# Qualitative and Quantitative Analysis of the Major Constituents in Chinese Medical Preparation Lianhua-Qingwen Capsule by UPLC-DAD-QTOF-MS

Authors: Weina Jia; Chunhua Wang; Yuefei Wang; Guixiang Pan; Miaomiao Jiang; Zheng Li; Yan Zhu
Journal: The Scientific World Journal
 (2015)

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## Abstract

Lianhua-Qingwen capsule (LQC) is a commonly used Chinese medical preparation to treat viral influenza and especially played a very important role in the fight against severe acute respiratory syndrome (SARS) in 2002-2003 in China. In this paper, a rapid ultraperformance liquid chromatography coupled with diode-array detector and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF-MS) method was established for qualitative and quantitative analysis of the major constituents of LQC. A total of 61 compounds including flavonoids, phenylpropanoids, anthraquinones, triterpenoids, iridoids, and other types of compounds were unambiguously or tentatively identified by comparing the retention times and accurate mass measurement with reference compounds or literature data. Among them, twelve representative compounds were further quantified as chemical markers in quantitative analysis, including salidroside, chlorogenic acid, forsythoside E, cryptochlorogenic acid, amygdalin, sweroside, hyperin, rutin, forsythoside A, phillyrin, rhein, and glycyrrhizic acid. The UPLC-DAD method was evaluated with linearity, limit of detection (LOD), limit of quantification (LOQ), precision, stability, repeatability, and recovery tests. The results showed that the developed quantitative method was linear, sensitive, and precise for the quality control of LQC.


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## Body

## 1. Introduction

Lianhua-Qingwen capsule (LQC), developed from the two classical traditional Chinese medicine (TCM) formulaeMaxing-Shigan-Tang andYinqiao-San which have a long history of clinical application in the treatment of influenza [1], is a commonly used Chinese medical preparation to treat viral influenza and especially played an important role in the fight against severe acute respiratory syndrome (SARS) in 2002-2003 in China [2]. LQC is composed of 11 herbs including Fructus Forsythiae (Lianqiao), Flos Lonicerae Japonicae (Jinyinhua), Herba Ephedrae (Mahuang), Semen Armeniacae Amarum (Kuxingren), Radix Isatidis (Banlangen), Rhizoma Dryopteridis Crassirhizomatis (Mianmaguanzhong), Herba Houttuyniae (Yuxingcao), Herba Pogostemonis (Guanghuoxiang), Radixet Rhizoma Rhei (Dahuang), Radixet Rhizoma Rhodiolae Crenulatae (Hongjingtian), and Radixet Rhizoma Glycyrrhizae (Gancao), along with menthol and a traditional Chinese mineral medicine, Gypsum Fibrosum (Shigao). According to previous reports, LQC has a good clinical effect on influenza with the symptoms of high fever, aversion to cold, headache, pharyngalgia, cough, sneezing, muscle ache, and so on [3]. Modern pharmacological studies have shown that LQC also has the antiviral, antibacterial, and anti-inflammatory activities [4, 5]. Recently, the study on its bioactive ingredients and molecular mechanism of action has been gradually reported as well [6].Although some preliminary analytical methods have been developed for the quality control for LQC, including thin layer chromatography (TLC) [7], high performance liquid chromatography (HPLC) [8, 9], micellar electrokinetic capillary chromatography (MEKC) [10], and liquid chromatography tandem mass spectrometry (LC-MS/MS) [11], no systematical and comprehensive study on the chemical profiling and quality control method for LQC has been reported so far. For a classical and complex Chinese medical preparation, the comprehensive quality evaluation method should be based on its multiple chemical constituents. Therefore, it is necessary to develop a rapid and sensitive method to identify and quantify the chemical constituents in LQC, which will be beneficial to investigate the effectiveness and evaluate the quality of LQC.In this study, a reliable, sensitive, and simple ultraperformance liquid chromatography coupled with diode-array detector and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF-MS) method which was more systematical and comprehensive than the earlier ones was established for characterization and quantification of the major chemical constituents of LQC. A total of 61 compounds were unambiguously or tentatively identified by comparing the retention times, exact molecular masses, and MS/MS spectral data with reference compounds or literature data. Furthermore, twenty-seven compounds were confirmed by comparing with the standards. Among them, twelve representative compounds were quantified as chemical markers in quantitative analysis, including salidroside, chlorogenic acid, forsythoside E, cryptochlorogenic acid, amygdalin, sweroside, hyperin, rutin, forsythoside A, phillyrin, rhein, and glycyrrhizic acid. This is the first systematical and comprehensive study on the qualitative and quantitative analysis of LQC.


## 2. Experimental

### 2.1. Reagents, Chemicals, and Materials

Methanol and acetonitrile (HPLC grade) were purchased from Sigma Aldrich (St. Louis, MO, USA). Formic acid (HPLC grade) was purchased from Tianjin Damao chemical reagent factory (Tianjin, China). Water (HPLC grade) for UPLC analysis was produced by the Milli-Q water purification system (Millipore, USA). Salidroside, chlorogenic acid, forsythoside E, cryptochlorogenic acid, amygdalin, sweroside, hyperin, rutin, forsythoside A, phillyrin, rhein, and glycyrrhizic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). The purity of standard substances was above 98%. Ten batches of LQC were provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, China).


### 2.2. UPLC Analysis

The UPLC analysis was performed on a Waters ACQUITY UPLC instrument (Waters Corporation, MA, USA) coupled with a binary pump, a sample manager, an autosampler, a column compartment, and diode-array detector (DAD). The separation of samples was performed on a Waters ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 μm) column with the column temperature at 50°C. The analysis was completed in 30 min with a gradient elution of 0.1% formic acid aqueous solution (A) and methanol (B) at the flow rate of 0.3 mL/min. The gradient program was designed as follows: 0–11 min, 5–35% B; 11–18 min, 35–55% B; 18–22 min, 55–75% B; 22–24 min, 75–90% B; 24-25 min, 90–100% B; and 25–30 min, 100% B. The injection volume was 5 μL. The detection wavelengths of DAD were set at 210 nm, 225 nm, and 254 nm.


### 2.3. UPLC-DAD-QTOF-MS Analysis

The Waters ACQUITY UPLC instrument (Waters, MA, USA) coupled with Waters Synapt HDMS G1 (Waters, Manchester, UK) via an electrospray ionization (ESI) interface. The UPLC analytical conditions were the same as the UPLC analysis described above. The full scan mass spectra data were acquired in positive and negative ion modes. Acquisition parameters are as follows: capillary voltage was 3000 V for ESI (+) and 2600 V for ESI (−); cone voltage was 45 V; the ESI source temperature was 100°C; the desolvation temperature was 350°C; the nitrogen (N2) was used as desolvation gas at flow rates of 600 L/h for both ESI (+) and ESI (−); and the range of full scan was set atm/z 150–1000 Da. The version of analysis software was Mass Lynx V4.1.


### 2.4. Sample and Standard Solutions Preparation

The powder of LQC (0.4 g) was accurately weighed and extracted with 60% methanol-water (v/v) solution (20 mL) in an ultrasonic water bath for 30 min at room temperature. The supernatant solution was diluted with the same amount of water and then centrifuged for 10 min at 14,000 r/min. All the obtained solutions were filtered through 0.22μm syringe filter before the UPLC analysis.Twelve standards were accurately weighed and dissolved in methanol to obtain stock solutions, respectively. A mixed stock solution of standards was prepared by adding a suitable volume of each stock solution to a 5 mL flask and diluted with 30% methanol-water solution at the concentration of 67.8μg/mL for salidroside, 109.65 μg/mL for chlorogenic acid, 77.64 μg/mL for forsythoside E, 106.47 μg/mL for cryptochlorogenic acid, 62.57 μg/mL for amygdalin, 31.96 μg/mL for sweroside, 3.21 μg/mL for hyperin, 8.5 μg/mL for rutin, 67.34 μg/mL for forsythoside A, 45.71 μg/mL for phillyrin, 55.49 μg/mL for rhein, and 84.35 μg/mL for glycyrrhizic acid, respectively. The mixed stock solution was then serially diluted with 30% methanol-water solution to obtain five appropriate concentrations used for plotting standard curves. The lowest concentration of the mixture stock solution was further diluted to give a series of different concentrations for investigating the limits of detection (LODs) and limits of quantification (LOQs) of the 12 chemical constituents. All solutions were stored at 4°C until analysis.


### 2.5. Validation of the Quantitative Analysis

The UPLC-DAD method was evaluated with linearity, LOD, LOQ, precision, stability, repeatability, and recovery tests. The calibration curves were constructed with five different concentrations of chemical markers in triplicate. The LODs and LOQs were measured under the UPLC analytical conditions at a signal-to-noise (S/N) ratio of 3 and 10, respectively. For intraday and interday precisions test, the samples were analyzed by six repetitive injections within one day and once a day for three successive days, respectively. At room temperature, the stability of sample solution was evaluated by replicate injection at 0, 1, 2, 4, 6, 8, 10, 14, 24, and 48 h. In order to check the repeatability, six samples from the same source were investigated. Accurate amounts of the reference standards were added to 0.20 g powder of sample in sextuplicate. The resultant sample solutions were then extracted and quantified with the described method. The relative standard deviation (RSD) was used to evaluate the results.


## 2.1. Reagents, Chemicals, and Materials

Methanol and acetonitrile (HPLC grade) were purchased from Sigma Aldrich (St. Louis, MO, USA). Formic acid (HPLC grade) was purchased from Tianjin Damao chemical reagent factory (Tianjin, China). Water (HPLC grade) for UPLC analysis was produced by the Milli-Q water purification system (Millipore, USA). Salidroside, chlorogenic acid, forsythoside E, cryptochlorogenic acid, amygdalin, sweroside, hyperin, rutin, forsythoside A, phillyrin, rhein, and glycyrrhizic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). The purity of standard substances was above 98%. Ten batches of LQC were provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, China).


## 2.2. UPLC Analysis

The UPLC analysis was performed on a Waters ACQUITY UPLC instrument (Waters Corporation, MA, USA) coupled with a binary pump, a sample manager, an autosampler, a column compartment, and diode-array detector (DAD). The separation of samples was performed on a Waters ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 μm) column with the column temperature at 50°C. The analysis was completed in 30 min with a gradient elution of 0.1% formic acid aqueous solution (A) and methanol (B) at the flow rate of 0.3 mL/min. The gradient program was designed as follows: 0–11 min, 5–35% B; 11–18 min, 35–55% B; 18–22 min, 55–75% B; 22–24 min, 75–90% B; 24-25 min, 90–100% B; and 25–30 min, 100% B. The injection volume was 5 μL. The detection wavelengths of DAD were set at 210 nm, 225 nm, and 254 nm.


## 2.3. UPLC-DAD-QTOF-MS Analysis

The Waters ACQUITY UPLC instrument (Waters, MA, USA) coupled with Waters Synapt HDMS G1 (Waters, Manchester, UK) via an electrospray ionization (ESI) interface. The UPLC analytical conditions were the same as the UPLC analysis described above. The full scan mass spectra data were acquired in positive and negative ion modes. Acquisition parameters are as follows: capillary voltage was 3000 V for ESI (+) and 2600 V for ESI (−); cone voltage was 45 V; the ESI source temperature was 100°C; the desolvation temperature was 350°C; the nitrogen (N2) was used as desolvation gas at flow rates of 600 L/h for both ESI (+) and ESI (−); and the range of full scan was set atm/z 150–1000 Da. The version of analysis software was Mass Lynx V4.1.


## 2.4. Sample and Standard Solutions Preparation

The powder of LQC (0.4 g) was accurately weighed and extracted with 60% methanol-water (v/v) solution (20 mL) in an ultrasonic water bath for 30 min at room temperature. The supernatant solution was diluted with the same amount of water and then centrifuged for 10 min at 14,000 r/min. All the obtained solutions were filtered through 0.22μm syringe filter before the UPLC analysis.Twelve standards were accurately weighed and dissolved in methanol to obtain stock solutions, respectively. A mixed stock solution of standards was prepared by adding a suitable volume of each stock solution to a 5 mL flask and diluted with 30% methanol-water solution at the concentration of 67.8μg/mL for salidroside, 109.65 μg/mL for chlorogenic acid, 77.64 μg/mL for forsythoside E, 106.47 μg/mL for cryptochlorogenic acid, 62.57 μg/mL for amygdalin, 31.96 μg/mL for sweroside, 3.21 μg/mL for hyperin, 8.5 μg/mL for rutin, 67.34 μg/mL for forsythoside A, 45.71 μg/mL for phillyrin, 55.49 μg/mL for rhein, and 84.35 μg/mL for glycyrrhizic acid, respectively. The mixed stock solution was then serially diluted with 30% methanol-water solution to obtain five appropriate concentrations used for plotting standard curves. The lowest concentration of the mixture stock solution was further diluted to give a series of different concentrations for investigating the limits of detection (LODs) and limits of quantification (LOQs) of the 12 chemical constituents. All solutions were stored at 4°C until analysis.


## 2.5. Validation of the Quantitative Analysis

The UPLC-DAD method was evaluated with linearity, LOD, LOQ, precision, stability, repeatability, and recovery tests. The calibration curves were constructed with five different concentrations of chemical markers in triplicate. The LODs and LOQs were measured under the UPLC analytical conditions at a signal-to-noise (S/N) ratio of 3 and 10, respectively. For intraday and interday precisions test, the samples were analyzed by six repetitive injections within one day and once a day for three successive days, respectively. At room temperature, the stability of sample solution was evaluated by replicate injection at 0, 1, 2, 4, 6, 8, 10, 14, 24, and 48 h. In order to check the repeatability, six samples from the same source were investigated. Accurate amounts of the reference standards were added to 0.20 g powder of sample in sextuplicate. The resultant sample solutions were then extracted and quantified with the described method. The relative standard deviation (RSD) was used to evaluate the results.


## 3. Results and Discussion

### 3.1. Optimization of the Extraction and Chromatographic Conditions

A single-factor method was used to investigate the extraction effect of the extraction solvent (30%, 60%, and 90% methanol-water solution), extraction solvent ratio (1 : 50, 1 : 100, and 1 : 200 (w/v)), and extraction time (15 min, 30 min, and 45 min), respectively. By analyzing the extraction efficiency, 60% methanol-water solution, extraction solvent ratio at 1 : 100, and 30 min of ultrasonic time were selected as the eventual extraction conditions. The results are described in Table1.Table 1
Quantitative results of 12 compounds in LQC extracted by different methods.
Content (μg/g)
Methods
30%a
60%
90%
60%
60%
60%
60%
60%
60%
30 minb
30 min
30 min
30 min
30 min
30 min
15 min
30 min
45 min
1 : 100c
1 : 100
1 : 100
1 : 50
1 : 100
1 : 200
1 : 100
1 : 100
1 : 100
Salidroside
1726.28
1701.25
1622.02
1522.31
1701.25
1656.32
1711.54
1701.25
1688.54
Chlorogenic acid
2444.97
2492.15
2216.89
2285.43
2492.15
2289.27
2492.62
2492.15
2552.17
Forsythoside E
1583.93
1620.78
451.91
1462.09
1620.78
1402.53
1627.01
1620.78
1579.63
Cryptochlorogenic acid
1862.98
1851.64
667.50
1703.05
1851.64
1771.79
1837.89
1851.64
1857.95
Amygdalin
1424.11
1455.39
1442.56
1268.93
1455.39
1298.11
1395.97
1455.39
1431.38
Sweroside
816.19
813.18
789.32
747.06
813.18
772.67
812.51
813.18
808.24
Hyperin
135.10
151.73
167.26
140.84
151.73
140.80
152.72
151.73
157.67
Rutin
122.00
121.17
115.22
106.11
121.17
116.67
117.09
121.17
116.62
Forsythoside A
2484.60
2536.34
2661.79
2285.76
2536.34
2396.35
2543.87
2536.34
2521.10
Phillyrin
1660.26
1521.45
1551.59
1410.19
1521.45
1390.12
1523.01
1521.45
1551.52
Rhein
803.13
1102.06
1370.11
937.63
1102.06
932.05
956.38
1102.06
1054.77
Glycyrrhizic acid
1530.49
1680.43
1594.37
1437.40
1680.43
1619.99
1674.81
1680.43
1665.42
aExtracting solvent: 30%, 60%, and 90% methanol-water solution.
bUltrasonic time: 15 min, 30 min, and 45 min.
cExtraction solvent multiples: 1 : 50, 1 : 100, and 1 : 200 expressed 50, 100, and 200 times per gram of sample.Due to the existence of acidic constituents in sample solutions, formic acid was added into the mobile phase which could inhibit the ionization of these acidic ingredients to improve the peak shape. The mobile phase systems (methanol-formic acid aqueous solution and acetonitrile-formic acid aqueous solution) and column temperature (40°C and 50°C) were investigated, which showed that methanol-0.1% formic acid aqueous solution as mobile phase with column temperature at 50°C could obtain the best chromatographic peak shape. Because the maximum absorptions of 12 reference compounds were different, three detection wavelengths were finally selected in order to achieve the goal of high detection sensitivity and little interference. Forsythoside E (peak 7), cryptochlorogenic acid (peak 9), amygdalin (peak 33), and phillyrin (peak 39) had satisfactory sensitivity at 210 nm, salidroside (peak 6), chlorogenic acid (peak 8), and rhein (peak 54) at 225 nm, and sweroside (peak 13), hyperin (peak 26), rutin (peak 29), forsythoside A (peak 30), and glycyrrhizic acid (peak 57) at 254 nm. The chromatograms are presented in Figure1.UPLC-DAD chromatograms of standard solution of 210 nm (a), 225 nm (b), 254 nm (c), negative sample solution (d), and sample solution of 210 nm (e), 225 nm (f), and 254 nm (g).
(a)
(b)
(c)
(d)
(e)
(f)
(g)


### 3.2. UPLC-DAD-QTOF-MS Analysis of Reference Compounds and LQC Samples

As shown in Table2, a total of 61 compounds were unambiguously or tentatively identified by comparing the retention times and accurate mass measurement with references or literature data. These compounds were divided into six types according to their structural characteristics including flavonoids, phenylpropanoids, anthraquinones, triterpenoids, iridoids, and other types. The structures of identified compounds are listed in Figure 3. Among them, twenty-seven compounds were further confirmed by comparing with standards. The total ion chromatograms are shown in Figure 2.Table 2
Characterization of compounds in LQC by UPLC-DAD-QTOF-MS.
Peaknumber
T
R (min)
λ
max
⁡ (nm)
Formula
ES− (m/z)
ES+ (m/z)
Identification
Reference
[M–H]− (ppm)
MS2b
[M + H]+(ppm)
[M + Na]+
MS2b
1
0.817
C7H12O6
191.0555 (−0.5)
Quinic acid
[12]
2
0.982
223, 287
C6H8O7
191.0198 (3.1)
173.0099 [M–H–H2O]−, 111.0088 [M–H–2H2O–CO2]−
Citric acid
[13]
3
a
1.545
216, 271
C7H6O5
169.0142 (3.0)
125.0241 [M–H–CO2]−
Gallic acid
[14]
4
a
3.010
325
C16H17O9
353.0878 (1.4)375.0695 [M–2H + Na]−, 707.1830 [2M–H]−
191.0497 [M–H–Ca]−, 179.0312 [Caffeic acid–H]−, 135.0359 [Caffeic acid–H–CO2]−
Neochlorogenic acid
[13]
5
a
3.639
254
C16H24O10
375.1302 (2.9),751.2667 [2M–H]−
213.0773 [M–H–Glc]−, 151.0878 [M–H–Glc–CO2–H2O]−, 125.0721 [M–H–Glc–2CO2]−
Loganic acid
[15]
6
a
4.048
224,278
C14H20O7
299.1133 (0.7)
179.0447 [M–H–C8H8O]−, 119.0246 [M–H–Glc–H2O]−
Salidroside
[16]
7
a
4.557
C20H30O12
461.1674 (3.3)
315.1348 [M–H–Rha]−, 297.1589 [M–H–Rha–H2O]−, 153.0644 [M–H–Rha–Glc]−, 135.0573 [M–H–Rha–Glc–H2O]−
463.1220 (−4.3)
485.1568
137.0599 [M + H–Rha–Glc–H2O]+
Forsythoside E
[17]
8
a
4.749
327
C16H17O9
353.0880 (2.0)
191.0556 [M–H–Ca]−, 179.0224 [Caffeic acid–H]−
355.1028 (−0.3)
377.0857
Chlorogenic acid
[18]
9
a
5.281
325
C16H17O9
353.0880 (2.0)
179.0348 [Caffeic acid–H]−, 173.0461 [Quinic acid–H–H2O]−, 191.0417 [M–H–Ca]−
Cryptochlorogenic acid
[18]
10
6.435
270
C17H26O10
389.1096 (3.1)
391.2176 (−0.8)
413.1067
395.2354 [M + Na–H2O]+ 229.0835 [M + H–Glc]+
Loganin
[19]
11
6.749
C14H23O9
335.0776 (2.7)
173.0459 [M–H–Glc]−, 161.0373 [Glc–H–H2O]−, 133.0408 [Glc–H–H2O–CO]−
337.0883 (5.3)
359.0686
163.0419 [Glc + H–H2O]+
Rengynic acid-1′-O-β-D-glucoside
[20]
12
7.222
C21H20O9
415.1261 (5.1)
439.1207
255.0879 [M + H–Glc]+
Isomer of chrysophanolglucoside
[21]
13
a
7.428
245
C
16
H
22
O
9
357.1208 (6.2), 403.1263[M + HCOO]-, 393.0938 [M + Cl]-
359.1337 (−1.4)
381.1154
Sweroside
[15]
14
7.859
C14H23O9
335.0871 (4.2)
193.0506, 161.0377, 133.0423
337.0774 (0.9)
359.0225
163.0416 [Glc + H–H2O]+
Isomer of rengynic acid-1′-O-β-D-glucoside
15
8.106
306
C20H22O8
389.1458 (2.6), 435.1519 [M + HCOO]−
227.0943 [M–H–Glc]−
Polydatin
[22]
16
8.222
C29H36O16
639.2219 (−1.7)
477.1894 [M–H–Glc]−, 179.0546 [M–Rha–C17H14O6]−, 161.0328 [Glc–H–H2O]−,
R-suspensaside
[23]
17
a
8.320
C21H20O9
415.1253 (3.1)
253.0542 [M–H–Glc]−
417.1394 (−0.7)
439.1200
255.0864 [M + H–Glc]+
Chrysophanol glucoside
[21]
18
8.874
C29H36O16
639.2002 (−2.7)
161.0328 [Glc–H–H2O]−, 179.0349 [M–Rha–C17H14O6]−
S-suspensaside
[24]
19
9.054
C29H36O15
623.1999 (−1.2)
311.2176
625.2137 (0.8)
647.1942
479.1562, 471.1479, 325.0925, 163.0394
Isomer of forsythoside A
20
a
9.470
235
C17H24O11
403.1261 (5.2)
405.1331 (−1.7)
427.1203
243.0871 [M + H–Glc]+
Secoxyloganin
[19]
21
9.552
276, 313
C21H22O9
417.1205 (4.6), 835.2331 [2M–H]−
255.0652 [M–H–Glc]−,
441.1132
257.0811 [M + H–Glc]+
Liquiritin
[25]
22
a
9.886
277, 312
C26H30O13
549.1627 (3.5)
551.2823 (4.7)
573.1737
257.0822 [M + H–Api–Glc]+
Liquiritin apioside
[26]
23
9.995
C26H30O13
549.1629 (3.8)
551.3403 (−5.1)
573.1735
257.0815 [M + H–Api–Glc]+
Isomer of liquiritin apioside
[26]
24
10.088
C27H30O16
609.1844 (−1.8)
633.1712
Rhodiosin
[27]
25
a
10.384
250, 300
C22H22O9
429.1410 (3.0)
453.1176
269.0974 [M + H–Glc]+
Ononin
[28, 29]
26
a
10.722
360
C21H20O12
463.0905 (2.8)
301.0464 [M–H–Glc]−
465.1033 (5.2)
487.0333
303.0515 [M + H–Glc]+
Hyperin
[24]
27
a
10.819
326
C25H24O12
515.1205 (2.9)
353.0751 [M–H–Ca]−
517.1296 (1.7)
539.1156
499.1187 [M + H–H2O]+, 163.0395 [M + H–Ca–Quinic acid]+
3,5-Dicaffeoylquinic acid
[30]
28
10.931
C29H36O15
623.1999 (−1.9)
461.1378, 161.0185
647.1941
471.1497, 325.0925, 163.0415
Isomer of forsythoside A
29
a
11.10
256, 354
C27H30O16
609.1465 (1.5)
611.1573 (3.3)
633.1404
465.0982 [M + H–Rha]+ 303.0495 [M + H–Rut]+
Rutin
[31]
30
a
11.220
280
C29H36O15
623.2001 (−1.6)
461.1920 [M–H–Ca]−, 161.0328 [Caffeic acid–H–H2O]−
647.1927
163.0386 [Caffeic acid + H–H2O]+
Forsythoside A
[32]
31
11.589
283
C27H30O15
593.1534 (−1.3)
285.0677 [M–H–Rut]−
595.1694 (−0.7)
287.0936 [M + H–Rut]+
Kaempferol-3-O-rutinoside
[13]
32
11.692
C19H35O16
519.1884 (3.5), 565.1953 [M + HCOO]−
357.1366 [M–H–Glc]−
543.1855
359.1454 [M + H–Glc]+, 341.1411 [M + H–Glc–H2O]+
(+)-Pinoresinol-β-D-glucoside
[33]
33
a
12.213
210, 262
C20H27NO11
456.1523 (3.7)
458.1641 (−4.6)
296.1124 [M + H–Glc]+, 162.0862 [M + H–Glc–HCT]+
Amygdalin
[13]
34
a
12.659
326
C25H24O12
515.1162 (−5.4)
353.0786 [M–H–Ca]−
517.1348 (0.4)
539.1193
499.1248 [M + H–H2O]+, 163.0387 [M + H–Ca–Quinic acid]+
3,4-Dicaffeoylquinic acid
[30]
35
a
13.082
276
C15H12O4
255.0654 (−1.2)
257.0659 (−0.8)
147.0621 [M + H–RL]+, 137.0422 [M + H–VP]+, 119.0691 [M + H–RL–CO]+
Liquiritigenin
[34]
36
a
13.251
243, 372
C26H30O13
549.1621 (2.4)
551.1725 (3.4)
573.1563
419.1360 [M + H–Api]+, 257.0814 [M + H–Api–Glc]+
Isoliquiritin apioside
[35]
37
a
13.300
242, 362
C21H22O9
417.1198 (2.9)
255.0518 [M–H–Glc]−
419.1314 (−6.7)
441.1150
257.0823 [M + H–Glc]+, 229.1357 [M + H–Glc–CO]+
Isoliquiritin
[26]
38
13.479
280
C27H34O11
579.2103 [M + HCOO]−
557.1987
552.2463 [M + NH4]+, 355.1516 [M + H–Glc–H2O]+
Arctiin
[36]
39
a
14.161
277
C27H34O11
579.2106[M + HCOO]−
371.1826 [M–H–Glc]−, 356.1547 [M–H–Glc–H2O]−
552.2444[M + NH4]+
557.1991
373.1563 [M + H–Glc]+, 355.1544 [M + H–Glc–H2O]+
Phillyrin
[37, 38]
40
14.678
372, 255
C15H10O7
301.0355 (2.3)
151.0137 [M–H–C8H6O3]−
303.0520 (4.9)
Quercetin
[39]
41
15.039
284
C28H34O15
609.1522 (1.3)
301.0582 [M–H–Rut]−
Hesperidin
[13]
42
15.402
265, 366
C15H10O6
285.0406 (2.5)
287.0721 (4.5)
153.0469 [M + H–HEP]+
Kaempferol
[13]
43
16.627
255
C15H10O4
253.0507 (2.4)
225.0741 [M–H–CO]−, 210.0494 [M–H–CO–CH3]−, 182.0542 [M–H–2CO–CH3]−, 154.0553 [M–H–3CO–CH3]−
Chrysophanol
[40]
44
17.035
C15H10O4
253.0512 (4.3)
225.0752, 210.0474, 182.0485,154.0531
Isomer of chrysophanol
[40]
45
a
17.347
254, 287, 426
C21H20O10
431.0989 (2.6)
269.0673 [M–H–Glc]−, 241.0719 [M–H–Glc–CO]−, 225.0753 [M–H–Glc–CO2]−, 210.0578 [M–H–Glc–CO2–CH3]−, 197.0771 [M–H–Glc–CO–CO2]−, 182.0486 [M–H–Glc–CO2–CH3–CO]−
455.1010
271.0715 [M + H–Glc]+
Emodin-8-O-glucoside
[41]
46
17.396
240, 330, 395
C15H12O4
255.0639 (−7.1)
257.0812 (−0.8)
Isoliquiritigenin
[26]
47
a
18.385
250, 304
C16H12O4
267.0653 (−1.5)
269.0827 (4.8)
291.0751
Formononetin
[42]
48
18.804
895.4082 (2.8)
719.3849 [M–H–GlcA]− 351.0630 [2GlcA–H]−
897.4107 (1.2)
919.3925
22β-Acetoxy licorice saponin B2/uralsaponin F
[43]
49
19.194
271, 421
C
22
H
2
2
O
10
445.1168 (−5.6)
283.0632 [M–H–Glc]−
469.1128
285.0775 [M + H–Glc]+
Physcion-8-O-β-D-glucopyranoside
[41]
50
19.603
248
C42H60O16
819.3843 (0.6)
351.0699 [2GlcA–H]−
821.3994 (−0.1)
843.3996
645.3612 [M + H–GlcA]+, 469.3302 [M + H–2GlcA]+, 451.3211 [M + H–2GlcA–H2O]+
Licorice saponin E2
[35]
51
19.704
C16H24O7
327.2180 (2.8)
164.9664 [M–H–Glc]−
351.2133
Rhododendrol-4′-O-β-D-glucopyranoside
[44]
52
20.000
254
C44H64O18
879.4062 (1.3)
351.0806 [M–H–22AG]−
881.4170 (−0.1)
705.3897 [M + H–GlcA]+, 529.3588 [M + H–2GlcA]+, 511.3395 [M + H–2GlcA–H2O]+
22-Acetoxyglycyrrhizin
[42]
53
20.573
352
C17H14O7
329.2341 (3.9)
353.2280
301.1443 [M + H–CH2O]+, 287.2002 [M + H–CO2]+, 259.1248 [M + H–CO2–CO]+
Tricin
[45]
54
a
21.199
260
C15H8O6
283.0262 (6.7)
239.0359 [M–H–CO2]−
285.0515 (−13.0)
Rhein
[46]
55
21.540
253
837.3977 (1.2)
819.4404 [M–H–H2O]−, 661.4100 [M–H–GlcA]−, 351.0815 [2GlcA–H]−, 289.0840 [2GlcA–H–H2O–CO2]−
839.4061 (−0.5)
861.3890
663.3868 [M + H–GlcA]+, 487.3395 [M + H–2GlcA]+, 469.3286 [M + H–2GlcA–H2O]+
Licorice saponin G2
[42]
56
21.626
236, 327, 370, 384
C21H20O6
367.1208 (7.1)
309.1566 [M–H–CO–CH2O]−, 265.0635 [M–H–CO–CH2O–CO2]−, 221.0221 [M–H–CO–CH2O–2CO2]−
369.1369 (−7.6)
391.1176
Glycycoumarin
[47]
57
22.087
249
C42H62O16
821.3977 (1.7)
803.4501 [M–H–H2O]−, 759.4542 [M–H–H2O–CO2]−, 645.4102 [M–H–GlcA]−, 627.4000 [M–H–GlcA–H2O]−, 469.3705 [M–H–2GlcA]−, 351.0809 [2GlcA–H]−, 289.0758 [2GlcA–H–H2O–CO2]−
823.4128 (1.5)
845.4557
647.3797 [M + H–GlcA]+, 471.3480 [M + H–2GlcA]+, 453.3365 [M + H–2GlcA–H2O]+
Glycyrrhizic acid
[48]
58
22.188
252
C42H62O16
821.3986 (−1.1)
351.0768 [M–H–GA]−
823.4134 (−2.1)
845.4127
647.3823 [M + H–GlcA]+, 471.3365 [M + H–2GlcA]+, 453.3324 [M + H–2GlcA–H2O]+
Licorice saponin H2
[26]
59
22.389
248
C42H64O15
807.4197 (−0.6)
745.4601 [M–H–Glc]−, 631.4210 [M–H–GlcA]−, 351.0699 [M–H–GA]−
809.4410 (−0.9)
831.4138
Licorice saponin B2
[49]
60
22.623
252
C42H62O16
821.3997 (0.2)
351.0837 [M–H–GA]−
823.4105 (−1.3)
845.3900
647.3838 [M + H–GlcA]+, 471.3288 [M + H–2GlcA]+, 453.3279 [M + H–2GlcA–H2O]+
Licorice saponin K2
[50]
61
a
23.230
288, 439
C15H10O5
269.0462 (4.5)
241.0669 [M–H–CO]−, 225.0723 [M–H–CO2]−, 210.0427 [M–H–CO2–CH3]−, 197.0695 [M–H–CO–CO2]−, 182.0477 [M–H–CO2–CH3–CO]−, 154.0552 [M–H–CO2–CH3–2CO]−
Emodin
[40]
aCompared with reference compounds.
b22AG: 22-acetoxyglycyrrhizin; Api: apiose; Ca: caffeoyl; GA: glycyrrhetinic acid; Glc: glucose; GlcA: glucuronic acid; HCT: 4-(hydroxymethyl)cyclobutane-1,2,3-triol; HEP: 4-(hydroxyethynyl) phenol; Rha: rhamnose; RL: resorcinol; Rut: rutinose; VP: 4-vinylphenol.Figure 2
UPLC-QTOF-MS chromatograms of sample solution from negative ion mode and positive ion mode.Chemical structures of the compounds identified from LQC (except for the 7 isomers).
(a)
(b)
(c)
(d)
(e)
(f)


#### 3.2.1. Flavonoids

Seventeen flavonoids (Figure3(a)) in LQC including flavone aglycones and glycosides were identified. They were mainly obtained from Lianqiao, Jinyinhua, Gancao, Hongjingtian, and Mahuang. Amongst them, liquiritin apioside (22), ononin (25), hyperin (26), rutin (29), liquiritigenin (35), isoliquiritin apioside (36), isoliquiritin (37), and formononetin (47) were unambiguously identified via the standards.In negative and positive ion modes, flavone aglycones mainly gain fragment ions by the reverse Diels-Alder (RDA) reaction and the loss of CO (28 Da). The characteristic fragmentation behavior of compound35 is shown in Figure 4(a) with high abundant fragmentation [M+H–VP (4-vinylphenol)]+ atm/z 137.0422. The abundance of fragment ions [M+H–RL (resorcinol)]+ atm/z 147.0621 and [M+H–RL–CO]+ atm/z 119.0691 is relatively lower. Compounds24,40,42, and46were tentatively identified via comparing their exact molecular masses, MS/MS spectra data, and retention behaviors with literature data [13, 26, 39]. Flavone glycosides have the similar fragmentation pathways of simultaneous or successive loss of glucose (162 Da), rhamnose (146 Da), or apiose (132 Da). The fragmentation pathway of compound26 was exemplified in Figure 4(b) in negative ion mode. Compounds21,23,31,41, and53were tentatively identified by comparing the molecular mass and MS/MS data with literature data [25, 45].The MS spectra and fragmentation pathway of compound35 (a), compound26 (b), compound8 (c), compound30 (d), compound39 (e), compound61 (f), compound45 (g), compound57 (h), and compound5 (i).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)


#### 3.2.2. Phenylpropanoids

Fourteen phenylpropanoids (Figure3(b)) including phenylpropionic acids, lignans, and coumarins were identified in LQC. They were mainly obtained from Lianqiao, Jinyinhua, and Gancao. Neochlorogenic acid (4), chlorogenic acid (8), cryptochlorogenic acid (9), 3,5-dicaffeoylquinic acid (27), forsythoside A (30), 3,4-dicaffeoylquinic acid (34), and phillyrin (39) were unambiguously characterized by comparing with standards.In negative ion mode, phenylpropionic acids have the similar fragmentation pathways of simultaneous or successive loss of H2O (18 Da), CO (28 Da), and CO2 (44 Da). The fragmentation pathway of compound8[18], as the representative of phenylpropionic acids, is shown in Figure 4(c). Compound30 produced [M–H–Ca (caffeoyl)]− atm/z 461.1920 and [Caffeic–H–H2O]− atm/z 161.0328, as displayed in Figure 4(d). Lignans primarily generated [M+HCOO]− in negative ion mode and further elimination of glucose (162 Da) produced aglycone. As shown in Figure 4(e), compound39produced characteristic fragments atm/z 371.1826 andm/z 356.1547 corresponding to [M–H–Glc (glucose)]− and [M–H–Glc–CH3]−, respectively. Compounds16,18,19,28,32,38,and56were tentatively identified on the basis of the exact molecular formulae matching, fragmentation information, and retention behaviors as well as literature data [23, 24, 33, 36, 47].


#### 3.2.3. Anthraquinones

Eight anthraquinones (Figure3(c)) in LQC were definitely or tentatively identified. All of them were derived from Dahuang. Chrysophanol glucoside (17), emodin-8-O-glucoside (45), rhein (54), and emodin (61) were confirmed via comparing with standard substances.The characteristic fragmentation behavior of anthraquinones was the loss of CO2 (44 Da), CH3 (15 Da), and CO (28 Da) in negative ion mode. Typical compound61was used to explain the fragmentation pathway of anthraquinones presented in Figure 4(f). Compound45, as shown in Figure 4(g), produced characteristic fragments atm/z 269.0673,m/z 241.0719,m/z 225.0753,m/z 197.0771, andm/z 182.0486 which corresponded to [M–H–Glc]−, [M–H–Glc–CO]−, [M–H–Glc–CO2]−, [M–H–CO–CO2]−, and [M–H–CO2–CH3–CO]−, respectively. Compounds12,43,44, and49 were tentatively identified by comparing their accurate molecular masses and MS/MS fragment data with literature data [21, 40, 41].


#### 3.2.4. Triterpenoids

Eight triterpenoids (Figure3(d)) in LQC were unambiguously or tentatively identified. All of them were derived from Gancao. Glycyrrhizic acid (57) was confirmed by comparing with standards. In negative ion mode, representative compound57yielded [M–H–H2O–CO2]− atm/z 759.4524, [M–H–GlcA (glucuronic acid)]− atm/z 645.4102, [M–H–2GlcA]− atm/z 469.3705, [2GlcA–H]− atm/z 351.0809, and [2GlcA–H–H2O–CO2]− atm/z 289.0758, as shown in Figure 4(h). Compounds48,50,52,55,58,59,and60were tentatively identified by comparing their exact molecular masses and MS/MS spectral data with the literature data [51].


#### 3.2.5. Iridoids

Four iridoids (Figure3(e)) in LQC including iridoid glycosides and secoiridoid glycosides were identified. All of them were derived from Jinyinhua. Loganic acid (5), sweroside (13), and secoxyloganin (20) were unambiguously identified via the standards.The fragment1,4F (fragment generated from the fracture of 1/4 bonds of iridoids) in the negative ion mode was identified as the characteristic fragment ion of iridoid glycosides. Meanwhile, iridoid glycosides would lose the functional groups such as H2O (18 Da), CO2 (44 Da), and glucose (162 Da). Typical compound5 gave [M–H–Glc–CO2–H2O]− atm/z 151.0878, [M–H–Glc–2CO2]− atm/z 125.0712, and [1,4F–H2O]− atm/z 93.0423 [52], as presented in Figure 4(i). Compound10 was tentatively identified by comparing its exact molecular mass and MS/MS spectral data with the literature data [19].


#### 3.2.6. Other Types of Compounds

Compounds1,2,11,14,15, and51were tentatively identified by comparing their exact molecular masses and MS/MS spectral data with the literature data except gallic acid (3), salidroside (6), forsythoside E (7), and amygdalin (33) which were identified via the standards (Figure 3(f)) [12, 53].


### 3.3. Methodological Validation of the Quantitative Analysis

As shown in Table3, twelve standards were of good linearity with high correlation coefficient values over 0.9993. The LODs and LOQs were 0.051–1.71 μg/mL and 0.16–5.69 μg/mL, independently. Twelve analytes in sample solution were stable at room temperature within 48 h with the RSD less than 2.76%. The RSD values of intraday and interday precisions were less than 1.10% and 2.92%, respectively. The RSD of repeatability was less than 2.39%. The average recovery rates of 12 compounds ranged from 92.99% to 103.95% with the RSD less than 3.54%. All the results showed that the assay was satisfactory with high accuracy, good reproducibility, and high sensitivity which were beneficial to the analytical investigation and quality control for LQC.Table 3
Linear regression, LODs and LOQs, intraday and interday precisions, repeatability, stability, and recovery for 12 compounds.
Compound
Regression equationa  (n=3)
R
2
Linear range (μg/mL)
LODb (μg/mL)
LOQc (μg/mL)
Intraday  (RSD, %) (n=6)
Interday  (RSD, %) (n=3)
Repeatability (n=6)
Stability(RSD, %)
Recovery (n = 6)
Mean (μg/g)
RSD (%)
Original(μg)
Spiked(μg)
Detected(μg)
Recovery(%)
RSD(%)
Salidroside
y
=
13980
x
-
11265
0.9999
5.27–84.35
1.06
3.50
0.12
0.26
1779.55
±
10.92
0.61
1.38
355.91
354.20
707.50
99.28
1.24
Chlorogenic acid
y
=
26510
x
+
3673.3
0.9998
6.85–109.56
1.71
5.69
0.77
2.12
2473.71
±
12.74
0.51
2.54
494.74
493.85
959.90
94.19
2.42
Forsythoside E
y
=
11793
x
+
5016.8
0.9995
4.85–77.64
1.21
4.04
0.67
2.25
1754.03
±
18.72
1.07
2.67
351.81
351.05
677.26
92.99
3.54
Cryptochlorogenic acid
y
=
21498
x
-
5018.9
0.9997
6.65–106.47
1.66
5.53
1.10
1.63
2029.49
±
20.65
1.02
2.46
405.90
406.60
784.00
93.10
1.43
Amygdalin
y
=
10530
x
-
3464.7
0.9998
3.91–62.57
0.98
3.26
0.79
0.93
1485.38
±
25.85
1.74
2.48
297.08
298.62
576.39
93.58
3.32
Sweroside
y
=
20593
x
-
6003.4
0.9999
2.00–31.96
0.25
0.80
0.11
0.33
798.06 ±2.93
0.37
0.48
159.61
159.51
325.43
103.95
0.41
Hyperin
y
=
27773
x
-
879.41
0.9999
0.20–3.21
0.051
0.16
0.78
0.62
92.03 ±0.80
0.87
0.83
18.41
18.24
37.07
102.29
1.82
Rutin
y
=
30768
x
-
3783.7
0.9993
0.53–8.50
0.072
0.21
0.41
2.24
184.15
±
3.06
1.66
1.57
36.83
36.81
72.50
96.90
2.34
Forsythoside A
y
=
8546.6
x
-
6024.5
0.9999
4.21–67.34
0.30
1.05
0.20
2.92
2484.99
±
18.43
0.74
1.95
497.00
495.95
997.33
100.89
1.49
Phillyrin
y
=
30390
x
+
5282.8
0.9999
2.86–45.71
0.71
2.36
0.79
1.63
1577.80
±
10.11
0.64
2.76
315.56
316.72
623.99
100.25
1.60
Rhein
y
=
7473.6
x
-
5172.7
0.9996
3.47–55.49
0.87
2.88
0.40
0.28
681.92
±
16.32
2.39
0.97
136.38
136.27
273.48
100.61
1.61
Glycyrrhizic acid
y
=
10296
x
-
6531.3
0.9999
5.27–84.35
0.092
0.33
0.34
2.09
1622.75
±
17.59
1.08
2.15
324.55
323.939
652.16
101.31
3.27
a
y is the peak area; x is the concentration of standard solutions.
bLOD refers to the limits of detection, S/N=3.
cLOQ refers to the limits of quantity, S/N=10.


### 3.4. Sample Analysis

Twelve representative compounds in 10 batches of LQC were quantified through the developed UPLC-DAD analytical method described above. The results are summarized in Table4, which showed that the total concentrations of 12 quantitative compounds in different batches of the LQC varied narrowly; moreover, the 12 components differed greatly in their contents, which may be affected by the source of medicinal materials, the quality of the plant material, or the preparation technology. Among them, forsythoside A showed the highest amount (3164.55–2089.22 μg/g) followed by amygdalin (2594.75–1623.14 μg/g) and hyperin had the lowest amount at 100.80–62.56 μg/g.Table 4
Contents of the 12 compounds in 10 batches.
Sample
Compound (mean ± SD) (μg/g)
Total
RSD (%)
Salidroside
Chlorogenic acid
Forsythoside E
Cryptochlorogenic acid
Amygdalin
Sweroside
Hyperin
Rutin
Forsythoside A
Phillyrin
Rhein
Glycyrrhizic acid
Lot.1
1740.57
±
30.70
2159.11
±
12.47
1888.99
±
7.95
1464.88
±
10.22
2109.85
±
31.27
830.23
±
8.44
67.06
±
0.68
156.22
±
1.11
3163.84
±
33.14
1599.20
±
12.29
570.70
±
2.23
1028.45
±
4.21
16779.10
10.03
Lot.2
1697.04
±
12.54
1810.69
±
11.73
1424.67
±
38.04
1167.41
±
8.35
1623.14
±
25.14
650.95
±
7.21
68.07
±
1.37
106.85
±
2.82
2497.34
±
27.21
1220.59
±
35.57
554.26
±
8.27
837.16
±
11.65
13658.17
Lot.3
1456.36
±
22.08
1941.91
±
44.94
1477.93
±
29.66
1232.30
±
29.88
1641.25
±
34.31
681.53
±
6.35
72.66
±
2.05
110.31
±
2.16
2089.22
±
36.09
1314.99
±
31.21
617.91
±
10.70
904.75
±
15.73
13541.12
Lot.4
1520.75
±
8.91
1993.38
±
50.86
1501.09
±
42.60
1409.15
±
6.32
2147.01
±
60.37
743.98
±
18.00
62.56
±
1.28
100.86
±
2.39
2652.53
±
67.03
1139.47
±
24.08
696.42
±
18.76
985.60
±
22.85
14925.80
Lot.5
1593.12
±
35.00
2081.89
±
50.77
1626.62
±
48.29
1523.11
±
43.22
2282.76
±
46.19
777.98
±
4.18
75.89
±
1.69
121.06
±
2.41
3072.10
±
44.89
1477.18
±
25.48
686.35
±
16.44
984.79
±
29.13
16302.85
Lot.6
1821.34
±
48.55
2436.69
±
31.00
2025.28
±
43.31
1494.76
±
12.65
2377.04
±
63.64
860.29
±
13.37
100.80
±
1.87
145.77
±
2.94
3680.72
±
92.85
1991.09
±
18.29
648.86
±
3.91
1057.29
±
30.41
18639.93
Lot.7
1474.32
±
36.85
2222.61
±
38.27
1372.97
±
36.85
1727.36
±
16.40
2594.75
±
50.67
854.30
±
19.31
77.25
±
2.14
156.90
±
4.65
2987.54
±
36.76
1168.12
±
12.70
551.26
±
5.18
1227.74
±
20.24
16415.12
Lot.8
1598.66
±
19.51
2183.54
±
15.11
1589.79
±
19.71
1721.26
±
11.36
2494.81
±
19.61
824.71
±
4.47
81.26
±
0.69
174.62
±
0.86
3164.55
±
36.81
1277.14
±
11.04
716.94
±
5.74
1220.87
±
9.89
17048.15
Lot.9
1535.78
±
30.36
1961.72
±
22.33
1789.55
±
28.12
1247.34
±
36.59
1824.87
±
35.66
696.40
±
7.08
73.33
±
0.46
136.23
±
2.51
2823.09
±
15.19
1495.85
±
17.76
625.10
±
11.44
891.45
±
10.60
15100.71
Lot.10
1836.41
±
29.14
2216.36
±
8.18
1500.75
±
8.06
1670.72
±
23.19
2281.74
±
14.32
809.41
±
3.13
75.33
±
0.85
134.19
±
1.24
3010.34
±
12.64
1204.92
±
28.26
891.01
±
9.99
1176.72
±
6.68
16807.90


## 3.1. Optimization of the Extraction and Chromatographic Conditions

A single-factor method was used to investigate the extraction effect of the extraction solvent (30%, 60%, and 90% methanol-water solution), extraction solvent ratio (1 : 50, 1 : 100, and 1 : 200 (w/v)), and extraction time (15 min, 30 min, and 45 min), respectively. By analyzing the extraction efficiency, 60% methanol-water solution, extraction solvent ratio at 1 : 100, and 30 min of ultrasonic time were selected as the eventual extraction conditions. The results are described in Table1.Table 1
Quantitative results of 12 compounds in LQC extracted by different methods.
Content (μg/g)
Methods
30%a
60%
90%
60%
60%
60%
60%
60%
60%
30 minb
30 min
30 min
30 min
30 min
30 min
15 min
30 min
45 min
1 : 100c
1 : 100
1 : 100
1 : 50
1 : 100
1 : 200
1 : 100
1 : 100
1 : 100
Salidroside
1726.28
1701.25
1622.02
1522.31
1701.25
1656.32
1711.54
1701.25
1688.54
Chlorogenic acid
2444.97
2492.15
2216.89
2285.43
2492.15
2289.27
2492.62
2492.15
2552.17
Forsythoside E
1583.93
1620.78
451.91
1462.09
1620.78
1402.53
1627.01
1620.78
1579.63
Cryptochlorogenic acid
1862.98
1851.64
667.50
1703.05
1851.64
1771.79
1837.89
1851.64
1857.95
Amygdalin
1424.11
1455.39
1442.56
1268.93
1455.39
1298.11
1395.97
1455.39
1431.38
Sweroside
816.19
813.18
789.32
747.06
813.18
772.67
812.51
813.18
808.24
Hyperin
135.10
151.73
167.26
140.84
151.73
140.80
152.72
151.73
157.67
Rutin
122.00
121.17
115.22
106.11
121.17
116.67
117.09
121.17
116.62
Forsythoside A
2484.60
2536.34
2661.79
2285.76
2536.34
2396.35
2543.87
2536.34
2521.10
Phillyrin
1660.26
1521.45
1551.59
1410.19
1521.45
1390.12
1523.01
1521.45
1551.52
Rhein
803.13
1102.06
1370.11
937.63
1102.06
932.05
956.38
1102.06
1054.77
Glycyrrhizic acid
1530.49
1680.43
1594.37
1437.40
1680.43
1619.99
1674.81
1680.43
1665.42
aExtracting solvent: 30%, 60%, and 90% methanol-water solution.
bUltrasonic time: 15 min, 30 min, and 45 min.
cExtraction solvent multiples: 1 : 50, 1 : 100, and 1 : 200 expressed 50, 100, and 200 times per gram of sample.Due to the existence of acidic constituents in sample solutions, formic acid was added into the mobile phase which could inhibit the ionization of these acidic ingredients to improve the peak shape. The mobile phase systems (methanol-formic acid aqueous solution and acetonitrile-formic acid aqueous solution) and column temperature (40°C and 50°C) were investigated, which showed that methanol-0.1% formic acid aqueous solution as mobile phase with column temperature at 50°C could obtain the best chromatographic peak shape. Because the maximum absorptions of 12 reference compounds were different, three detection wavelengths were finally selected in order to achieve the goal of high detection sensitivity and little interference. Forsythoside E (peak 7), cryptochlorogenic acid (peak 9), amygdalin (peak 33), and phillyrin (peak 39) had satisfactory sensitivity at 210 nm, salidroside (peak 6), chlorogenic acid (peak 8), and rhein (peak 54) at 225 nm, and sweroside (peak 13), hyperin (peak 26), rutin (peak 29), forsythoside A (peak 30), and glycyrrhizic acid (peak 57) at 254 nm. The chromatograms are presented in Figure1.UPLC-DAD chromatograms of standard solution of 210 nm (a), 225 nm (b), 254 nm (c), negative sample solution (d), and sample solution of 210 nm (e), 225 nm (f), and 254 nm (g).
(a)
(b)
(c)
(d)
(e)
(f)
(g)


## 3.2. UPLC-DAD-QTOF-MS Analysis of Reference Compounds and LQC Samples

As shown in Table2, a total of 61 compounds were unambiguously or tentatively identified by comparing the retention times and accurate mass measurement with references or literature data. These compounds were divided into six types according to their structural characteristics including flavonoids, phenylpropanoids, anthraquinones, triterpenoids, iridoids, and other types. The structures of identified compounds are listed in Figure 3. Among them, twenty-seven compounds were further confirmed by comparing with standards. The total ion chromatograms are shown in Figure 2.Table 2
Characterization of compounds in LQC by UPLC-DAD-QTOF-MS.
Peaknumber
T
R (min)
λ
max
⁡ (nm)
Formula
ES− (m/z)
ES+ (m/z)
Identification
Reference
[M–H]− (ppm)
MS2b
[M + H]+(ppm)
[M + Na]+
MS2b
1
0.817
C7H12O6
191.0555 (−0.5)
Quinic acid
[12]
2
0.982
223, 287
C6H8O7
191.0198 (3.1)
173.0099 [M–H–H2O]−, 111.0088 [M–H–2H2O–CO2]−
Citric acid
[13]
3
a
1.545
216, 271
C7H6O5
169.0142 (3.0)
125.0241 [M–H–CO2]−
Gallic acid
[14]
4
a
3.010
325
C16H17O9
353.0878 (1.4)375.0695 [M–2H + Na]−, 707.1830 [2M–H]−
191.0497 [M–H–Ca]−, 179.0312 [Caffeic acid–H]−, 135.0359 [Caffeic acid–H–CO2]−
Neochlorogenic acid
[13]
5
a
3.639
254
C16H24O10
375.1302 (2.9),751.2667 [2M–H]−
213.0773 [M–H–Glc]−, 151.0878 [M–H–Glc–CO2–H2O]−, 125.0721 [M–H–Glc–2CO2]−
Loganic acid
[15]
6
a
4.048
224,278
C14H20O7
299.1133 (0.7)
179.0447 [M–H–C8H8O]−, 119.0246 [M–H–Glc–H2O]−
Salidroside
[16]
7
a
4.557
C20H30O12
461.1674 (3.3)
315.1348 [M–H–Rha]−, 297.1589 [M–H–Rha–H2O]−, 153.0644 [M–H–Rha–Glc]−, 135.0573 [M–H–Rha–Glc–H2O]−
463.1220 (−4.3)
485.1568
137.0599 [M + H–Rha–Glc–H2O]+
Forsythoside E
[17]
8
a
4.749
327
C16H17O9
353.0880 (2.0)
191.0556 [M–H–Ca]−, 179.0224 [Caffeic acid–H]−
355.1028 (−0.3)
377.0857
Chlorogenic acid
[18]
9
a
5.281
325
C16H17O9
353.0880 (2.0)
179.0348 [Caffeic acid–H]−, 173.0461 [Quinic acid–H–H2O]−, 191.0417 [M–H–Ca]−
Cryptochlorogenic acid
[18]
10
6.435
270
C17H26O10
389.1096 (3.1)
391.2176 (−0.8)
413.1067
395.2354 [M + Na–H2O]+ 229.0835 [M + H–Glc]+
Loganin
[19]
11
6.749
C14H23O9
335.0776 (2.7)
173.0459 [M–H–Glc]−, 161.0373 [Glc–H–H2O]−, 133.0408 [Glc–H–H2O–CO]−
337.0883 (5.3)
359.0686
163.0419 [Glc + H–H2O]+
Rengynic acid-1′-O-β-D-glucoside
[20]
12
7.222
C21H20O9
415.1261 (5.1)
439.1207
255.0879 [M + H–Glc]+
Isomer of chrysophanolglucoside
[21]
13
a
7.428
245
C
16
H
22
O
9
357.1208 (6.2), 403.1263[M + HCOO]-, 393.0938 [M + Cl]-
359.1337 (−1.4)
381.1154
Sweroside
[15]
14
7.859
C14H23O9
335.0871 (4.2)
193.0506, 161.0377, 133.0423
337.0774 (0.9)
359.0225
163.0416 [Glc + H–H2O]+
Isomer of rengynic acid-1′-O-β-D-glucoside
15
8.106
306
C20H22O8
389.1458 (2.6), 435.1519 [M + HCOO]−
227.0943 [M–H–Glc]−
Polydatin
[22]
16
8.222
C29H36O16
639.2219 (−1.7)
477.1894 [M–H–Glc]−, 179.0546 [M–Rha–C17H14O6]−, 161.0328 [Glc–H–H2O]−,
R-suspensaside
[23]
17
a
8.320
C21H20O9
415.1253 (3.1)
253.0542 [M–H–Glc]−
417.1394 (−0.7)
439.1200
255.0864 [M + H–Glc]+
Chrysophanol glucoside
[21]
18
8.874
C29H36O16
639.2002 (−2.7)
161.0328 [Glc–H–H2O]−, 179.0349 [M–Rha–C17H14O6]−
S-suspensaside
[24]
19
9.054
C29H36O15
623.1999 (−1.2)
311.2176
625.2137 (0.8)
647.1942
479.1562, 471.1479, 325.0925, 163.0394
Isomer of forsythoside A
20
a
9.470
235
C17H24O11
403.1261 (5.2)
405.1331 (−1.7)
427.1203
243.0871 [M + H–Glc]+
Secoxyloganin
[19]
21
9.552
276, 313
C21H22O9
417.1205 (4.6), 835.2331 [2M–H]−
255.0652 [M–H–Glc]−,
441.1132
257.0811 [M + H–Glc]+
Liquiritin
[25]
22
a
9.886
277, 312
C26H30O13
549.1627 (3.5)
551.2823 (4.7)
573.1737
257.0822 [M + H–Api–Glc]+
Liquiritin apioside
[26]
23
9.995
C26H30O13
549.1629 (3.8)
551.3403 (−5.1)
573.1735
257.0815 [M + H–Api–Glc]+
Isomer of liquiritin apioside
[26]
24
10.088
C27H30O16
609.1844 (−1.8)
633.1712
Rhodiosin
[27]
25
a
10.384
250, 300
C22H22O9
429.1410 (3.0)
453.1176
269.0974 [M + H–Glc]+
Ononin
[28, 29]
26
a
10.722
360
C21H20O12
463.0905 (2.8)
301.0464 [M–H–Glc]−
465.1033 (5.2)
487.0333
303.0515 [M + H–Glc]+
Hyperin
[24]
27
a
10.819
326
C25H24O12
515.1205 (2.9)
353.0751 [M–H–Ca]−
517.1296 (1.7)
539.1156
499.1187 [M + H–H2O]+, 163.0395 [M + H–Ca–Quinic acid]+
3,5-Dicaffeoylquinic acid
[30]
28
10.931
C29H36O15
623.1999 (−1.9)
461.1378, 161.0185
647.1941
471.1497, 325.0925, 163.0415
Isomer of forsythoside A
29
a
11.10
256, 354
C27H30O16
609.1465 (1.5)
611.1573 (3.3)
633.1404
465.0982 [M + H–Rha]+ 303.0495 [M + H–Rut]+
Rutin
[31]
30
a
11.220
280
C29H36O15
623.2001 (−1.6)
461.1920 [M–H–Ca]−, 161.0328 [Caffeic acid–H–H2O]−
647.1927
163.0386 [Caffeic acid + H–H2O]+
Forsythoside A
[32]
31
11.589
283
C27H30O15
593.1534 (−1.3)
285.0677 [M–H–Rut]−
595.1694 (−0.7)
287.0936 [M + H–Rut]+
Kaempferol-3-O-rutinoside
[13]
32
11.692
C19H35O16
519.1884 (3.5), 565.1953 [M + HCOO]−
357.1366 [M–H–Glc]−
543.1855
359.1454 [M + H–Glc]+, 341.1411 [M + H–Glc–H2O]+
(+)-Pinoresinol-β-D-glucoside
[33]
33
a
12.213
210, 262
C20H27NO11
456.1523 (3.7)
458.1641 (−4.6)
296.1124 [M + H–Glc]+, 162.0862 [M + H–Glc–HCT]+
Amygdalin
[13]
34
a
12.659
326
C25H24O12
515.1162 (−5.4)
353.0786 [M–H–Ca]−
517.1348 (0.4)
539.1193
499.1248 [M + H–H2O]+, 163.0387 [M + H–Ca–Quinic acid]+
3,4-Dicaffeoylquinic acid
[30]
35
a
13.082
276
C15H12O4
255.0654 (−1.2)
257.0659 (−0.8)
147.0621 [M + H–RL]+, 137.0422 [M + H–VP]+, 119.0691 [M + H–RL–CO]+
Liquiritigenin
[34]
36
a
13.251
243, 372
C26H30O13
549.1621 (2.4)
551.1725 (3.4)
573.1563
419.1360 [M + H–Api]+, 257.0814 [M + H–Api–Glc]+
Isoliquiritin apioside
[35]
37
a
13.300
242, 362
C21H22O9
417.1198 (2.9)
255.0518 [M–H–Glc]−
419.1314 (−6.7)
441.1150
257.0823 [M + H–Glc]+, 229.1357 [M + H–Glc–CO]+
Isoliquiritin
[26]
38
13.479
280
C27H34O11
579.2103 [M + HCOO]−
557.1987
552.2463 [M + NH4]+, 355.1516 [M + H–Glc–H2O]+
Arctiin
[36]
39
a
14.161
277
C27H34O11
579.2106[M + HCOO]−
371.1826 [M–H–Glc]−, 356.1547 [M–H–Glc–H2O]−
552.2444[M + NH4]+
557.1991
373.1563 [M + H–Glc]+, 355.1544 [M + H–Glc–H2O]+
Phillyrin
[37, 38]
40
14.678
372, 255
C15H10O7
301.0355 (2.3)
151.0137 [M–H–C8H6O3]−
303.0520 (4.9)
Quercetin
[39]
41
15.039
284
C28H34O15
609.1522 (1.3)
301.0582 [M–H–Rut]−
Hesperidin
[13]
42
15.402
265, 366
C15H10O6
285.0406 (2.5)
287.0721 (4.5)
153.0469 [M + H–HEP]+
Kaempferol
[13]
43
16.627
255
C15H10O4
253.0507 (2.4)
225.0741 [M–H–CO]−, 210.0494 [M–H–CO–CH3]−, 182.0542 [M–H–2CO–CH3]−, 154.0553 [M–H–3CO–CH3]−
Chrysophanol
[40]
44
17.035
C15H10O4
253.0512 (4.3)
225.0752, 210.0474, 182.0485,154.0531
Isomer of chrysophanol
[40]
45
a
17.347
254, 287, 426
C21H20O10
431.0989 (2.6)
269.0673 [M–H–Glc]−, 241.0719 [M–H–Glc–CO]−, 225.0753 [M–H–Glc–CO2]−, 210.0578 [M–H–Glc–CO2–CH3]−, 197.0771 [M–H–Glc–CO–CO2]−, 182.0486 [M–H–Glc–CO2–CH3–CO]−
455.1010
271.0715 [M + H–Glc]+
Emodin-8-O-glucoside
[41]
46
17.396
240, 330, 395
C15H12O4
255.0639 (−7.1)
257.0812 (−0.8)
Isoliquiritigenin
[26]
47
a
18.385
250, 304
C16H12O4
267.0653 (−1.5)
269.0827 (4.8)
291.0751
Formononetin
[42]
48
18.804
895.4082 (2.8)
719.3849 [M–H–GlcA]− 351.0630 [2GlcA–H]−
897.4107 (1.2)
919.3925
22β-Acetoxy licorice saponin B2/uralsaponin F
[43]
49
19.194
271, 421
C
22
H
2
2
O
10
445.1168 (−5.6)
283.0632 [M–H–Glc]−
469.1128
285.0775 [M + H–Glc]+
Physcion-8-O-β-D-glucopyranoside
[41]
50
19.603
248
C42H60O16
819.3843 (0.6)
351.0699 [2GlcA–H]−
821.3994 (−0.1)
843.3996
645.3612 [M + H–GlcA]+, 469.3302 [M + H–2GlcA]+, 451.3211 [M + H–2GlcA–H2O]+
Licorice saponin E2
[35]
51
19.704
C16H24O7
327.2180 (2.8)
164.9664 [M–H–Glc]−
351.2133
Rhododendrol-4′-O-β-D-glucopyranoside
[44]
52
20.000
254
C44H64O18
879.4062 (1.3)
351.0806 [M–H–22AG]−
881.4170 (−0.1)
705.3897 [M + H–GlcA]+, 529.3588 [M + H–2GlcA]+, 511.3395 [M + H–2GlcA–H2O]+
22-Acetoxyglycyrrhizin
[42]
53
20.573
352
C17H14O7
329.2341 (3.9)
353.2280
301.1443 [M + H–CH2O]+, 287.2002 [M + H–CO2]+, 259.1248 [M + H–CO2–CO]+
Tricin
[45]
54
a
21.199
260
C15H8O6
283.0262 (6.7)
239.0359 [M–H–CO2]−
285.0515 (−13.0)
Rhein
[46]
55
21.540
253
837.3977 (1.2)
819.4404 [M–H–H2O]−, 661.4100 [M–H–GlcA]−, 351.0815 [2GlcA–H]−, 289.0840 [2GlcA–H–H2O–CO2]−
839.4061 (−0.5)
861.3890
663.3868 [M + H–GlcA]+, 487.3395 [M + H–2GlcA]+, 469.3286 [M + H–2GlcA–H2O]+
Licorice saponin G2
[42]
56
21.626
236, 327, 370, 384
C21H20O6
367.1208 (7.1)
309.1566 [M–H–CO–CH2O]−, 265.0635 [M–H–CO–CH2O–CO2]−, 221.0221 [M–H–CO–CH2O–2CO2]−
369.1369 (−7.6)
391.1176
Glycycoumarin
[47]
57
22.087
249
C42H62O16
821.3977 (1.7)
803.4501 [M–H–H2O]−, 759.4542 [M–H–H2O–CO2]−, 645.4102 [M–H–GlcA]−, 627.4000 [M–H–GlcA–H2O]−, 469.3705 [M–H–2GlcA]−, 351.0809 [2GlcA–H]−, 289.0758 [2GlcA–H–H2O–CO2]−
823.4128 (1.5)
845.4557
647.3797 [M + H–GlcA]+, 471.3480 [M + H–2GlcA]+, 453.3365 [M + H–2GlcA–H2O]+
Glycyrrhizic acid
[48]
58
22.188
252
C42H62O16
821.3986 (−1.1)
351.0768 [M–H–GA]−
823.4134 (−2.1)
845.4127
647.3823 [M + H–GlcA]+, 471.3365 [M + H–2GlcA]+, 453.3324 [M + H–2GlcA–H2O]+
Licorice saponin H2
[26]
59
22.389
248
C42H64O15
807.4197 (−0.6)
745.4601 [M–H–Glc]−, 631.4210 [M–H–GlcA]−, 351.0699 [M–H–GA]−
809.4410 (−0.9)
831.4138
Licorice saponin B2
[49]
60
22.623
252
C42H62O16
821.3997 (0.2)
351.0837 [M–H–GA]−
823.4105 (−1.3)
845.3900
647.3838 [M + H–GlcA]+, 471.3288 [M + H–2GlcA]+, 453.3279 [M + H–2GlcA–H2O]+
Licorice saponin K2
[50]
61
a
23.230
288, 439
C15H10O5
269.0462 (4.5)
241.0669 [M–H–CO]−, 225.0723 [M–H–CO2]−, 210.0427 [M–H–CO2–CH3]−, 197.0695 [M–H–CO–CO2]−, 182.0477 [M–H–CO2–CH3–CO]−, 154.0552 [M–H–CO2–CH3–2CO]−
Emodin
[40]
aCompared with reference compounds.
b22AG: 22-acetoxyglycyrrhizin; Api: apiose; Ca: caffeoyl; GA: glycyrrhetinic acid; Glc: glucose; GlcA: glucuronic acid; HCT: 4-(hydroxymethyl)cyclobutane-1,2,3-triol; HEP: 4-(hydroxyethynyl) phenol; Rha: rhamnose; RL: resorcinol; Rut: rutinose; VP: 4-vinylphenol.Figure 2
UPLC-QTOF-MS chromatograms of sample solution from negative ion mode and positive ion mode.Chemical structures of the compounds identified from LQC (except for the 7 isomers).
(a)
(b)
(c)
(d)
(e)
(f)


### 3.2.1. Flavonoids

Seventeen flavonoids (Figure3(a)) in LQC including flavone aglycones and glycosides were identified. They were mainly obtained from Lianqiao, Jinyinhua, Gancao, Hongjingtian, and Mahuang. Amongst them, liquiritin apioside (22), ononin (25), hyperin (26), rutin (29), liquiritigenin (35), isoliquiritin apioside (36), isoliquiritin (37), and formononetin (47) were unambiguously identified via the standards.In negative and positive ion modes, flavone aglycones mainly gain fragment ions by the reverse Diels-Alder (RDA) reaction and the loss of CO (28 Da). The characteristic fragmentation behavior of compound35 is shown in Figure 4(a) with high abundant fragmentation [M+H–VP (4-vinylphenol)]+ atm/z 137.0422. The abundance of fragment ions [M+H–RL (resorcinol)]+ atm/z 147.0621 and [M+H–RL–CO]+ atm/z 119.0691 is relatively lower. Compounds24,40,42, and46were tentatively identified via comparing their exact molecular masses, MS/MS spectra data, and retention behaviors with literature data [13, 26, 39]. Flavone glycosides have the similar fragmentation pathways of simultaneous or successive loss of glucose (162 Da), rhamnose (146 Da), or apiose (132 Da). The fragmentation pathway of compound26 was exemplified in Figure 4(b) in negative ion mode. Compounds21,23,31,41, and53were tentatively identified by comparing the molecular mass and MS/MS data with literature data [25, 45].The MS spectra and fragmentation pathway of compound35 (a), compound26 (b), compound8 (c), compound30 (d), compound39 (e), compound61 (f), compound45 (g), compound57 (h), and compound5 (i).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)


### 3.2.2. Phenylpropanoids

Fourteen phenylpropanoids (Figure3(b)) including phenylpropionic acids, lignans, and coumarins were identified in LQC. They were mainly obtained from Lianqiao, Jinyinhua, and Gancao. Neochlorogenic acid (4), chlorogenic acid (8), cryptochlorogenic acid (9), 3,5-dicaffeoylquinic acid (27), forsythoside A (30), 3,4-dicaffeoylquinic acid (34), and phillyrin (39) were unambiguously characterized by comparing with standards.In negative ion mode, phenylpropionic acids have the similar fragmentation pathways of simultaneous or successive loss of H2O (18 Da), CO (28 Da), and CO2 (44 Da). The fragmentation pathway of compound8[18], as the representative of phenylpropionic acids, is shown in Figure 4(c). Compound30 produced [M–H–Ca (caffeoyl)]− atm/z 461.1920 and [Caffeic–H–H2O]− atm/z 161.0328, as displayed in Figure 4(d). Lignans primarily generated [M+HCOO]− in negative ion mode and further elimination of glucose (162 Da) produced aglycone. As shown in Figure 4(e), compound39produced characteristic fragments atm/z 371.1826 andm/z 356.1547 corresponding to [M–H–Glc (glucose)]− and [M–H–Glc–CH3]−, respectively. Compounds16,18,19,28,32,38,and56were tentatively identified on the basis of the exact molecular formulae matching, fragmentation information, and retention behaviors as well as literature data [23, 24, 33, 36, 47].


### 3.2.3. Anthraquinones

Eight anthraquinones (Figure3(c)) in LQC were definitely or tentatively identified. All of them were derived from Dahuang. Chrysophanol glucoside (17), emodin-8-O-glucoside (45), rhein (54), and emodin (61) were confirmed via comparing with standard substances.The characteristic fragmentation behavior of anthraquinones was the loss of CO2 (44 Da), CH3 (15 Da), and CO (28 Da) in negative ion mode. Typical compound61was used to explain the fragmentation pathway of anthraquinones presented in Figure 4(f). Compound45, as shown in Figure 4(g), produced characteristic fragments atm/z 269.0673,m/z 241.0719,m/z 225.0753,m/z 197.0771, andm/z 182.0486 which corresponded to [M–H–Glc]−, [M–H–Glc–CO]−, [M–H–Glc–CO2]−, [M–H–CO–CO2]−, and [M–H–CO2–CH3–CO]−, respectively. Compounds12,43,44, and49 were tentatively identified by comparing their accurate molecular masses and MS/MS fragment data with literature data [21, 40, 41].


### 3.2.4. Triterpenoids

Eight triterpenoids (Figure3(d)) in LQC were unambiguously or tentatively identified. All of them were derived from Gancao. Glycyrrhizic acid (57) was confirmed by comparing with standards. In negative ion mode, representative compound57yielded [M–H–H2O–CO2]− atm/z 759.4524, [M–H–GlcA (glucuronic acid)]− atm/z 645.4102, [M–H–2GlcA]− atm/z 469.3705, [2GlcA–H]− atm/z 351.0809, and [2GlcA–H–H2O–CO2]− atm/z 289.0758, as shown in Figure 4(h). Compounds48,50,52,55,58,59,and60were tentatively identified by comparing their exact molecular masses and MS/MS spectral data with the literature data [51].


### 3.2.5. Iridoids

Four iridoids (Figure3(e)) in LQC including iridoid glycosides and secoiridoid glycosides were identified. All of them were derived from Jinyinhua. Loganic acid (5), sweroside (13), and secoxyloganin (20) were unambiguously identified via the standards.The fragment1,4F (fragment generated from the fracture of 1/4 bonds of iridoids) in the negative ion mode was identified as the characteristic fragment ion of iridoid glycosides. Meanwhile, iridoid glycosides would lose the functional groups such as H2O (18 Da), CO2 (44 Da), and glucose (162 Da). Typical compound5 gave [M–H–Glc–CO2–H2O]− atm/z 151.0878, [M–H–Glc–2CO2]− atm/z 125.0712, and [1,4F–H2O]− atm/z 93.0423 [52], as presented in Figure 4(i). Compound10 was tentatively identified by comparing its exact molecular mass and MS/MS spectral data with the literature data [19].


### 3.2.6. Other Types of Compounds

Compounds1,2,11,14,15, and51were tentatively identified by comparing their exact molecular masses and MS/MS spectral data with the literature data except gallic acid (3), salidroside (6), forsythoside E (7), and amygdalin (33) which were identified via the standards (Figure 3(f)) [12, 53].


## 3.2.1. Flavonoids

Seventeen flavonoids (Figure3(a)) in LQC including flavone aglycones and glycosides were identified. They were mainly obtained from Lianqiao, Jinyinhua, Gancao, Hongjingtian, and Mahuang. Amongst them, liquiritin apioside (22), ononin (25), hyperin (26), rutin (29), liquiritigenin (35), isoliquiritin apioside (36), isoliquiritin (37), and formononetin (47) were unambiguously identified via the standards.In negative and positive ion modes, flavone aglycones mainly gain fragment ions by the reverse Diels-Alder (RDA) reaction and the loss of CO (28 Da). The characteristic fragmentation behavior of compound35 is shown in Figure 4(a) with high abundant fragmentation [M+H–VP (4-vinylphenol)]+ atm/z 137.0422. The abundance of fragment ions [M+H–RL (resorcinol)]+ atm/z 147.0621 and [M+H–RL–CO]+ atm/z 119.0691 is relatively lower. Compounds24,40,42, and46were tentatively identified via comparing their exact molecular masses, MS/MS spectra data, and retention behaviors with literature data [13, 26, 39]. Flavone glycosides have the similar fragmentation pathways of simultaneous or successive loss of glucose (162 Da), rhamnose (146 Da), or apiose (132 Da). The fragmentation pathway of compound26 was exemplified in Figure 4(b) in negative ion mode. Compounds21,23,31,41, and53were tentatively identified by comparing the molecular mass and MS/MS data with literature data [25, 45].The MS spectra and fragmentation pathway of compound35 (a), compound26 (b), compound8 (c), compound30 (d), compound39 (e), compound61 (f), compound45 (g), compound57 (h), and compound5 (i).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)


## 3.2.2. Phenylpropanoids

Fourteen phenylpropanoids (Figure3(b)) including phenylpropionic acids, lignans, and coumarins were identified in LQC. They were mainly obtained from Lianqiao, Jinyinhua, and Gancao. Neochlorogenic acid (4), chlorogenic acid (8), cryptochlorogenic acid (9), 3,5-dicaffeoylquinic acid (27), forsythoside A (30), 3,4-dicaffeoylquinic acid (34), and phillyrin (39) were unambiguously characterized by comparing with standards.In negative ion mode, phenylpropionic acids have the similar fragmentation pathways of simultaneous or successive loss of H2O (18 Da), CO (28 Da), and CO2 (44 Da). The fragmentation pathway of compound8[18], as the representative of phenylpropionic acids, is shown in Figure 4(c). Compound30 produced [M–H–Ca (caffeoyl)]− atm/z 461.1920 and [Caffeic–H–H2O]− atm/z 161.0328, as displayed in Figure 4(d). Lignans primarily generated [M+HCOO]− in negative ion mode and further elimination of glucose (162 Da) produced aglycone. As shown in Figure 4(e), compound39produced characteristic fragments atm/z 371.1826 andm/z 356.1547 corresponding to [M–H–Glc (glucose)]− and [M–H–Glc–CH3]−, respectively. Compounds16,18,19,28,32,38,and56were tentatively identified on the basis of the exact molecular formulae matching, fragmentation information, and retention behaviors as well as literature data [23, 24, 33, 36, 47].


## 3.2.3. Anthraquinones

Eight anthraquinones (Figure3(c)) in LQC were definitely or tentatively identified. All of them were derived from Dahuang. Chrysophanol glucoside (17), emodin-8-O-glucoside (45), rhein (54), and emodin (61) were confirmed via comparing with standard substances.The characteristic fragmentation behavior of anthraquinones was the loss of CO2 (44 Da), CH3 (15 Da), and CO (28 Da) in negative ion mode. Typical compound61was used to explain the fragmentation pathway of anthraquinones presented in Figure 4(f). Compound45, as shown in Figure 4(g), produced characteristic fragments atm/z 269.0673,m/z 241.0719,m/z 225.0753,m/z 197.0771, andm/z 182.0486 which corresponded to [M–H–Glc]−, [M–H–Glc–CO]−, [M–H–Glc–CO2]−, [M–H–CO–CO2]−, and [M–H–CO2–CH3–CO]−, respectively. Compounds12,43,44, and49 were tentatively identified by comparing their accurate molecular masses and MS/MS fragment data with literature data [21, 40, 41].


## 3.2.4. Triterpenoids

Eight triterpenoids (Figure3(d)) in LQC were unambiguously or tentatively identified. All of them were derived from Gancao. Glycyrrhizic acid (57) was confirmed by comparing with standards. In negative ion mode, representative compound57yielded [M–H–H2O–CO2]− atm/z 759.4524, [M–H–GlcA (glucuronic acid)]− atm/z 645.4102, [M–H–2GlcA]− atm/z 469.3705, [2GlcA–H]− atm/z 351.0809, and [2GlcA–H–H2O–CO2]− atm/z 289.0758, as shown in Figure 4(h). Compounds48,50,52,55,58,59,and60were tentatively identified by comparing their exact molecular masses and MS/MS spectral data with the literature data [51].


## 3.2.5. Iridoids

Four iridoids (Figure3(e)) in LQC including iridoid glycosides and secoiridoid glycosides were identified. All of them were derived from Jinyinhua. Loganic acid (5), sweroside (13), and secoxyloganin (20) were unambiguously identified via the standards.The fragment1,4F (fragment generated from the fracture of 1/4 bonds of iridoids) in the negative ion mode was identified as the characteristic fragment ion of iridoid glycosides. Meanwhile, iridoid glycosides would lose the functional groups such as H2O (18 Da), CO2 (44 Da), and glucose (162 Da). Typical compound5 gave [M–H–Glc–CO2–H2O]− atm/z 151.0878, [M–H–Glc–2CO2]− atm/z 125.0712, and [1,4F–H2O]− atm/z 93.0423 [52], as presented in Figure 4(i). Compound10 was tentatively identified by comparing its exact molecular mass and MS/MS spectral data with the literature data [19].


## 3.2.6. Other Types of Compounds

Compounds1,2,11,14,15, and51were tentatively identified by comparing their exact molecular masses and MS/MS spectral data with the literature data except gallic acid (3), salidroside (6), forsythoside E (7), and amygdalin (33) which were identified via the standards (Figure 3(f)) [12, 53].


## 3.3. Methodological Validation of the Quantitative Analysis

As shown in Table3, twelve standards were of good linearity with high correlation coefficient values over 0.9993. The LODs and LOQs were 0.051–1.71 μg/mL and 0.16–5.69 μg/mL, independently. Twelve analytes in sample solution were stable at room temperature within 48 h with the RSD less than 2.76%. The RSD values of intraday and interday precisions were less than 1.10% and 2.92%, respectively. The RSD of repeatability was less than 2.39%. The average recovery rates of 12 compounds ranged from 92.99% to 103.95% with the RSD less than 3.54%. All the results showed that the assay was satisfactory with high accuracy, good reproducibility, and high sensitivity which were beneficial to the analytical investigation and quality control for LQC.Table 3
Linear regression, LODs and LOQs, intraday and interday precisions, repeatability, stability, and recovery for 12 compounds.
Compound
Regression equationa  (n=3)
R
2
Linear range (μg/mL)
LODb (μg/mL)
LOQc (μg/mL)
Intraday  (RSD, %) (n=6)
Interday  (RSD, %) (n=3)
Repeatability (n=6)
Stability(RSD, %)
Recovery (n = 6)
Mean (μg/g)
RSD (%)
Original(μg)
Spiked(μg)
Detected(μg)
Recovery(%)
RSD(%)
Salidroside
y
=
13980
x
-
11265
0.9999
5.27–84.35
1.06
3.50
0.12
0.26
1779.55
±
10.92
0.61
1.38
355.91
354.20
707.50
99.28
1.24
Chlorogenic acid
y
=
26510
x
+
3673.3
0.9998
6.85–109.56
1.71
5.69
0.77
2.12
2473.71
±
12.74
0.51
2.54
494.74
493.85
959.90
94.19
2.42
Forsythoside E
y
=
11793
x
+
5016.8
0.9995
4.85–77.64
1.21
4.04
0.67
2.25
1754.03
±
18.72
1.07
2.67
351.81
351.05
677.26
92.99
3.54
Cryptochlorogenic acid
y
=
21498
x
-
5018.9
0.9997
6.65–106.47
1.66
5.53
1.10
1.63
2029.49
±
20.65
1.02
2.46
405.90
406.60
784.00
93.10
1.43
Amygdalin
y
=
10530
x
-
3464.7
0.9998
3.91–62.57
0.98
3.26
0.79
0.93
1485.38
±
25.85
1.74
2.48
297.08
298.62
576.39
93.58
3.32
Sweroside
y
=
20593
x
-
6003.4
0.9999
2.00–31.96
0.25
0.80
0.11
0.33
798.06 ±2.93
0.37
0.48
159.61
159.51
325.43
103.95
0.41
Hyperin
y
=
27773
x
-
879.41
0.9999
0.20–3.21
0.051
0.16
0.78
0.62
92.03 ±0.80
0.87
0.83
18.41
18.24
37.07
102.29
1.82
Rutin
y
=
30768
x
-
3783.7
0.9993
0.53–8.50
0.072
0.21
0.41
2.24
184.15
±
3.06
1.66
1.57
36.83
36.81
72.50
96.90
2.34
Forsythoside A
y
=
8546.6
x
-
6024.5
0.9999
4.21–67.34
0.30
1.05
0.20
2.92
2484.99
±
18.43
0.74
1.95
497.00
495.95
997.33
100.89
1.49
Phillyrin
y
=
30390
x
+
5282.8
0.9999
2.86–45.71
0.71
2.36
0.79
1.63
1577.80
±
10.11
0.64
2.76
315.56
316.72
623.99
100.25
1.60
Rhein
y
=
7473.6
x
-
5172.7
0.9996
3.47–55.49
0.87
2.88
0.40
0.28
681.92
±
16.32
2.39
0.97
136.38
136.27
273.48
100.61
1.61
Glycyrrhizic acid
y
=
10296
x
-
6531.3
0.9999
5.27–84.35
0.092
0.33
0.34
2.09
1622.75
±
17.59
1.08
2.15
324.55
323.939
652.16
101.31
3.27
a
y is the peak area; x is the concentration of standard solutions.
bLOD refers to the limits of detection, S/N=3.
cLOQ refers to the limits of quantity, S/N=10.


## 3.4. Sample Analysis

Twelve representative compounds in 10 batches of LQC were quantified through the developed UPLC-DAD analytical method described above. The results are summarized in Table4, which showed that the total concentrations of 12 quantitative compounds in different batches of the LQC varied narrowly; moreover, the 12 components differed greatly in their contents, which may be affected by the source of medicinal materials, the quality of the plant material, or the preparation technology. Among them, forsythoside A showed the highest amount (3164.55–2089.22 μg/g) followed by amygdalin (2594.75–1623.14 μg/g) and hyperin had the lowest amount at 100.80–62.56 μg/g.Table 4
Contents of the 12 compounds in 10 batches.
Sample
Compound (mean ± SD) (μg/g)
Total
RSD (%)
Salidroside
Chlorogenic acid
Forsythoside E
Cryptochlorogenic acid
Amygdalin
Sweroside
Hyperin
Rutin
Forsythoside A
Phillyrin
Rhein
Glycyrrhizic acid
Lot.1
1740.57
±
30.70
2159.11
±
12.47
1888.99
±
7.95
1464.88
±
10.22
2109.85
±
31.27
830.23
±
8.44
67.06
±
0.68
156.22
±
1.11
3163.84
±
33.14
1599.20
±
12.29
570.70
±
2.23
1028.45
±
4.21
16779.10
10.03
Lot.2
1697.04
±
12.54
1810.69
±
11.73
1424.67
±
38.04
1167.41
±
8.35
1623.14
±
25.14
650.95
±
7.21
68.07
±
1.37
106.85
±
2.82
2497.34
±
27.21
1220.59
±
35.57
554.26
±
8.27
837.16
±
11.65
13658.17
Lot.3
1456.36
±
22.08
1941.91
±
44.94
1477.93
±
29.66
1232.30
±
29.88
1641.25
±
34.31
681.53
±
6.35
72.66
±
2.05
110.31
±
2.16
2089.22
±
36.09
1314.99
±
31.21
617.91
±
10.70
904.75
±
15.73
13541.12
Lot.4
1520.75
±
8.91
1993.38
±
50.86
1501.09
±
42.60
1409.15
±
6.32
2147.01
±
60.37
743.98
±
18.00
62.56
±
1.28
100.86
±
2.39
2652.53
±
67.03
1139.47
±
24.08
696.42
±
18.76
985.60
±
22.85
14925.80
Lot.5
1593.12
±
35.00
2081.89
±
50.77
1626.62
±
48.29
1523.11
±
43.22
2282.76
±
46.19
777.98
±
4.18
75.89
±
1.69
121.06
±
2.41
3072.10
±
44.89
1477.18
±
25.48
686.35
±
16.44
984.79
±
29.13
16302.85
Lot.6
1821.34
±
48.55
2436.69
±
31.00
2025.28
±
43.31
1494.76
±
12.65
2377.04
±
63.64
860.29
±
13.37
100.80
±
1.87
145.77
±
2.94
3680.72
±
92.85
1991.09
±
18.29
648.86
±
3.91
1057.29
±
30.41
18639.93
Lot.7
1474.32
±
36.85
2222.61
±
38.27
1372.97
±
36.85
1727.36
±
16.40
2594.75
±
50.67
854.30
±
19.31
77.25
±
2.14
156.90
±
4.65
2987.54
±
36.76
1168.12
±
12.70
551.26
±
5.18
1227.74
±
20.24
16415.12
Lot.8
1598.66
±
19.51
2183.54
±
15.11
1589.79
±
19.71
1721.26
±
11.36
2494.81
±
19.61
824.71
±
4.47
81.26
±
0.69
174.62
±
0.86
3164.55
±
36.81
1277.14
±
11.04
716.94
±
5.74
1220.87
±
9.89
17048.15
Lot.9
1535.78
±
30.36
1961.72
±
22.33
1789.55
±
28.12
1247.34
±
36.59
1824.87
±
35.66
696.40
±
7.08
73.33
±
0.46
136.23
±
2.51
2823.09
±
15.19
1495.85
±
17.76
625.10
±
11.44
891.45
±
10.60
15100.71
Lot.10
1836.41
±
29.14
2216.36
±
8.18
1500.75
±
8.06
1670.72
±
23.19
2281.74
±
14.32
809.41
±
3.13
75.33
±
0.85
134.19
±
1.24
3010.34
±
12.64
1204.92
±
28.26
891.01
±
9.99
1176.72
±
6.68
16807.90


## 4. Conclusion

LQC is a commonly used Chinese medical preparation to treat viral influenza. To date, there has not been a systematical and comprehensive study on the chemical profiling and quality control method for LQC. Therefore, an accurate, sensitive, and reliable quality control procedure for LQC is in urgent need to be established. In our study, the chemical profile of LQC was thoroughly and systematically investigated by UPLC-DAD-QTOF-MS for the first time. Sixty-one compounds were unambiguously or tentatively identified. Based on the qualitative analysis, a UPLC-DAD method was established for quantitative analysis of 12 representative compounds in LQC, which has been demonstrated to be effective for the analysis of 10 batches of LQC. This developed method could be applied as an effective quality control procedure for LQC. In addition, this study would be a powerful reference for the identification of similar compounds presented here, such as flavonoids, phenylpropanoids, anthraquinones, triterpenoids, and iridoids by MS spectra.



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*Source: 731765-2015-01-08.xml*