Development and Validation of Ultrahigh-Performance Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometry Method for Quantitative Determination of Ten Active Compounds in Ge-Gen-Jiao-Tai-Wan

A rapid, accurate, and sensitive method for the simultaneous determination of 10 main components, namely puerarin, daidzin, coptisine, epiberberine, jatrorrhizine, berberine, palmatine, coumarin, daidzein, and cinnamic acid in Ge-Gen-Jiao-Tai-Wan, was developed based on ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry. Analysis was performed on an Agilent 1290 Infinity II series UHPLC system, equipped with a Waters ACQUITY UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm) by using (A) 0.1% acetic acid and (B) methanol as mobile phase. The flow rate was 0.3 mL/min, and the injection volume was 1 μL. Mass spectrometry was operated in multiple reaction monitoring mode using an Agilent 6460 triple quadrupole mass spectrometer equipped with an AJS-ESI ion source. Agilent Mass Hunter Work Station Software was employed for data acquisition and processing. All calibration curves showed excellent linear regressions (R2 > 0.9992). The precision, repeatability, and stability of the ten compounds were below 4.56% in terms of relative standard deviation. The average extraction recovery ranged from 96.53% to 102.69% with a relative standard deviation of 1.14–3.78% for all samples. This study potently contributes to the quantitative evaluation of Ge-Gen-Jiao-Tai-Wan, thereby providing a scientific basis for further studies and clinical application of Ge-Gen-Jiao-Tai-Wan.


Introduction
Traditional Chinese medicine (TCM) is gaining attention from the international community because of the holistic concept of the TCM theory and historical clinical practice. TCM, not only acts as an extremely important part of China's medical and health causes, but is also a shining pearl in the treasure house of human traditional medicine culture. As China's unique medical characteristics, TCM owns rich clinical experience and huge potential therapeutic value in the long-term practice process.
e Ge-Gen-Jiao-Tai-Wan (GGJTW) formula, derived from the prior and well-known TCM formula designated Jiao-Tai-Wan, is composed of Kudzu root (Ge-Gen in Chinese), Rhizoma coptidis (Huang-Lian in Chinese), and Cinnamon (Rou-Gui in Chinese), and it has been a classic formula for the treatment of type 2 diabetes mellitus (T2DM) in Central South University, Xiangya Hospital for many years. Our previous studies have proved that GGJTW contributes greatly to the amelioration of hyperglycaemia in T2DM [1,2]. Kudzu root, Rhizoma coptidis, and cinnamon, first recorded in the ancient TCM book "Shen-Nong-Ben-Cao-Jing" (Han Dynasty), are nowadays famous Chinese herbs documented in the Pharmacopoeia of the People's Republic of China (2015 edition).
e chemical components contained in TCM compounds are complex with uneven content, and those effective components are the material basis of the biological effect of the whole formula.
rough the simultaneous qualitative and quantitative analysis, the content of various chemical components and the proportion of each component can be determined, hence realizing the quality control of TCM compounds [21][22][23][24][25][26]. However, to the best of our knowledge, the methods for determining components used in previous reports only focused on one or two herbs of the whole preparation of GGJTW [1,[27][28][29]. Fang et al. developed a method based on the high-performance capillary electrophoresis (HPCE) method with diode array detection (DAD) for the separation and determination of isoflavonoids in Kudzu root [27]. Kong et al. simultaneously quantified five active alkaloids and chemical fingerprint analysis for quality control of Rhizoma coptidis chinensis based on UPLC-PAD combined with chemometrics methods [28]. Foudah et al. reported the determination of cinnamaldehyde and eugenol in cinnamon using a sustainable/green HPTLC technique [29]. In our previous study, we only determined the content of puerarin and berberine in the respective herbs and in the final GGJTW extracts by UPLC-PAD, lacking the identification of multiple compounds in the whole formula [1]. As previously mentioned, there have been no reports specifically focusing on quantitative determination of the whole GGJTW formula. erefore, a rapid, accurate, and sensitive method is emergent to quantify the compounds in GGJTW, which is conducive for the quality control, searching the multiple active compositions of the classical TCM formula, and illustrating the deeper meaning of combined use of single herbs. Nowadays, ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS) has become a pivotal and potent instrument in analyzing substances with a lower content of complicated TCM formulas because of its high resolution and outstanding sensitivity [30][31][32]. In this regard, our present study conducted a systematic and comprehensive analysis by UHPLC-QQQ-MS to fully illuminate the chemical constituents of GGJTW for the first time.
Herein, we successfully established a UHPLC-QQQ-MS analytical method for simultaneous determining ten active compounds (puerarin, daidzin, coptisine, epiberberine, jatrorrhizine, berberine, palmatine, coumarin, daidzein, and cinnamic acid) in GGJTW, and the method was well validated for quality evaluation based on specificity, linearity, precision, recovery, repeatability, and stability. e proposed methodology of quantitative determination contributed greatly for the detection of the ten targeted compounds in the GGJTW extract and can be used for further quality control, thereby establishing a forceful basis for the further study of its efficacy and safety in clinical applications.

Preparation of GGJTW Extracts.
e extraction of GGJTW was based on our previous literature report [1] and strictly followed the drug production standard. e extraction was approved by professor Yanmei Peng, Institute of Traditional Chinese Medicine, Hunan University of Chinese Medicine (Hunan, China) and was completely implemented in Hunan Guo-Hua Pharmaceuticals Ltd. (Hunan, China). e main ingredient of cinnamon is volatile oil, thus the extraction procedure of the formula is to extract cinnamon oil first and then mix the liquid for further waterextraction [33]. e final calculated extract yield of GGJTW was 18.37%, and the content of volatile oil was 1.38% (v/w, mL/g) in cinnamon, the total puerarin content in Kudzu root was 2.5% (w/w), and the total berberine content in Rhizoma coptidis was 5.8% (w/w). us, these major components were compliant with the herb quality standards of the Chinese Pharmacopoeia (2015 edition). e GGJTW extracts were stored in a 4°C refrigerator for use.

Chromatographic
Conditions. An ultrahigh-performance liquid chromatograph was implemented on a 1290 Infinity II series UHPLC System (Agilent Technologies).

Mass Spectrometry
Conditions. An Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies), equipped with an AJS electrospray ionization (AJS-ESI) interface, was applied to perform mass spectrometry in multiple reaction monitoring (MRM) mode. Typical ion source parameters were as follows: capillary voltage � +4000/-3500 V, nozzle voltage � +500/-500 V, atomizing gas (N2) temperature � 300°C, atomizing gas (N2) flow rate � 5 L/min, sheath gas (N2) temperature � 250°C, sheath gas (N2) flow rate � 11 L/min, and nebulizer � 45 psi. e MRM parameters for each of the targeted analytes were optimized using flow injection analysis by injecting the standard solutions of the individual analytes into the API source of the mass spectrometer. Several most sensitive transitions were used in the MRM scan mode to optimize the collision energy for each Q1/Q3 pair (Table 1). Among the optimized MRM transitions per analyte, the Q1/Q3 pairs that showed the highest sensitivity and selectivity were selected as "quantifier" for quantitative monitoring. e additional transitions acted as "qualifier" for the purpose of verifying the identity of the target analytes. Agilent Mass-Hunter Work Station Software (B.08.00, Agilent Technologies) was employed for MRM data acquisition and processing [34].

Preparation of Standard and Quality Control (QC)
Solutions. Puerarin 4.096 mg, daidzin 3.836 mg, coptisine 1.788 mg, epiberberine 0.942 mg, jatrorrhizine 1.812 mg, berberine 0.955 mg, palmatine 0.974 mg, coumarin 1.468 mg, daidzein 0.611 mg, and 7.390 mg of cinnamic acid were accurately weighed and were separately placed in a 10 mL brown volumetric flask. Methanol was added and then dissolved by ultrasound to obtain the reserve solution of ten reference substances. e concentrations were 409.6, 383.6, 178.8, 94.2, 181.2, 95.5, 97.4, 146.8, 61.1, and 739.0 μg/mL, respectively. Another 10 mL brown flask was taken, and 5 mL of methanol and 50 μL of each of the ten reference substances reserve solutions were added, respectively, and diluted to 10 mL with methanol. e concentration of each analyte in standard mixture solutions was as follows: 2.0480 μg/mL for puerarin, 1.9180 μg/ mL for daidzin, 0.8940 μg/mL for coptisine, 0.4710 μg/mL for epiberberine, 0.9060 μg/mL for jatrorrhizine, 0.4775 μg/mL for berberine, 0.4870 μg/mL for palmatine, 0.7340 μg/mL for coumarin, 0.3055 μg/mL for daidzein, and 3.6950 μg/mL for cinnamic acid. en, the standard mixture solution was successively diluted by 2 times to prepare a series of standard solutions containing 9 concentration points to establish calibration curves. QC samples were prepared at three concentration levels containing puerarin (0.1307, 0.5440, and 2.0480 μg/mL), daidzin (0.1440, 0.5753, and 1.9180 μg/mL), coptisine   Journal of Analytical Methods in Chemistry 2.6. Preparation of GGJTW Solution. 1.2 g of GGJTW extract (crude herb, 6.53 g) was accurately weighed and added to 40 mL of deionized water, followed by ultrasound for 10 min to a concentration of 30 mg/mL of GGJTW solution. A 10 μL aliquot of the sample was precisely transferred to an Eppendorf tube. After the addition of 990 μL of 75% methanol, the samples were vortexed for 30 s, and centrifuged at 12000 rpm (4°C) for 15 min. Finally, a 60 μL aliquot of the clear supernatant was transferred to an auto-sampler vial and 1 μL of volume was sucked into the system for UHPLC-QQQ-MS analysis (dilution factor � 100). us, the sample was diluted by 100 times for the determination of coumarin, daidzein, and cinnamic acid. In addition, the supernatant was further diluted 10 times with 75% methanol for the determination of puerarin, daidzin, coptisine, epiberberine, jatrorrhizine, berberine, and palmatine (dilution factor � 1000).

Optimization of the UHPLC Chromatographic
Conditions. e UHPLC conditions were principally determined by selecting columns and optimizing the compositions of the mobile phase and gradient elution programs for the rapid and effective separation of the chemical constituents in GGJTW. It was tested that the Waters ACQUITY HSS T3 column (100 × 2.1 mm, 1.8 μm) from Waters Co. (USA) achieved high column efficiency and excellent separation of multiple compounds in this study. Moreover, in our preliminary test, we investigated the mobile phase systems of acetonitrile-water and methanol-water. It was found that methanol-water could well separate the chromatographic peaks in the GGJTW prescription, and the peak capacity was large. Phosphoric acid and acetic acid were also added to the mobile phase for adjusting the pH or polarity. A good peak shape can be obtained when the concentration of acetic acid was 0.1%, and the measured components can be separated from the interference peak to the baseline, meanwhile, most of the components had good response by mass spectrum. As a result, 0.1% acetic acid aqueous solution and methanol were chosen as the preferred mobile phases. Furthermore, because of the complexity of the compounds and the significant differences between respective contents, gradient elution was used to effectively separate the compounds to the greatest extent. By optimizing the gradient elution conditions, the UHPLC system employed a gradient elution of 0.1% acetic acid aqueous solution (A) and methanol (B) (0-1 min, 70-70% A; 1-6 min, 70-5% A; 6-10 min, 5-5% A; 10-10.5 min, 5-70% A; 10.5-14 min, 70-70% A) with a 1 μL injection volume at a mobile phase flow rate of 0.3 mL/min, obtaining good resolution and peak shape. And the temperature of the sample tray was set at 4°C, the column temperature was kept at 40°C. e typical extracted ion chromatograms (EIC) of the ten components are presented in Figure 2.

Optimization of MS Conditions.
Both positive and negative ion scanning modes were implemented for qualitative analysis of the compounds due to the variety of chemical constituents in the extracts and their different response modes. By adjusting the fragment voltage of the mass spectrum, we found that most of the excimer ion peaks of the chemical components are stable when the fragment voltage is between 80-120 V, and a certain amount of fragments will be generated, which is conducive to the qualitative analysis of the compounds. Several parentdaughter ion pairs (transitions) with the highest signal intensity were selected to optimize the MRM parameters, with atomizer pressure determined to be 45 psi, atomizing gas flow rate to be 5 L/min, and sheath flow rate at 11 L/min. e capillary voltage is 4000 V for the positive ion mode and 3500 V for the negative ion mode as conventional value.

Method Validation.
e developed method was assessed in terms of specificity, linearity, LOD, LOQ, precision, accuracy, stability, and repeatability, and followed with Guidance for Industry-Bioanalytical Method Validation by the U.S. Food and Drug Administration (FDA).

3.3.2.
Linearity. Taking the measured standard peak area of each component y as the ordinate and the concentration of the target compound x as the abscissa, linear regression was carried out by using the least square method, and the weight was set as 1/x to obtain the linear range and regression equation of each component. As illustrated in Table 2, linear regression analysis of the 10 components in the respective concentration range showed good linearity for each analyte (R 2 > 0.9992), which allowed for the acquisition of reliable and effective data for the analyzed samples.

Precision.
e intra and interday variations were for determining the precision of the developed method. Relative standard deviation (RSD) was utilized as a measurement of precision. Intra and interday precision were  determined on 6 replicates of QC samples at 3 different concentrations within 1 day or 3 consecutive days, respectively. e peak area of each component was recorded for UHPLC-QQQ-MS analysis, and the regression equation was employed to calculate the concentration of 10 components in each injection. Results concerning the precision of this developed method are exhibited in Table S1 (Supplementary Material). Overall, the RSD of intraday precision ranged from 1.02% to 3.66%, and interday precision ranged from 1.14% to 3.39%.

3.3.5.
Accuracy. e accuracy of the analytical method was evaluated by using the recovery test. Recoveries of 10 compounds on six replicates were investigated by spiking with the authentic standards to the GGJTW samples. Peak areas of each analyte in six GGJTW samples at three different concentration levels (80%, 100%, and 120% of the known amounts) were recorded. en, the concentrations of the 10 compounds in the GGJTW samples after spiking were calculated according to the peak area using the calibration curve. e average recovery percentage was calculated by the following formula: recovery (%) � (total amount after spiking−original amount in sample)/spiked amount × 100%. e average extraction recovery ranged from 96.53% to 102.69%, and the RSDs were below 3.78% (Table S2, Supplementary Material), indicating that the method could ensure the acquisition of accurate and consistent data for all the constituents.

Stability.
e stability of the analytical GGJTW solution at environmental temperature was studied by detecting the sample solution at 0, 2, 4, 8, 12, and 24 h. e RSD values of peak areas were taken for assessment. Table S3 (Supplementary  Material) shows the stability tests. e RSD values of the 10 compounds were below 4.56%. e data confirmed that there was no significant degradation and all the 10 compounds in the GGJTW solution had good stability within 0-24 h.

Repeatability.
In order to verify the repeatability of the method, six different sample solutions prepared from the same GGJTW sample were analyzed in parallel by the method described in Section 2.3-2.4. Table S4 (Supplementary Material) exhibited that the RSD values of the ten chemical constituents were below 4.44%, which confirmed the high reproducibility of the method.
ere were significant differences in the content of the 10 components. Puerarin is the most abundant constituent, accounting for 5.73% of the total content. Daidzin and berberine followed with 2.14% and 1.09%, respectively. e proportions of coptisine, epiberberine, jatrorrhizine, and palmatine were relative closer, significantly lower than those of puerarin, daidzin, and berberine. e content of coumarin, daidzein, and cinnamic acid was similar at a lower level, and daidzein was the lowest, only accounting for 0.04% of the total GGJTW content.

Conclusions
is work demonstrated a detailed research for quantitative determination in GGJTW. To the best of our knowledge, this is the first report using the UHPLC-QQQ-MS method for simultaneously qualitative and quantitative determination of puerarin, daidzin, coptisine, epiberberine, jatrorrhizine, berberine, palmatine, coumarin, daidzein, and cinnamic acid in GGJTW extract. e established method could well identify the contents of the abovementioned ten components. In terms of methodology, the standard curves of the ten compounds were successfully established, and the linearity, precision, recovery rate, stability, and reproducibility of the method were fully validated. is effective, sensitive, rugged, and safe method can be used for further quality control of GGJTW. Our systematic study of the chemical constituents of GGJTW extract by the developed UHPLC-QQQ-MS method provides a basis for further scientific studies on GGJTW and a foundation for the effective development and application of GGJTW.

Data Availability
e data used to support the findings of this study are included within the article, and any further information is available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.