Qualitative analysis of chemical components in Lianhua Qingwen capsule by HPLC-Q Exactive-Orbitrap-MS coupled with GC-MS.

The Lianhua Qingwen (LHQW) capsule is a popular traditional Chinese medicine for the treatment of viral respiratory diseases. In particular, it has been recently prescribed to treat infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, due to its complex composition, little attention has been directed toward the analysis of chemical constituents present in the LHQW capsule. This study presents a reliable and comprehensive approach to characterizing the chemical constituents present in LHQW by high-performance liquid chromatography-Q Exactive-Orbitrap mass spectrometry (HPLC-Q Exactive-Orbitrap-MS) coupled with gas chromatography-mass spectrometry (GC-MS). An automated library alignment method with a high mass accuracy (within 5 ppm) was used for the rapid identification of compounds. A total of 104 compounds, consisting of alkaloids, flavonoids, phenols, phenolic acids, phenylpropanoids, quinones, terpenoids, and other phytochemicals, were successfully characterized. In addition, the fragmentation pathways and characteristic fragments of some representative compounds were elucidated. GC-MS analysis was conducted to characterize the volatile compounds present in LHQW. In total, 17 compounds were putatively characterized by comparing the acquired data with that from the NIST library. The major constituent was menthol, and all the other compounds were terpenoids. This is the first comprehensive report on the identification of the major chemical constituents present in the LHQW capsule by HPLC-Q Exactive-Orbitrap-MS, coupled with GC-MS, and the results of this study can be used for the quality control and standardization of LHQW capsules.

Introduction

Lianhua Qingwen capsule (LHQW), produced by Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, China), is a patented traditional Chinese medicine (TCM). It was developed to treat patients infected with the severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) in China. LHQW is based on two TCM formulas, i.e., Maxing-Shigan-Tang and Yinqiao-San, which were originally mentioned in the classical Chinese books ShangHanLun and WenBingTiaoBian, respectively [1]. Both Maxing-Shigan-Tang and Yinqiao-San are prescribed to treat fever, inflammation, and seasonal influenza [2,3]. LHQW is composed of 11 herbs [4]: Forsythiae Fructus, Lonicerae Japonicae Flos, Ephedrae Herba, Armeniacae Semen Amarum, Isatidis Radix, Dryopteridis Crassirhizomatis Rhizoma, Houttuyniae Herba, Pogostemonis Herba, Rhei Radix Et Rhizoma, Rhodiolae Crenulatae Radix Et Rhizoma and Glycyrrhizae Radix Et Rhizoma. In addition, it contains Gypsum Fibrosum, a mineral and menthol, extracted from Menthae Haplocalycis Herba.

In China, LHQW has been widely used to treat the symptoms of respiratory diseases. Various reports have established the good anti-influenza activity of LHQW against H7N9 [1] and H1N1 [5] viruses. In particular, a randomized, double-blind, and positive clinical trial indicated that effectiveness of LHQW against the H1N1 virus is similar to that of Oseltamivir [5]. Another recent study has reported that LHQW is also effective against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [6], the pathogen that causes the coronavirus disease (COVID-19). Thus, LHQW has been included as a representative TCM prescription in the Guideline for the Diagnosis and Treatment of COVID-19 Pneumonia issued by the National Heath Commission of the People's Republic of China [7]. However, the use of LHQW is limited to China. For LHQW to be globally accepted, its chemical composition and mechanism of action must be well understood. Thus, it is essential to determine the chemical composition of LHQW, which can further enable its quality control and standardization.

Several analytical methods have been developed for the quality control of LHQW. For example, Zhang et al. [8] and Chen et al. [9] developed ultra-performance liquid chromatography (UPLC) fingerprinting methods for the quality control of LHQW. Qiao et al. [10] and Jia et al. [11] developed methods based on gas chromatography with flame-ionization detection and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile constituents and raw materials of an LHQW capsule, respectively. Jia et al. [12] developed a method based on a combination of UPLC with a diode-array detector and quadrupole time-of-flight mass spectrometry to determine the major chemical constituents of an LHQW capsule. Nevertheless, a detailed analysis of the chemical composition of an LHQW capsule has not yet been reported.

Orbitrap mass spectrometry (MS) has become a powerful tool to characterize the chemical components in TCMs because of its high sensitivity, high mass resolution, and high mass accuracy [13,14]. This method also helps analyze MSn fragments, thus facilitating the structural elucidation of a compound. Gas chromatography-mass spectrometry (GC-MS) has been widely applied for the detection of volatile components in a TCM [15]. In this paper, we present the unprecedented comprehensive analysis of the major chemical components in an LHQW capsule by high-performance liquid chromatography (HPLC)-Q Exactive-Orbitrap-MS coupled with GC-MS. In total, 104 compounds including 6 alkaloids, 33 flavonoids, 7 phenols, 9 phenolic acids, 16 phenylpropanoids, 7 quinones, 12 terpenoids and 14 other phytochemicals, were putatively identified via HPLC-Q Exactive-Orbitrap-MS. In addition, 17 volatile compounds were putatively identified via GC-MS. Thus, a total of 120 compounds were successfully extracted and characterized.

Materials and methods

Chemicals and materials

LHQW capsules (Lot No. B2001158) were obtained from Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, China). Acetonitrile (ACN, HPLC grade) and methanol (HPLC grade) were obtained from Merck KGaA (Darmstadt, Germany). Watsons-distilled water was purchased from Jingdong Mall (Beijing, China). Formic acid (FA), acetone (HPLC grade), and hexane (HPLC grade) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ethyl acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Sample preparation

For the HPLC-Q Exactive-Orbitrap-MS analysis, the raw material (brown powder; 0.4 g) of the LHQW capsule was accurately weighed, dissolved in 60% methanol (V/V; 20 mL), and sonicated for 30 min. The solution was centrifuged at 12,000 r/min and the supernatant was filtered through a 0.22 μm membrane.

For GC-MS analysis, three different extraction solvents were used: hexane, acetone, and ethyl acetate. The raw material (1 g) was accurately weighed, dissolved in the extracted solvent (10 mL), and sonicated for 20 min. The resulting extracts were centrifuged at 12,000 r/min and the supernatants were analyzed.

HPLC-Q Exactive-Orbitrap-MS analysis

An HPLC-high resolution mass spectrometer (HPLC-HRMS; Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a reversed-phase Hypersil Gold aQ C18 column (2.1 mm × 150 mm, 3 μm; Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the chemical constituents present in the LHQW sample. The column temperature was maintained at 40 °C. The mobile phases included 0.1% FA in ACN (A) and 0.1% FA in H2O (B). A constant flow rate was maintained (0.2 mL/min). The elution gradient was maintained at 5% A for 1 min, increased to 95% A over 41 min, and maintained at this phase for 4.9 min. The column was then re-equilibrated at 5% A for 3 min. The samples were maintained at room temperature and the injection volume was 5 μL.

The Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated electrospray ionization interface was operated under both electrospray ionization (ESI) negative and ESI positive modes. The instrument was calibrated with the calibration solutions provided by the manufacturer. Data were acquired using Xcalibur 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA), and all obtained data were processed by the Compound Discoverer (CD) 3.0 (Thermo Fisher Scientific, Waltham, MA, USA) and Xcalibur 4.1 software packages. The source parameters were optimized with a spray voltage of 3.5 kV (+)/3.2 kV (−). The other parameters were set as follows: capillary temperature, 320 °C; auxiliary gas heater temperature, 350 °C; sheath gas pressure, 40 arb; auxiliary gas pressure, 15 arb; sweep gas pressure, 0 arb; S-lens RF level, 50 V.

The Q Exactive detector was operated in full scan and data-dependent MS2 (dd MS2) modes. In full scan mode, the resolution was set at 70,000. The automatic gain control (AGC) target and maximum injection time (IT) were 1 × 106 ions capacity and 100 ms, respectively. For the dd MS2 mode, the acquisition parameters were: resolution, 17,500; AGC target, 2 × 105 ions capacity; maximum IT, 50 ms; scan range, 100–1500 m/z; loop count, 3; normalized collision energy (stepped), 20%, 40%, 60%; isolation window, 1.2 Da; apex trigger, 5–15 s; dynamic exclusion, 5 s. The Top N (N = number of top abundant ions for fragmentation) was set to 5.

GC-MS analysis

The GC-MS analysis was conducted on a Trace GC Ultra system equipped with an AS 3000 auto-sampler, a split/splitless injector, and TSQ Quantum XLS MS detector with triple quadrupole (Thermo Fisher Scientific, Waltham, MA, USA). The TR-5 MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

The injection temperature and ion source temperature were 250 °C. The oven temperature program was maintained at 50 °C for 1 min, increased to 200 °C (rate: 8 °C/min), maintained at 200 °C for 5 min, increased to 280 °C (rate: 10 °C/min), and maintained for 5 min. Helium was used as the carrier gas (flow rate: 1 mL/min). Sample injection volume and split ratio were 1 μL and 50:1, respectively. The ionizing energy was 70 eV. The chromatograms were obtained by collecting the total ion currents in the scan range of m/z 50–550. Data were acquired using the Xcalibur 2.2 software.

Data processing and compound identification

The raw data acquired from the HPLC-Q Exactive-Orbitrap-MS analysis were processed by the CD 3.0 software. The data matrices of the molecular masses, retention time, fragments, and peak areas from both ESI positive and negative modes were extracted and aligned to the mzVault library, which was integrated in the CD software. The mzVault spectral library (Thermo Fisher Scientific, Waltham, MA, USA) contained the retention time, precise mass ions, and MS2 fragments of 1200 commercial reference standards, which were analyzed by Q Exactive-Orbitrap-MS. The CD software equipped with the mzVault library identified peaks with a high mass accuracy (< 5 ppm) and an isotope pattern variation within 85%. The molecular compositions adhered to the hydrogen to carbon ratio rules, and were matched to potential compounds using rings and double-bonds equivalents. The MS2 spectra were compared with the reference spectra from the mzVault library. Compound identification was accepted only when the matching score was greater than 85 (total score = 100). In addition, the accuracy of the compound identification was improved by comparing the obtained data and possible fragmentation patterns with those reported in the literature.

Results and discussion

The base peak chromatograms of the LHQW capsule in positive and negative ion modes are depicted in Fig. 1. In total, 104 compounds, including alkaloids, flavonoids, phenols, phenolic acids, phenylpropanoids, quinones, terpenoids, and other phytochemicals, were putatively identified (as listed in Table 1 [[16], [17], [18], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44]]) using the library alignment method. According to previous studies [[16], [17], [18], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44]], the identified compounds are present in the herbs used in the LHQW formulation. No phytochemical was identified from Dryopteridis Crassirhizomatis Rhizoma. The compound identification of several specific compounds is presented below.

Fig. 1

Base peak chromatogram of the Lianhua Qingwen (LHQW) capsule analyzed using HPLC-Q Exactive-Orbitrap-MS analysis in (A) electrospray ionization (ESI) positive mode and (B) ESI negative mode.

Table 1

Identification of the chemical constituents in the LHQW capsule using HPLC-Q Exactive-Orbitrap-MS.

No.	tR (min)	Error (ppm)	Formula	[M+H]+		[M−H]-		MS2		Alignmentscore	Potentialcompounds	Herb	Refs.
				Theo.m/z	Exp.m/z	Theo.m/z	Exp.m/z	ESI (+)	ESI (−)
1	2.07	1.7	C6H14O6			181.0717	181.0714		101.0239, 71.0134b, 59.0135	86.5	Mannitol	IR	[16]
2	2.09	1.5	C12H22O11			341.1089	341.1084		101.0238, 71.0134, 59.0135b	95.6	Sucrose	IR	[17]
3	2.12	1.6	C7H12O6			191.0561	191.0558		191.0556b, 127.0394, 85.0289	94.8	Quinic acid	LJ	[18]
4	2.13	−2.6	C5H9NO2	116.0706	116.0709			70.0659b		86.4	Proline	HH	[19]
5	2.7	−2.4	C6H5NO2	124.0393	124.0396			124.0396b, 96.0449,81.0534		89.3	Nicotinic acid	IR	[17]
6	2.7	1.6	C9H12N2O6			243.0622	243.0618		110.0242b, 82.0294	93.1	Uridine	IR	[20]
7	2.71	−1.5	C5H5N5	136.0617	136.0619			136.0619b, 119.0356		89.8	Adenine	IR	[21]
8	2.71	2.1	C6H8O7			191.0197	191.0193		111.0082b, 87.0082	93.9	Citric acid	RR	[17]
9	2.9	−0.7	C10H13N5O4	268.1040	268.1042			163.0619b		92.6	Adenosine	IR	[20]
10	2.97	−0.4	C10H13N5O5	284.0989	284.0990			152.0569b		87.7	Guanosine	IR	[20]
11	3.5	3.0	C7H6O5			169.0142	169.0137		125.0239b	92.3	Gallic acid	RR	[22]
12	5.57	3.3	C8H10O3			153.0557	153.0552		123.0447, 109.0289b	87.3	3,4-Dihydroxyphenylethanol	FF	[17]
13	5.6	2.6	C7H6O4			153.0193	153.0189		109.0289b	88.6	Protocatechuic acid	LJ	[18]
14	6	−1.5	C5H7NOS	130.0321	130.0323			70.0659b		86.5	Epigoitrin	IR	[23]
15a	6.8	0.8	C16H24O10			375.1296	375.1293		213.0765, 151.0758, 125.0602b	89.4	Loganic acid	LJ	[24]
16	7.7	3.6	C7H6O3			137.0244	137.0239		137.0238b	92.1	Protocatechualdehyde
17	7.72	1.0	C14H20O7			299.1136	299.1133		137.0605, 119.0496, 89.0239, 71.0134,59.0135b	96.6	Salidroside	RC	[25]
18a	8.06	0.5	C16H22O10			373.1140	373.1138		149.0602, 123.0446, 59.0135b	91.1	Geniposidic acid
19	8.3	0	C20H30O12			461.1664	461.1664		205.0709, 135.0446b	95.7	Forsythoside E	FF	[26]
20	8.59	2.2	C8H8O5			183.0298	183.0294		183.0930b, 168.0066	88.5	Methylgallate
21	9.23	−0.5	C10H8O4	193.0495	193.0496			119.0495, 95.0497b, 91.0549		88.5	4-Methyl-6,7-dihydroxycoumarin
22	9.55	0.4	C20H27NO11			456.1511	456.1509		323.0979, 161.0450, 113.0239, 101.0239,71.0134, 59.0135b	89.4	Amygdalin	AS	[27,28]
23	9.64	0.6	C16H18O9			353.0878	353.0876		191.0556, 173.0449, 136.0446b	97	Cryptochlorogenic acid	LJ	[29]
24	9.8	2.4	C9H6O4			177.0193	177.0189		177.0288b, 133.0289, 105.0341	90.5	Esculetin
25	9.96	3.3	C7H6O2			121.0295	121.0291		121.0290b	89.8	p-Hydroxybenzaldehyde	IR	[17]
26	10.75	−0.3	C16H22O9	359.1336	359.1335			197.0810, 127.0392b, 179.0704, 151.0755		96.2	Sweroside	LJ	[24]
27	11.18	0	C18H19NO4	314.1386	314.1386			265.0860b, 237.0911, 222.0678		88.1	Norisoboldine
28	11.2	0.5	C27H30O15			593.1511	593.1508		473.1089, 383.0769, 353.0664b	91.7	Vicenin II	GR	[17]
29	11.5	0	C8H8O3	153.0546	153.0546			125.0600, 111.0445b		85.2	2-Hydroxy-4-methoxybenzaldehyde
30	11.63	1.0	C17H24O11			403.1246	403.1242		121.0289b	93.4	Secoxyloganin	LJ	[24]
31	11.7	2.0	C9H10O5			197.0455	197.0451		169.0138, 140.0111, 125.0239b	94	Ethyl gallate	RC	[25]
32	11.7	0.9	C32H42O16			681.2400	681.2394		357.1340,151.0395b, 136.0161	89.7	Pinoresinol diglucoside	FF	[30]
33a	11.8	0	C19H21NO4	328.1543	328.1543			297.1124b, 282.0889, 265.0862, 237.0912, 220.0677		85.9	Boldine
34	11.8	1.6	C26H28O14			563.1406	563.1397	383.0769, 353.0662b, 297.0763		94.1	Isoschaftoside	GR	[31]
35	12.2	2.5	C9H8O3			163.04	163.0396		119.0496b	92.9	p-Hydroxy-cinnamic acid
36	12.4	−0.4	C21H20O11			447.0932	447.0934		357.0611, 327.0506b, 133.0289	90.7	Homoorientin
37	12.4	−0.5	C9H10O4	183.0651	183.0652			155.0704, 140.0470, 123.0444, 95.0497b		87.4	Syringaldehydes	EH	[17]
38a	13.2	0.8	C15H12O4	257.0808	257.0806			147.0442, 137.0235b, 119.0495		95.3	Isoliquiritigenin	GR	[31]
39a	13.2	1.1	C9H8O4	181.0495	181.0493			163.0391b, 135.0443		85.9	Caffeic acid	LJ	[29]
40a	13.2	1.0	C29H36O15			623.1981	623.1975		161.0238b, 133.0289	96.3	Forsythoside I	FF	[26]
41	13.3	0	C27H32O15			595.1668	595.1668		459.1141, 151.0031b, 135.0447	88.9	Eriocitrin	MH	[32]
42	13.3	−0.2	C27H30O14	579.1708	579.1709			433.1133 313.0708 283.0603b		87.5	Vitexin-2-O-rhamnoside
43a	13.34	1.3	C26H30O13			549.1617	549.1610		255.0659b, 153.0878, 135.0083, 119.0497	93.9	Liguiritigenin-7-O-β-D-apiosyl-4′-O-β-D-glucoside
44a	13.35	0.7	C21H22O9			417.1191	417.1188		135.0082, 119.0496b, 91.0184	93.6	Liquiritin	GR	[31]
45	13.4	0.3	C27H30O16			609.1461	609.1459		300.0271b,271.0246, 255.0296, 243.0295, 151.0032	94.2	Rutin	LJ	[29]
46a	13.5	0.9	C21H20O10			431.0983	431.0979		311.0557, 283.0606, 269.0451b	86	Vitexin	EH	[33]
47a	13.6	0	C21H20O12			463.0881	463.0881		271.0246b, 255.0296, 151.0032	96.4	Hyperoside	LJHH	[18,34]
48	13.6	0.7	C14H6O8			300.9989	300.9987		300.9987b, 283.9956, 229.1361	94.1	Ellagic acid
49a	13.8	0.3	C27H30O15			593.1512	593.1510		285.0401b	95.1	Lonicerin	LJ	[24]
50	13.8	0.2	C21H20O11			447.0932	447.0931		285.0401b	94	Cynaroside	LJ	[18]
51	13.8	0	C21H18O12			461.0725	461.0725		285.0402b, 133.0289	90.2	Luteolin 7-glucuronide	MH	[35]
52a	13.8	0.6	C23H26O11			477.1402	477.1399		161.0239b, 133.0289	95.7	Calceolarioside B
53	14.07	0.6	C16H18O9	355.1023	355.1021			163.0392b, 135.0443		85.4	Chlorogenic acid	LJ	[24]
54	14.09	1.2	C25H24O12			515.1195	515.1189		353.0875, 119.0556, 179.0344, 173.0449, 135.0446b	96.3	Isochlorogenic acid B	LJ	[24]
55	14.18	3.6	C7H6O3			137.0244	137.0239		93.0340b	87.6	4-Hydroxybenzoic acid	LJ	[17]
56	14.3	0.8	C26H32O11			519.1871	519.1867		151.0395b, 136.0159	90.9	Pinoresinol-4-O-glucoside	FF	[30]
57	14.4	1.0	C25H24O12			515.1195	515.1190		353.0873, 191.0556b, 135.0446	96.3	3,5-Dicaffeoylquinic acid	LJ	[24]
58	14.7	0	C20H23NO4	342.1699	342.1699			296.1046, 280.1095b, 265.0861, 237.0912		85.2	Corydine
59a	14.8	0.7	C15H10O7	303.0499	303.0497			303.0497b, 229.0497, 153.0183		94.2	Quercetin	LJ	[24]
60	14.89	0.4	C21H20O11			447.0932	447.0930		271.0244b, 255.0295, 151.0031	95.6	Quercitrin	HH	[34]
61	14.9	0	C27H30O14	579.1708	579.1708			271.0603b, 153.0833		91.9	Rhoifolin	LJ	[18]
62	14.9	2.2	C9H10O4			181.0506	181.0502		153.0188, 109.0289b	93.3	Ethyl protocatechuate
63	14.9	2.1	C9H16O4			187.0975	187.0971		125.0966b, 97.0653	95.2	Azelaic acid
64	15.15	1.0	C25H24O12			515.1195	515.1190		191.0555, 173.0449, 135.0446b	95.7	Isochlorogenic acid C	LJ	[24]
65	15.27	0	C28H32O15	609.1813	609.1813			463.1235, 301.0709b, 286.0474		91.2	Diosmin	MH	[32]
66	15.4	−0.2	C22H22O11	463.1234	463.1235			301.0708b, 286.0473, 258.0523		86.4	Diosmetin-7-O-β-D-glucopyranoside
67a	16.13	0	C15H10O6	287.0550	287.0550			287.0552b, 153.0184		91	Kaempferol	KJ	[18]
68	16.3	0	C22H22O9	431.1337	431.1337			269.0809b		95.2	Ononin	GR	[36]
69a	16.5	0.2	C21H22O9			417.1191	417.1190		255.0659, 148.0161b, 119.0497	93.5	Isoliquiritin	GR	[31]
70a	16.89	1.1	C15H20O4	265.1434	265.1431			219.1356, 204.1150b		87.7	Abscisic acid	IR	[37]
71	16.9	2.8	C16H14O5			285.0768	285.0760		270.0530, 177.0189, 285.0760, 150.0316b	89.9	Licochalcone B	GR	[31]
72a	16.9	0.8	C15H12O4			255.0663	255.0661		135.0082, 119.0497b, 91.0184	92.3	Liquiritigenin	GR	[31]
73	16.9	0.7	C15H12O6			287.0561	287.0559		151.0032, 135.0447b, 107.0132	93.4	Eriodictyol	EHMH	[17]
74a	17	1.3	C27H30O15	595.1659	595.1651			287.0552b		90.7	Kaempferol-3-O-rutinoside	LJ	[18]
75	17.12	−0.4	C27H34O11	535.2173	535.2175			337.1434, 201.0912, 189.0913,175.0756, 151.0756, 137.0599b		91.8	Forsythin	FF	[29]
76	17.55	0.8	C28H32O14	593.1864	593.1859			447.1286, 285.0752b		89.9	Linarin	MH	[38]
77	17.8	1.8	C16H12O5	285.0757	285.0752			285.0759b, 270.0525,134.0364		85.6	Calycosin	GR	[31]
78	17.9	0.7	C15H10O6			285.0404	285.0402		285.0402b, 133.0289	95.3	Luteolin	LJ	[31]
79	17.9	1.4	C11H12O4			207.0662	207.0659		179.0344, 161.0238, 135.0446b	94.4	Ethyl caffeate	LJ	[39]
80a	18.1	0.6	C20H22O6	359.1489	359.1487			187.0756, 137.0599b		87.7	(+)-Pinoresinol	FF	[26]
81a	18.4	0.2	C21H20O9			415.1034	415.1033		253.0504b, 225.0553	94.9	Chrysophanol-8-O-β-D-glucoside
82a	18.4	0.8	C15H10O4			253.0506	253.0504		253.0503, 225.0553b	88.1	Rubiadin
83a	19.02	0.3	C15H12O5			271.06119	271.0611		151.0031, 119.0496b, 107.0133	94.6	Naringenin	GR	[31]
84	19.6	0.4	C16H14O4	271.0964	271.0963			229.0861, 177.0547,121.0288b, 111.0443		86.2	Retrochalcone	GR	[31]
85	19.9	−0.4	C30H46O3	455.3519	455.3521			409.3471, 189.1642, 95.0861b		85.7	Oleanonic acid	PH	[40]
86	20.04	0.4	C16H12O5			283.0611	283.0610		240.0425b	86.7	Physcion	RR	[17]
87a	21.6	0	C16H12O4	269.0808	269.0808			269.0809b, 254.0575		94	Formononetin	GR	[31]
88a	22.826	0	C30H46O4	471.3468	471.3468			471.3476b, 453.3369,189.1639		89.2	Glycyrrhetic acid	GR	[41]
89	22.9	0	C16H14O4	271.0964	271.0964			137.0599b, 109.0653		88.5	Medicarpin	GR	[36]
90	23.15	−0.4	C15H24O2	237.1849	237.1850			237.1853b, 219.1744,135.0807		88.1	Curdione
91	23.422	0.4	C15H10O5			269.0455	269.0454		269.0453b, 240.0424	91.8	Aloeemodin	RR	[22]
92	23.57	1.0	C42H62O16			821.3965	821.3957		351.0575, 193.0350, 113.0350b	93.1	Glycyrrhizic acid	GR	[31]
93	23.8	0.7	C15H8O6			283.0248	283.0246		239.0346, 229.0499, 183.0446b	92.8	Rhein	RR	[22]
94	23.831	1.3	C14H8O4			239.0349	239.0346		239.0346b, 211.0396, 183.0446	88.2	Antrapurol
95	26	0	C15H24O2	237.1849	237.1849			121.1016b		87.4	Dihydroartemisinic acid
96	26.69	−0.6	C21H22O4	339.159	339.1592			121.0288b		89.4	Licochalcone A	GR	[31]
97	27.75	0.4	C15H10O5			269.0455	269.0454		269.0454b, 241.0502, 225.0553, 197.0603	96.8	Emodin	RR	[42]
98	28.49	−0.4	C12H16O4	225.1121	225.1122			207.1019, 139.0392, 81.0706b		96	Pogostone	PH	[43]
99	28.5	−0.5	C15H22O	219.1743	219.1744			219.1745b, 95.0861		88.7	(+)-Nootkatone
100	29.6	−0.5	C25H28O4	393.206	393.2062			337.1431, 167.0342b, 149.0236		88.4	Kanzonol C	GR	[44]
101	30.5	−0.5	C15H22O	219.1743	219.1744			219.1746b, 137.0964		92.8	Germacrone
102	32.39	−0.8	C30H48O4	473.3625	473.3629			409.3465, 205.1592b, 189.1639		91	Maslinic acid	EH	[17]
103	36.3	−0.4	C18H30O2	279.2318	279.2319			109.1016, 95.0861, 81.0706, 67.0551b		92.4	α-Linolenic acid	IRAS	[17]
104a	39.8	−0.5	C30H48O	425.3777	425.3779			137.1327, 95.0862b		87.6	Lupenone	IR	[17]

tR: retention time.

a Representative retention time of this compound. More than one peak was identified as this compound.

b Base fragment ion. Theo. m/z: theoretical mass to charge ratio (m/z) of the compound. Exp. m/z: experimental m/z of the compound. FF: Forsythiae Fructus; LJ: Lonicerae Japonicae Flos; EH: Ephedrae Herba; AS: Armeniacae Semen Amarum; IR: Isatidis Radix; DC: Dryopteridis Crassirhizomatis Rhizoma; HH: Houttuyniae Herba; PH: Pogostemonis Herba; RR: Rhei Radix Et Rhizoma; RC: Rhodiolae Crenulatae Radix Et Rhizoma; GR: Glycyrrhizae Radix Et Rhizoma; MH: Menthae Haplocalycis Herba.

Flavonoids

The molecular formula of compound 71 was C16H14O5, as established by the [M−H]- peak at m/z 285.0760. The MS2 spectrum was similar to that previously reported [31] for licochalcone B. The MS2 spectrum and the possible fragmentation pathway are shown in Fig. S1A. The peak at m/z 270.0530 indicates the formation of [M–H–CH3]-. The base peak at m/z 150.0316 is attributed to the fragment generated by the cleavage of groups around the carbonyl carbon, followed by loss of CH3 unit. The fragment responsible for the peak at m/z 177.0189 is shown in Fig. S1A. The mzVault library alignment results (Fig. S1B; score: 89.9) revealed that the compound was licochalcone B, which is a phytochemical present in Glycyrrhizae Radix Et Rhizoma.

Terpenoids

The molecular formula of compound 26 was C16H22O9, as indicated by the molecular ion peak ([M+H]+) at m/z 359.1335. Fragmentation occurred via the loss of a neutral glucose (Glc) molecule (m/z 197.0810; [M+H–Glc]+), and the resulting ion was further fragmented to generate [M+H–Glc–H2O]+ (m/z 179.0704) and [M+H–Glc–H2O–CO]+ (m/z 151.0755). The base peak at m/z 127.0392 is attributed to the fragment that was generated by the retro-Diels-Alder fragmentation, as shown in Fig. S2A. The library alignment (Fig. S2B; score: 96.2) and comparison with literature data [24] revealed that compound 26 was sweroside, a phytochemical present in Lonicerae Japonicae Flos.

Quinones

The molecular formula of compound 97 was C15H10O5, as established by the [M−H]- peak at m/z 269.0454. Peaks at m/z 241.0502, 225.0553, and 197.0603 can be attributed to [M–H–CO]-, [M–H–CO–O]-, and [M–H–CO–O–CO]- fragments, respectively (Fig. S3A). Library alignment (Fig. S3B; score: 96.8) and comparison with literature data [42] revealed that the compound was emodin, a constituent of Rhei Radix Et Rhizoma.

Phenolic acids

Compound 31 had a molecular formula of C9H10O5, as evaluated from the molecular ion peak at m/z 197.0451 ([M−H]-) in the MS2 spectrum (Fig. S4A). The characteristic fragments were [M–H–C2H4]- (m/z 169.0138) and [M–H–C2H4–CO2]- (m/z 125.0239). Comparison with literature data [25] and library alignment (Fig. S4B; score: 94) revealed that compound 31 was ethyl gallate, a component of Rhodiolae Crenulatae Radix Et Rhizoma.

Phenylpropanoids

The peak at m/z 579.2079 in the MS spectrum of compound 75 is attributed to the [M+HCOO]- adduct, while the peak at m/z 533.2019 corresponds to [M−H]-. Characteristic peaks were m/z 371.1490, 356.1250, and 121.0288 in the MS2 spectrum. Library alignment (Fig. S5B) and a comparison with literature data [29] revealed that compound 75 was forsythin, a phytochemical present in Forsythiae Fructus. The possible fragmentation pathways and the characteristic fragments are shown in Fig. S5A.

Phenols

The peak at m/z 299.1133 in the MS2 spectrum of compound 17 is attributed to the [M−H]- ion. Some characteristic peaks and the corresponding fragments were at m/z 137.0605 ([M–H–Glc]-) and m/z 119.0496 ([M–H–Glc–H2O]-), and the data are in accordance with that of previous studies [25]. The possible fragmentation pathway is shown in Fig. S6A. Library alignment (Fig. S6B; score: 96.6) identified the compound as salidroside, a compound derived from Rhodiolae Crenulatae Radix Et Rhizoma.

Alkaloids

The molecular formula of compound 7 was C5H5N5. There were only two characteristic peaks in the MS2 spectrum (Fig. S7A): m/z 136.0619 ([M+H]+) and m/z 119.0356 ([M+H–NH3]+). Comparison with literature data [21] and the library alignment results (Fig. S7B) revealed that compound 7 was adenine, an alkaloid present in Isatidis Radix.

Miscellaneous

Compound 22 had a molecular formula of C20H27NO11, as indicated by the peaks at m/z 458.1675 ([M+H]+) and m/z 475.1922 ([M+NH4]+). The peak at m/z 296.1129 is attributed to [M+NH4–Glc]+, and the other characteristic fragments, along with the possible fragmentation pathway are shown in Fig. S8A. Comparison of the possible fragmentation pathway with reference data [27,28], along with the library alignment (Fig. S8B) of the retention time in ESI(−) mode indicated that compound 22 was amygdalin, a chemical constituent present in Armeniacae Semen Amarum.

The volatile constituents in the LHQW capsule were extracted in three different solvents, i.e., hexane, acetone, and ethyl acetate, and then detected using GC-MS. The total ion chromatograms (TICs; Fig. S9) of all three extracts were similar. The TIC of the LHQW capsule extracted in ethyl acetate is shown in Fig. 2. In total, 17 volatile compounds (Table 2) [43, [45], [46], [47], [48]] were putatively identified by comparing the obtained data with that from the NIST mass spectra library and previously reported literature [11]. The results showed that the major constituent was levomenthol with a relative percentage of 97.89%, as calculated by the peak area normalization method. The other most abundant components were patchouli alcohol (0.47%), β-pathchoulene (0.33%), α-guaiene (0.22%), and α-bulnesene (0.20%). Patchouli alcohol has anti-virus and anti-inflammatory activities reported by Kiyohara et al. [49]. Most of the components were terpenoids derived from Menthae Haplocalycis Herba and Pogostemonis Herba.

Fig. 2

(A) Total ion chromatogram (TIC) of the LHQW capsule (extracted in ethyl acetate) analyzed by GC-MS. (B) Enlarged image of the TIC from 4 to 24 min.

Table 2

Identification of the volatile components in the LHQW capsule (extracted in ethyl acetate) using GC-MS.

No.	tR (min)	Potential component	Formula	Relative amount（%）	Herb	Refs.
1	4.25	2,4-dimethylhept-1-ene	C9H18	0.07
2	6.8	α-phellandrene	C10H16	0.01
3	10.11	neoisopulegol	C10H18O	0.15
4	10.25	l-menthone	C10H18O	0.05	MH	[45]
5	10.43	isomenthone	C10H18O	0.02
6	10.5	d-menthol	C10H20O	0.07	MH	[46]
7	10.7	levomenthol	C10H20O	97.89	MH	[46]
8	14.25	β-patchoulene	C15H24	0.33	PH	[47]
9	14.68	ë-selinene	C15H24	0.02	PH	[47]
10	14.82	isocaryophillene	C15H24	0.05
11	14.98	α-guaiene	C15H24	0.22	PH	[48]
12	15.42	seychellene	C15H24	0.15	PH	[48]
13	15.59	α-patchoulene	C15H24	0.07	PH	[47]
14	15.63	10s,11s-Himachala-3(12),4-diene	C15H24	0.09
15	16.08	α-bulnesene	C15H24	0.20	PH	[47]
16	18.91	patchouli alcohol	C15H26O	0.47	PH	[48]
17a	19.36	pogostone	C12H16O4	0.14	PH	[43]

Compound was also detected by HPLC-Q Exactive-Orbitrap-MS. tR: retention time. MH: Menthae Haplocalycis Herba; PH: Pogostemonis Herba.

Conclusion

In this study, HPLC-Q Exactive-Orbitrap-MS coupled with GC-MS method was used for the first time to identify the chemical constituents in the LHQW capsule, a popular TCM prescribed to treat the symptoms of SARS-CoV-2 infections. In addition, the MS and MS2 spectrum library alignment method was found to be a convenient approach to rapidly identifying the components of a complex mixture. A total of 120 constituents, including alkaloids, flavonoids, phenols, phenolic acids, phenylpropanoids, quinones, terpenoids, and other phytochemicals, were successfully detected and putatively identified in this study. The findings of our study can contribute to future investigations on the active chemical constituents and the mechanism of action of the LHQW capsule.

Declaration of competing interest

The authors declare that there are no conflicts of interest.