Chemical Profiling and Quantification of Potential Bioactive Components in Gandouling Pill by Ultra-High Performance Liquid Chromatography Coupled with Diode Array Detector/Quadruple-Qrbitrap Mass Spectrometry

Gandouling (GDL) Pill is a novel Traditional Chinese medicinal drug to treat Wilson’s disease in clinics. It is composed of six separate herbal medicines, including Rhei Radix ET Rhizoma, Coptidis Rhizoma, Salviae Miltiorrhizae Radix ET Rhizoma, Spatholobi Caulis, Curcumae Rhizoma, and Curcumae Longae Rhizoma. In this study, a strategy was proposed to investigate the chemical constituents and to quantify the potential bioactive components in GDL Pill. Firstly, the mass fragmentation behaviors of representative compounds were investigated, and, in total, 69 compounds were characterized in GDL Pill using full scan/dd-MS2 scan mode by ultra-high-performance liquid chromatography (UPLC)/Q-Orbitrap mass spectrometry (MS). These compounds included 18 alkaloids, 18 ketones, 16 phenolic compounds, 11 organic acids, and 6 tanshinones. Seventeen of the compounds were unambiguously identified by comparison with reference standards. Secondly, the absorption components of GDL Pill in rat plasma were investigated by using target-Selected Ion Monitoring (t-SIM) scan mode built in Q-Orbitrap MS. A total of 18 components were detected, which were considered as potential bioactive components of GDL Pill. Thirdly, 10 major absorption components were simultaneously determined in six batches of samples by UPLC/diode array detector (DAD). The method was fully validated with respect to linearity, precision, repeatability, stability, and recovery. Alkaloids from Coptidis Rhizoma, such as coptisine (8), berberine (18), palmatine (19), were the most abundant bioactive compounds for GDL Pill that possess the potential be used as quality markers. The proposed strategy is practical and efficient for revealing the material basis of GDL Pill, and also provides a simple and accurate method for quality control.


Introduction
Traditional Chinese medicines (TCMs) are always used in the form of formulae in clinical practice [1]. They are demonstrated to be "complex matrix" in structure with a large array of compounds [2][3][4][5]. Fully understanding the chemicals, especially the potentially bioactive ones, is vital for the safety and efficacy evaluation of TCM formulae. In the past decades, various analytical technologies were developed for TCM formulae, such as liquid chromatography/diode array detector (LC/DAD) and liquid chromatography/mass spectrometry (LC/MS) [6]. Among them, LC/MS is a cost-effective tool to characterize a large number of compounds from TCM formulae. In our previous report, a total of 259 compounds were rapidly detected and characterized in the Xiaoer-Feire-Kechuan formula [3]. Multi-components determination also plays a key role for quality control

Optimization of the Extraction Method
According to the components of separate herbal medicines in GDL Pill, both hydrophilic compounds (e.g., alkaloids and flavonoid glycosides) and hydrophobic compounds (e.g., phenolic aglycones) may be involved. The extraction method was optimized to effectively extract both types of compounds. Different solvents (water, 50% methanol, 75% methanol, and methanol) were compared to fully extract the components in GDL Pill, and 75% methanol provided the best extraction efficiency for different types of compounds ( Figure S1, Table S1). For example, the alkaloids 18/19 and phenolic aglycones 58/64 exhibited higher recovery in 75% methanol. Therefore, 75% methanol was chosen to extract the chemicals in GDL Pill.
were optimized using the real sample. As shown in Figure S2, the Acquity CSH C18 column provided favorable resolution for alkaloids (8-11, 18, 19), as well as for other compounds (41,53,58,64). When comparing the peak shape using different types of mobile phases, it was illustrated that acidic additive was essential for baseline separation of alkaloids ( Figure S3), and 0.1% formic acid in water was used for the following study.

Characterization of Organic Acids
In total, 11 organic acids were characterized in GDL Pill, which were mainly from DS, HL, and DH. Due to the presence of carboxyl groups, organic acids are easily ionized in negative ion mode.   Figure 2D) [17]. Similarly, compound 65 was characterized as cryptotanshinone ( Figure 2D) [18]. In addition, 18 ketones were also characterized in GDL Pill, and their structures were also tentatively characterized using a similar method (Table 1).

Absorption Components of GDL Pill in Rat Plasma
Generally, the components that are absorbed in plasma after oral administration are always considered to be the bioactive ones for traditional Chinese medicines. Based on the chemical components that were characterized in GDL Pill, the plasma-absorption components were determined by a highly sensitive and selective targeted-selected reaction monitoring (t-SIM) scan mode when the GDL Pill was orally administered to rats. In total, 19 compounds were detected in rat plasma (Table 1, Figure 3). The extracted ion chromatograms of the 19 compounds are shown in Figure 4. These compounds included 9 alkaloids, 6 phenolic compounds, 2 organic acids, 2 tanshinones, and 1 ketone (Table 1). These compounds could be potential bioactive components of GDL Pill that could be used for quality control.

Method Validation
The calibration curves of 10 analytes were constructed by plotting the analyte peak area (y) against the concentration (x). All the 10 analytes showed good linearity (r 2 = 0.9973 − 1.0) ( Table 2). The stability was evaluated by analyzing the same sample solution at 0, 2, 4, 8, 12, and 24 h at room temperature (25 ± 2 °C). The RSD values for stability analysis ranged from 0.45% to 4.41%. The precision of the method was evaluated by analyzing the same reference solution six times continuously (intra-day) in the following three days (inter-day). The RSD values for intra-day and inter-day precisions ranged from 0.12% to 1.62% and 0.43% to 1.96%, respectively, indicating acceptable precision of the method. The

Method Validation
The calibration curves of 10 analytes were constructed by plotting the analyte peak area (y) against the concentration (x). All the 10 analytes showed good linearity (r 2 = 0.9973 − 1.0) ( Table 2). The stability was evaluated by analyzing the same sample solution at 0, 2, 4, 8, 12, and 24 h at room temperature (25 ± 2 • C). The RSD values for stability analysis ranged from 0.45% to 4.41%. The precision of the method was evaluated by analyzing the same reference solution six times continuously (intra-day) in the following three days (inter-day). The RSD values for intra-day and inter-day precisions ranged from 0.12% to 1.62% and 0.43% to 1.96%, respectively, indicating acceptable precision of the method. The repeatability was evaluated by injecting six independently prepared sample solutions. The reproducibility test showed a good consistency of the sample preparation process with RSD values ranging from 0.42%-4.26%. The accuracy was measured by spiking the reference standards at 100% level (equivalent to the concentrations in the sample solution) into sample solutions (n = 6). Recovery of the analytes varied from 96.4% to 106.2%, indicating acceptable accuracy of this method.

Sample Analysis
Contents of 10 potential bioactive compounds in 6 batches of GDL Pill were determined ( Figure 5). The total contents of these 10 compounds varied from 29.54 to 31.10 mg/g, suggesting good quality consistency. Alkaloids were the major components in GDL Pill with contents at 28.19 ± 1.41 mg/g. Among these, six alkaloids, berberine (18) and coptisine (8) were the predominant constituents. Their contents among the 6 batches of samples were also similar, i.e., 15.54 ± 0.78 mg/g for 18 and 4.16 ± 0.21 mg/g for 8. For 4 phenolic aglycones, chrysophanol (64) was the most abundant one, the contents of which varied from 1.11 to 1.22 mg/g. The total content of the other 3 phenolic aglycones (41, 53, 58

Sample Analysis
Contents of 10 potential bioactive compounds in 6 batches of GDL Pill were determined ( Figure 5). The total contents of these 10 compounds varied from 29.54 to 31.10 mg/g, suggesting good quality consistency. Alkaloids were the major components in GDL Pill with contents at 28.19 ± 1.41 mg/g. Among these, six alkaloids, berberine (18) and coptisine (8) were the predominant constituents. Their contents among the 6 batches of samples were also similar, i.e., 15.54 ± 0.78 mg/g for 18 and 4.16 ± 0.21 mg/g for 8. For 4 phenolic aglycones, chrysophanol (64) was the most abundant one, the contents of which varied from 1.11 to 1.22 mg/g. The total content of the other 3 phenolic aglycones (41, 53, 58) was 2.91 ± 0.15 mg/g. Figure 5. The contents of 10 potential bioactive components in GDL Pill (n = 6).

Preparation of Sample Solutions
For qualitative analysis, 200 mg of GDL Pill extracted in 20 mL of 75% methanol (v/v) for 30 min in an ultrasonic water bath (40 kHz, 500 W). Accurately, 300 mg of the HL, DH, DS, JXT, EZ, and JH powders were, respectively, extracted with 30 mL of 50% methanol (v/v) for 30 min in an ultrasonic water bath (40 kHz, 500 W). For quantitative analysis, 50 mg of GDL Pill extracted in 20 mL of 75% methanol (v/v) for 30 min in an ultrasonic water bath (40 kHz, 500 W).

Animal Experiments
Eight male SD rats weighing 220 ± 20 g were obtained from Beijing Weitong Lihua Experimental Animals Company (Beijing, China). The rats were housed in a controlled room at standard temperature (24 ± 2 • C) and humidity (70 ± 5%), and kept on a 12 h light/12 h dark regime. After a week acclimation, rats were randomly divided into two groups: Drug Group (n = 4) for test plasma; Control Group (n = 4) for blank plasma. They were fasted for 12 h with free access to water prior to the experiment. The animal protocols were approved by the institutional Animal Care and Use Committee at Anhui University of Chinese Medicine.
GDL Pill was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) solution. Rats in Drug Group were given a dose of 77.15 mg/kg body weight orally (equivalent to clinical dosage). 0.5% CMC-Na aqueous solution (2 mL) was administrated to rats in Control Group. Blood samples (0.5 mL) were taken from the suborbital venous plexus of rats at 0.5, 1, 2 and 4 h post-administration. All homogeneous biological samples from the same group were merged into a collective sample.

Mass Spectrometry
Mass spectrometry analysis was performed on a Q-Exactive Plus hybrid quadrupole Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization source (HESI). It was operated in both negative and positive ion modes. The other parameters were set as follows: spray voltage, ±3.5 kV; sheath gas flow rate, 35 arb; auxiliary gas, 10 arb; capillary temperature, 350 • C; auxiliary temperature, 400 • C; S-lens RF level, 60 V. Full Scan/dd-MS 2 was used to acquire the qualitative data. The resolution for MS and MS/MS was set as 70,000 and 17,500, respectively. The scan range was set as m/z 100-1500, and the normalized collision energies (NCE) were 35%. The five most abundant ions in each full scan were selected as precursor ions to obtain their MS/MS spectra. Data were processed using Xcalibur TM

Conclusions
In this study, an integrated strategy was proposed to reveal the chemical components for GDL Pill. Firstly, 69 compounds were characterized using Full Scan/dd-MS 2 scan mode built-in Q-Orbitrap MS, and 17 of them were unambiguously determined by comparison with reference standards. Secondly, 18 plasma-absorbed components were detected using t-SIM scan mode, which were considered to be potential bioactive components for GDL Pill. Finally, the contents of 10 major absorption components were simultaneously determined in six batches of samples by the UPLC/DAD method. Alkaloids from Coptidis Rhizoma, including coptisine (8), berberine (18), and palmatine (19), were the most abundant bioactive compounds for GDL Pill that could be used as quality markers. The established method is practical and efficient for the quality control of GDL Pill.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27238247/s1, Figure S1: Extraction efficiency of compounds in GDL Pills using different solvents; Figure S2: Separation efficiency of compounds in GDL Pills using different stationary phases; Figure S3: Separation efficiency of compounds in GDL Pills using different mobile phases; Figure S4: The LC/MS chromatograms of GDL Pill and separate herbs in the positive ion mode; Figure S5: The LC/MS chromatograms of GDL Pill and separate herbs in the negative ion mode; Figure S6: The MS/MS spectra of representative alkaloids identified in GDL Pill; Figure S7: The MS/MS spectra of representative organic acids identified in GDL Pill; Figure S8: The MS/MS spectra of representative phenolics identified in GDL Pill; Table S1 Comparison of peak areas of 6 major compounds in GDL Pill by using different kinds of extraction solvent.