.94 [23,24]. In compound 5, m/z 286.98 [M + H − Glc − Rham] + formed by removing glucose and rhamnose from the m/z 595.21 (Q1) peak was set as the Q3 peak [25]. The Q3 peaks of compounds 6 and 8 were observed as 485. 23

Verification of the LC-MS/MS Method
The LC-MS/MS method was validated for specificity, linearity, LOD, LOQ, accuracy (recovery), and precision. As shown in Figure S2, the specificity was confirmed as compounds were well separated without interference from each other when the retention times of all components were compared. The calibration curve showed the r 2 values of compounds 1-10 were between 0.9914 to 0.9997, showing good linearity, and the LOD and LOQ were calculated as 0.002-39.093 µg/L and 0.005-130.310 µg/L, respectively (Table 3). Recovery was 90.00-114.97% with RSD less than 20% and is shown in Table 4. The RSD values for the reproducibility of all marker analytes were measured as 0.21-0.90% for the retention time and 2.48-14.66% for the peak area. In addition, the RSD values of intraday, interday, and accuracy did not exceed 20% for any marker (Table 5). These verification results show that the LC-MS/MS MRM method developed is suitable for simultaneous analysis of marker compounds to assess the quality of the ISYPD samples.
The marker compounds in the ISYPD samples were quantified using MRM mode as shown in Table 2, and compounds 1-10 were detected at 2.40, 2.84, 4.16, 4.83, 6.23, 6.57, 7.57, 8.48, 9.09, and 9.25 min, respectively (Figure 1 and Figure S2). Compounds 1-10 were detected in the range 0.09-7.47 mg/g using the LC-MS/MS analysis method developed and validated using the ISYPD sample (Table 6). Among these components, compounds 1 and 2, which are maker compounds of Morus alba L. and Prunus armeniaca L., showed the highest levels at 7.47 mg/g and 6.12 mg/g, respectively (Table 6).

Conclusions
The LC-MS/MS MRM method is an accurate and fast way to control the quality of ISYPD efficiently, and is validated for specificity, linearity, LOD, LOQ, accuracy (recovery), and precision. This method can be successfully applied to the simultaneous quantification of marker compounds in ISYPD and may be useful to generate basic data to standardize not only ISYPD, but also other traditional herbal prescriptions.