Qualitative and Quantitative Analysis of the Major Constituents in WLJ Herbal Tea Using Multiple Chromatographic Techniques

Quality control of Chinese herbal tea remains a challenge due to our poor knowledge of their complex chemical profile. This study aims to investigate the chemical composition of one of the best-selling and famous brand of beverage in China, Wanglaoji Herbal Tea (WLJHT), via a full component quantitative analysis. In this paper, a total of thirty-four representative constituents were identified or tentatively characterized using ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Moreover, the quantitative analyses of fourteen constituents were performed by high performance liquid chromatography with a triple quadruple tandem mass spectrometry (HPLC-MS/MS) method and saccharide compositions of WLJHT were also quantitatively determined by high performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) on a Hilic column, separately. Using multiple chromatographic techniques presented a good precision, sensitivity, repeatability and stability, and was successfully applied to analyze 16 batches of WLJHT samples. Therefore, it would be a reliable and useful approach for the quality control of WLJHT.


Introduction
Traditional Chinese herbal tea, developed by Chinese people during the long-term for disease prevention and health care under the guidance of traditional Chinese medicine, has been approved as an intangible cultural heritage by the Chinese government in 2006 [1][2][3].
Wanglaoji Herbal Tea (WLJHT), founded in the Daoguang eighth year of Qing Dynasty (Ad 1828), is the earliest Cantonese herbal tea and recognized as the ancestor of herbal tea. It is consisted of seven traditional Chinese medicines (Chinese Mesona, PlumeriaeFlos, LoniceraeJaponicaeFlos, ChrysanthemiFlos, Prunellae Spica, Microctis Folium and Glycyrrhizae Radix et Rhizoma), and widely used for heat-clearing, detoxicating, engender liquid and allay thirst. Modern pharmacological studies have proven its protective effects on liver damage [2], improvement of cytotoxic T Lymphocytes activity in spleen [4], amelioration on lipids metabolism [5] and glucometabolism [6], and enhancement of immune functions of restrain-stress mice [7]. In our previous phytochemical study [8], several polyphenol constituents as phenolic acids and flavonoids were separated and structural elucidated from WLJHT.
As "King of herbal tea", WLJHT is lack of effective approach for quality control due to its chemical complexity. Three phenolic acids have been suggested to be employed for the quality assessment of WLJHT. However, as is known to all, multi-components in compositive herbs attribute comprehensive efficacy of herbal tea. Therefore, quality assessment based on a few of markers has been proven to be insufficient. In consideration of a mounts of unknown chemicals existed in herbal tea, LC/MS was widely used for the quality analyses of herbal medicines and herbal teas due to its powerful function on chemical structures [9]. Meanwhile, with the limitation of MS on carbohydrates, the evaporative light scattering detector (ELSD) has been used as an efficient method to identify and determine the little molecular carbohydrates in multi-herbals [10][11][12][13][14]. Therefore, complex application of multiple techniques for quality assessment of herbal tea seems to be more feasible and effective.
The aim of this study is to identify and quantify of the major constituents both the small molecules and saccharides in WLJHT, using ultra-high performance liquid chromatography (UPLC) coupled with quadrupole tandem time-of-flight mass spectrometry (Q-TOF-MS), and high performance liquid chromatography equipped with evaporative light scattering detector (HPLC-ELSD). 16 batches of samples were analyzed and the results were expected to provide comprehensive information for the quality control of WLJHT.

Identification of Constituents in WLJHT
Both positive and negative ion modes were detected for MS analysis depending on the different chemical properties of WLJHT as shown in Figure 1A,B, respectively. In the negative-ion ESI mode experiments, the deprotonated molecules [M-H] − were detected as the base peaks for most of the constituents.  The exact molecular weight of each constituent was easily calculated according to the experimental mass of the pseudo-molecular ions, and the molecular formulas of those were deduced from each exact molecular weight obtained by Q-TOF-MS. The fragmentation information of each constituent was also obtained by Q-TOF-MS/MS as shown in Supplementary Information, which was quite useful for the identification of each constituent. Table 1 lists the retention time (t R ), molecular formulas, experimental molecular weights, ESI-TOF-MS ions of thirty-four major peaks in the chromatograms came from WLJ herbal tea samples.
On the other hand, the saccharide profile was also shown in Figure 2, in which only four saccharides-fructose, a/β-D-glucose and sucrose-were identified by comparison with reference standards.
Molecules 2018, 23, x FOR PEER REVIEW 6 of 12 The exact molecular weight of each constituent was easily calculated according to the experimental mass of the pseudo-molecular ions, and the molecular formulas of those were deduced from each exact molecular weight obtained by Q-TOF-MS. The fragmentation information of each constituent was also obtained by Q-TOF-MS/MS as shown in Supplementary Information, which was quite useful for the identification of each constituent. Table 1 lists the retention time (tR), molecular formulas, experimental molecular weights, ESI-TOF-MS ions of thirty-four major peaks in the chromatograms came from WLJ herbal tea samples.
On the other hand, the saccharide profile was also shown in Figure 2, in which only four saccharides-fructose, a/β-D-glucose and sucrose-were identified by comparison with reference standards.

Limitation of Qualitative Analysis Solely Using Mass Spectrometry
WLJ herbal tea has two type components as non-sugar small molecules and little molecular carbohydrates. Although mass spectrometry is a powerful and sensitive analytical method as it can provide an accurate mass of the molecules and nanogram level of detection limit, solely using MS data in qualitative analysis is not suitable for weak electrolytes. For example, fructose, glucose and sucrose as the major carbohydrates of WLJHT, but they are too hard ionized to be detected by mass spectrometry. Therefore, it is suggested that the qualitative analysis is risky if only mass spectrometry is used. Moreover, the ionization mode plays an important role for MS analysis. The complex chemical composition of Chinese medicine requires a variety of ionization modes. Some components may be ignored if the single ionization mode is used. The comparison between two modes is necessary, which can avoid missing information.

Limitation of Qualitative Analysis Solely Using Mass Spectrometry
WLJ herbal tea has two type components as non-sugar small molecules and little molecular carbohydrates. Although mass spectrometry is a powerful and sensitive analytical method as it can provide an accurate mass of the molecules and nanogram level of detection limit, solely using MS data in qualitative analysis is not suitable for weak electrolytes. For example, fructose, glucose and sucrose as the major carbohydrates of WLJHT, but they are too hard ionized to be detected by mass spectrometry. Therefore, it is suggested that the qualitative analysis is risky if only mass spectrometry is used. Moreover, the ionization mode plays an important role for MS analysis. The complex chemical composition of Chinese medicine requires a variety of ionization modes. Some components may be ignored if the single ionization mode is used. The comparison between two modes is necessary, which can avoid missing information.

Method Validation
The linearity, ranges, regressions, LODs, LOQs and recoveries of the method are listed in Table 2. The data exhibited a satisfactory relationship between concentrations and peak areas of the analytes within the test ranges (R 2 ≥ 0.9992). The RSDs of intra-and inter-day variations for 14analytes were not beyond3.19% and 4.75%, respectively. The LODs and LOQs ranged from 0.2 to 20 µg/L and from 0.5 to 30 µg/L, respectively. The established method demonstrated acceptable accuracy with spike recovery of 96.75%-105.78% for all analytes; and the RSDs of the peak areas for 14analytes detected within 24 h were lower than 4.87%. These results indicated that the developed UPLC-MS method was efficient, accurate and sensitive for simultaneous quantitative determination of the 14 constituents in WLJHT.

Quantitative Determination of the Major Constituents in the WLJHT
The above HPLC-ELSD and HPLC-MS/MS methods were applied to quantify the contents of the 17 major constituents in 16 batches of WLJHT samples (Figure 3). All of the contents were calculated by the external standard method, and the mean values and SDs from the three parallel determinations of each sample are summarized in Table 3. In general, the total content of known chemical components reached 78.61%-90.06% of the dry weight of WLJHT samples. Among them, 14 representative non-sugar small molecules possessed 0.25‰-0.29‰, and monosaccharide/sucrose accounted for 78.58%-90.03%. These results exhibited a general feature of WLJHT's chemical profile: saccharides are the major components, and the non-sugar small molecules possess a very low content.

Preparation of Reference Solutions
Standard solutions of these 14 reference compounds were prepared in methanol-water (3:7, v/v) atthe known concentration (mg/mL):protocatechuid acid (

Preparation of Sample Solutions
The samples were stored in a refrigerator at 4 • C until used, and they warmed to room temperature, and filtered through a nylon membrane filter (0.22 µm, Phenomenex, USA) for qualitative analysis. For HPLC-MS/MS analysis, 1 mL of sample solution was transferred to 5-mL volumetric flask, brought up to volume with methanol-water (3:7, v/v) and filtered through a nylon membrane filter (0.22 µm, Phenomenex, USA) prior to use.

UPLC/Q-TOF-MS/MS Instrumentation and Methods
UPLC/Q-TOF-MS analysis was performed using an AB Sciex 5600 Triple-TOFTM mass spectrometer (AB Sciex, Redwood, CA, USA) coupled to a Shimadzu UPLC LC-30AD system (Kyoto, Japan) which were controlled with an Analyst ® TF 1.7 software (AB Sciex, Framingham, MA, USA). The chromatographic separations were accomplished on an ACQUITY UPLC BEH C18 column (Waters, Milford, MA, USA; 100 × 2.1 mm, 1.7 µm) with the column temperature kept at 25 • C. 0.1% formic acid (A) and acetonitrile (B) were served as the mobile phases at a flow rate of 0.35 mL/min under the following gradient elution mode: 0-2 min, 5% B; 2-3 min, 5%-10% B; 3-5 min, 10% B; 5-11 min, 10%-30% B; 11-20 min, 30%-60% B; 20-25 min, 60%-95% B. The injection volume was 5 µL, the column temperature was at room temperature. The mass spectrometer coupled with an electrospray ionization (ESI) sources was run in negative/positive ion and high sensitivity mode to acquire the TOF-MS. Meanwhile, accurate mass measurements were acquired with an automated calibration delivery system. After optimization, the nebulizer gas, heater gas and curtain gas were set at 55, 55 and 35 psi, respectively, and nitrogen was used as the source gases. The source temperature, ion spray voltage and declustering potential were set at 500 • C, ±4500 V and 100 V, respectively. For the IDA experiments, the collision energies were set at −45 eV and −25 eV, and the collision energy spread was set at 15 eV. TOF-MS spectra were obtained from 100 to 1500 Da followed by information dependent acquisition (IDA) scanning from 50 to 2000 Da. For further review of the mass spectrometric data for qualitative analysis, PeakView 2.0 Software (AB Sciex, Framingham, MA, USA) was used.

HPLC-ELSD Instrumentation and Methods
A previously reported HPLC-HILIC-ELSD method was used to determine the monosaccharides and oligosaccharides in TCM samples [2]. For quantitation of D-(−)-fructose, D-(+)-glucose and sucrose, an Agilent 1260liquidchromatography system (Agilent Technologies, Palo Alto, CA, USA), and Alltech3300 evaporative light scattering detector (Grace Alltech, Deerfield, IL, USA) coupled with a Merck ZIC-HILIC (4.6 mm × 200 mm, Merck, Tokyo, Germany) column at 30 • C were used. Water (A) and acetonitrile (B) were served as the mobile phases at a flow rate of 1.0 mL/min under the following gradient elution mode: 0-10 min, 85% B; 10-20 min, 85%-70% B; 20-40 min, 70%-55% B. The injection volume was 10 µL, the drift tube temperature of ELSD was set at 60 • C and the nitrogen flow rate of ELSD was set at 1.8 L/min. The gain number was equal to 1.

HPLC-MS/MS Instrumentation and Methods
For quantitation of 14 major constituents in the WLJHT, the HPLC-MS/MS analysis was performed using an Agilent 1260 HPLC system, equipped with G1312B 1260 Bin Pump, G1367E 1260 Hip ALS, G1316A 1260 TCC, Agilent 6460 LC/QQQ, Chemstation online workstation, electrospray ion source (ESI) and Agilent Poroshell 120 EC-C 18 column (3.0 mm × 50 mm, 2.7 µm). 0.1% formic acid (A) and acetonitrile (B) were served as the mobile phases at a flow rate of 0.25 mL/min under the following gradient elution mode: 0-3 min, 13%-25% B; 3-7 min, 25% B; 7-8 min, 25%-40% B; 8-12 min, 40%-54% B. The injection volume was 5 µL, the column temperature was 25 • C. For MS condition, the capillary voltage was set at 3500 V, nozzle voltage was 500 V. Nebulizer air, drying-gas and sheath gas were all nitrogen, the drying-gas temperature was 300 • C and the flow rate was 5 L/min, the nebulizer pressure was 45 psi, sheath gas temperature was 300 and its flow rate was 11 L/min, the mass scanning range was set from m/z 100 to 1500. The optimum parameters of triple quadruple mass spectrometry are given in Table 2.

Conclusions
In this study, UPLC/Q-TOF-MS/MS, HPLC-MS/MS and HPLC-ELSD methods were developed for the identification and determination of the major constituents in WLJHT. The UPLC coupled with MS quickly identified or tentatively characterized 34 compounds in WLJHT based on their determined exact molecular weights and fragmentation patterns. Accurate determinations of 17 major constituents in WLJHT were performed by HPLC-ELSD and HPLC-MS/MS methods, respectively. Compared to the reported method in the literature [20], the complex application of the above three methods showed good stability, reproducibility, comprehensiveness, and could be applied for the quality control of WLJHT.