Validation of the Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry Method for Simultaneous Analysis of Eighteen Compounds in the Traditional Herbal Prescription,

: Sanjoin-tang (SJIT) is an ancient oriental medicine prescription listed in the Jinguiyaolue that is mainly used for the treatment of primary insomnia. This study was conducted to develop and validate an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC– MS/MS) simultaneous analysis method for the quality control of SJIT using 18 target compounds. The 18 analytes were separated on an Acquity UPLC BEH C 18 column maintained at 45 ◦ C using a mobile phase composed of distilled water and acetonitrile. The MS system was used to simultaneously detect all analytes using the multiple reaction monitoring (MRM) method of Xevo TQ-XS coupled with an electrospray ionization source. The concentrations of the 18 analytes investigated in the SJIT samples ranged from below the limit of detection to 9.553 mg/g. In conclusion, the validated UPLC–MS/MS MRM analysis method can be used to obtain basic data to establish chemical-nonclinical linkage efﬁcacy and for the clinical research and quality evaluation of SJIT.


Introduction
Insomnia is a common symptom in menopausal women and is one of the factors that determine quality of life [1].Sanjoin-tang (SJIT, Suanzaoren-tang in Chinese, Sansoninto in Japanese) is one of the most frequently used prescriptions for treating insomnia in oriental medicine.SJIT was first recorded in Jinguiyaolue and consists of five traditional herbal medicines (Ziziphus jujuba Mill., Cnidium officinale Makino, Anemarrhena asphodeloides Bunge, Poria cocos Wolf, and Glycyrrhiza uralensis Fisch.)[1].As a principal herb in SJIT, Z. jujuba has been reported to have insomnia, hypnotic, tranquilizing, and antianxiety effects [2][3][4][5].In particular, jujubosides and spinosin, the main components of Z. jujuba seeds, have been reported to be effective for hypnosis and anxiety [2,6].In addition, G. uralensis and P. cocos, which are other medicinal herbs that make up SJIT, have been reported to be effective for insomnia [7][8][9][10].
Various types of components such as flavonoids [11,12], terpenoids [13][14][15], alkaloids [13], miscellaneous [16,17], phenylpropanoids [17], xanthones [18], and steroids [19] were isolated from each medicinal herb of SJIT.Quantitative and qualitative analysis methods using high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography, high-performance liquid chromatography coupled with quadrupole timeof-flight mass spectrometry, or ultra-performance liquid chromatography coupled with electrospray ionization (ESI) and quadrupole time-of-flight mass spectrometry have been developed for the efficient quality control of each herbal medicine based on the measurement of these isolated components [17,[20][21][22][23][24][25][26].As an analysis for the quality assurance of SJIT, Zhu et al. [27] reported a component profiling analysis using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and liquid chromatography-ion trap-mass spectrometry.Although the efficacy of SJIT and analysis methods for each component of herbal medicine have been reported, no simultaneous analysis method for the quality assurance of SJIT has been validated to date.
Ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) has high specificity, sensitivity, reproducibility, resolution, and accuracy, and its use for the quantitative analysis of traditional herbal medicines and herbal formulas has been increasing compared to other analytical instruments [28,29].
Ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) has high specificity, sensitivity, reproducibility, resolution, and accuracy, and its use for the quantitative analysis of traditional herbal medicines and herbal formulas has been increasing compared to other analytical instruments [28,29].

Chemicals and Reagents
The 18 standard compounds used for the evaluation of quality control of SJIT were purchased from professional high-purity phytochemical manufacturers: neomangiferin  S1).Solvents (acetonitrile, methanol, and distilled water) and reagent (formic acid) for analysis were LCMS grade.

Plant Materials and Preparation of the SJIT Water Extract
The five raw herbal medicines (Table S1) that make up SJIT were purchased from Kwangmyungdang Pharmaceutical (Ulsan, Korea) and used in the study after morphological identification by Dr. Goya Choi (Korea Institute of Oriental Medicine (KIOM), Naju, Republic of Korea).Each raw herbal medicine (KE-90-1 to KE-90-5) and SJIT water extract (KE90) were deposited at the Korean Medicine Science Research Division, KIOM (Daejeon, Republic of Korea).
After the SJIT water extract (SJIT-1) was mixed well according to the amount shown in Table S2 (total 5000.0 g: Z. jujuba 2666.67 g, C. officinale 666.67 g, A. asphodeloides 666.67 g, P. cocos 666.67 g, and G. uralensis 333.32),50 L of primary distilled water was added and the mixture was extracted at 100 °C for 2 h using an electric extractor, COSMOS-660 (Kyungseo E&P, Incheon, Korea).The extract solution was filtered through a sieve (53 µm  S1).Solvents (acetonitrile, methanol, and distilled water) and reagent (formic acid) for analysis were LC-MS grade.

Plant Materials and Preparation of the SJIT Water Extract
The five raw herbal medicines (Table S1) that make up SJIT were purchased from Kwangmyungdang Pharmaceutical (Ulsan, Korea) and used in the study after morphological identification by Dr. Goya Choi (Korea Institute of Oriental Medicine (KIOM), Naju, Republic of Korea).Each raw herbal medicine (KE-90-1 to KE-90-5) and SJIT water extract (KE90) were deposited at the Korean Medicine Science Research Division, KIOM (Daejeon, Republic of Korea).

Preparation of Standard Stock and Sample Solutions
Standard stock solutions of the 18 reference standards were prepared at a concentration of 1000 µg/mL using methanol and stored in a refrigerator (approximately 4 • C) until use.To prepare the sample solution of the SJIT samples, about 50 mg of the prepared SJIT water extract or commercially available SJIT granules was accurately taken and made to a concentration of 5 mg/mL using 70% methanol, followed by ultrasonic extraction (for 5.0 min) and vortexing (for 1.0 min).The extracted sample solution was filtered with a 0.2 µm hydrophobic filter (SSOLKOREA Co., Ltd., Daejeon, Korea) before UPLC-MS/MS analysis.

Validation of the UPLC-MS/MS MRM Analytical Method
The UPLC-MS/MS analytical method developed in this study was validated with respect to specificity, linearity, sensitivity, recovery, and precision evaluation [30].That is, specificity was confirmed by verifying the presence or absence of an interference peak in the chromatogram of each component.The linearity was evaluated by the coefficient of determination (r 2 ) value in the regression equation of the calibration curve prepared at different concentrations.The sensitivity of the method was verified by limit of detection (LOD) and limit of quantitation (LOQ) values and calculated as signal-to-noise (S/N) ratios of 3 and 10, respectively.Recovery (%) was evaluated by the standard addition method, which was evaluated by adding three different levels (low, medium, and high) of standard solutions to the known sample.This parameter was calculated using the following equation: Recovery (%) = found amount/spiked amount × 100.Precision was demonstrated by evaluating relative standard deviation (RSD) values of intraday (within a day) and interday (three consecutive days) precisions and repeatability.This parameter was calculated using the following equation: RSD (%) = standard deviation/mean × 100.

Multiple Reaction Monitoring Conditions for UPLC-MS/MS Analysis of SJIT Samples
As mentioned in Section 2.3, MRM conditions for the simultaneous analysis of the 18 marker components in SJIT were explored using a UPLC-MS/MS system.An ESI attachment was used as the ion source.Eight compounds (1, 2, 5, 6, 14, 15, 17, and 18) showed high intensity in the [M-H] − form, and eight compounds (4, 7-11, 13, and 16) showed good intensity in the [M+H] + form.Magnoflorine (3) and jujuboside A (12) showed an [M] + peak and an adduct peak in the form of [M+H 2 O] + , respectively (Figure 1).In case of jujuboside A, the adduct peak ([M+H 2 O] + ) observed at m/z 1225.1 was detected more strongly than [M+H] + during MS measurement (Figure S2).
To simultaneously quantify the 18 components in SJIT samples using the UPLC-MS/MS MRM analysis mode, parameters such as precursor ion (Q1) peak and product ion (Q3) peak, cone voltage, and collision energy for each component were set as shown in Table 1.The Q3 peaks of xanthone types, neomangiferin, and mangiferin were set to m/z 331.0 and 301.0 in the form of [M-H-Glu-C 4 H 8 O 4 ] − and [M-H-C 4 H 8 O 4 ] − , respectively [31,32].These components showed a common feature of the Q3 peak being formed due to the cleavage of the sugar ring.For magnoflorine and alkaloids, a Q3 peak was observed at m/z 297.2 ([M-(CH 3 ) 2 NH] + ), corresponding to the loss of two methyl groups, and an amine group was lost from the ion generating the Q1 peak [33].For spinosin, 6 -feruloyl spinosin, and liquiritigenin, Q3 peaks were observed at m/z 327.[33,34].In both liquiritin and ononin, Q3 peaks were generated as [M-H-Glu] − and [M+H-Glu] + with one molecule of glucose removed [33,35].In liquiritin apioside, the Q3 peak was detected in the form of an aglycone, in which apiose and glucose were removed from the Q1 peak [33].The Q3 peaks of ferulic acid (phenylpropanoid-type) and Z-ligustilide (benzoquinone derivative) were set to m/z 177.0 ([M+H-H 2 O] + ) and m/z 91 ([M+H-H 2 O-CO-C 4 H 6 ] + ), respectively [36].In the MRM transition of isoliquiritin and isoliquiritigenin, which are chalcone-type compounds, Q3 peaks were observed at m/z 257.0 ([M+H-Glu] + ) and m/z 137.0 ([M+H-C 8 H 7 O] + ), respectively, where m/z 257.0 was produced by the release of one molecule of glucose from the Q1 peak, and m/z 137.0 was generated by the cleavage of the B ring in the molecular structure [37].The Q3 peak of jujuboside A, triterpenoids, was observed at m/z 473.5 in the form of aglycone in which five sugars were cleaved from the Q1 peak [38], whereas the Q3 peak of glycyrrhizin was observed at m/z 351.2 in the form of two glucuronic acid conjugates, excluding the aglycone [39].For jujuboside B, the Q3 peak was observed at m/z 911.5 ([M-H-Xyl] − ), where one molecule of xylose was removed from the Q1 peak [40].In dehydropachymic acid and pachymic acid, the highest intensities were observed at m/z 59.0 and 465.4,respectively, as each Q1 peak was fragmented by cone voltage and collision energy, and these were set as Q3 peaks [24].Under the optimized UPLC-MS/MS MRM analysis conditions, all components were completely eluted within 13.0 min; representative total ion chromatograms (TICs) are shown in Figure 1.The TIC of the other samples (SJIT-2 to SJIT-4) is shown in Figure S3.In addition, the extracted ion chromatograms of the 18 reference markers and samples (SJIT-1 ~SJIT-4) are presented in Figure S4.5), liquiritin (6), ferulic acid ( 7), 6‴-feruloyl 5pinosyn (8), isoliquiritin (9), ononin (10), liquiritigenin (11), jujuboside A (12), isoliquiritigenin (13), glycyrrhizin (14), jujuboside B (15), Z-ligustilide ( 16), dehydropachymic acid (17), and pachymic acid (18).The concentration of each compound in the standard mixtures (A) was 100.00 µg/L.

Conclusions
In the present study, we developed a simultaneous analysis method using UPLC-MS/MS to quantify 18 components for the efficient quality control of SJIT, which is a prescription medicine used for insomnia.The developed assay was validated with respect to linearity, LOD, LOQ, recovery, and precision.The established analytical method was then applied to the simultaneous analysis of real SJIT samples, and the content of the 18 target components was analyzed simultaneously.Among them, neomangiferin, mangiferin, liquiritin apioside, glycyrrhizin, and Z-ligustilide, which are the main components of A. asphodeloides, G. uralensis, and C. officinale, were found to be relatively abundant compared with the other components.The method is therefore concluded to be suitable for collecting basic data for future clinical and efficacy studies.

Table 1 .
UPLC-MS/MS MRM analytical conditions for simultaneous analysis of the 18 analytes in SJIT samples.
2, 327.1, and 137.0, arising from the fragmentation of the Q1 peak to [M+H-Glu-C 4 H 8 O 4 ] + , with the loss of a glucose and a C 4 H 8 O 4 molecule; [M+H-Feryloyl-Glu-C 4 H 8 O 4 ] + , with the loss of a feruloyl functional group, one glucose, and a C 4 H 8 O 4 molecule; and [M+H-C 8 H 8 O 8 ] + , with the loss of a C 8 H 8 O 8 molecule, respectively

Table 3 .
Recovery (%) of the 18 target components using the UPLC-MS/MS MRM method.

Table 4 .
Precision test of the 18 target components using the UPLC-MS/MS MRM method.