Rapid Profiling of Chemical Constituents in Qingfei Paidu Granules Using High Performance Liquid Chromatography Coupled with Q Exactive Mass Spectrometry

Abstract

Qingfei Paidu (QFPD) granules have played a critical role during the Coronavirus Disease 2019 (COVID-19) in China. However, worldwide acceptance has been a problem because of the complex ingredients and unique theory of treatment. In this study, high-performance liquid chromatography (HPLC)-Q Exactive Orbitrap-mass spectrometry (MS) and the Orbitrap traditional Chinese medicine library (OTCML) were used to investigate the chemical constituents of QFPD granules. By comparing retention times, masses, isotope ion patterns, and MS² profiles, 108 compounds were putatively identified using the OTCML combined with manual verification, including 12 alkaloids, 49 flavonoids, 13 terpenoids, 14 phenylpropanoids, 4 phenolic acids, 5 phenols, and 11 other phytochemicals. Of these compounds, 17 were confirmed using reference standards. In addition, representative compounds of these different chemical types were used as examples to analyze the fragmentation pathways and characteristic product ions. Moreover, 20 herbs within the QFPD granules were also identified to establish the sources of these chemical components. This is the first rapid profiling of the chemical constituents of QFPD granules using HPLC-Q Exactive Orbitrap-MS and yields valuable information for further quality control and mechanistic studies of QFPD granules.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10337-021-04085-0.

Introduction

Qingfei Paidu (QFPD) granules and decoctions are effective traditional Chinese medicines (TCMs) that are included in the Guidelines for Diagnosis and Treatment of COVID-19 Pneumonia, issued by the National Heath Commission of the People’s Republic of China [1]. QFPD granules and decoctions are based on the following four formulae: Maxing-Shigan-Tang, Wuling-San, Xiaocaihu-Tang, and Shegan-Mahuang-Tang [2], which are different forms of prescription QFPD. QFPD granules contain 20 herbs: Ephedrae Herba, Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle, Armeniacae Semen Amarum, Cinnamomi Ramulus, Pogostemonis Herba, Alismatis Rhizoma, Polyporus, Atractylodis Macrocephalae Rhizoma, Poria, Bupleuri Radix, Scutellariae Radix, Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine, Zingiberis Rhizoma Recens, Asteris Radix Et Rhizoma, Farfarae Flos, Belamcandae Rhizoma, Asari Radix Et Rhizoma, Dioscoreae Rhizoma, Aurantii Fructus Immaturus, and Citri Reticulatae Pericarpium. In addition, QFPD contains the mineral Gypsum Fibrosum.

In China, QFPD granules and decoctions have been widely used to treat patients infected with SARS-CoV-2 owing to positive treatment results. Early treatment with prescription QFPD was associated with favorable patient outcomes and may be an effective strategy for epidemic control [1]. Functional network pharmacology analysis units showed that QFPD protected against COVID-19 through anti-viral and anti-inflammatory activities [2]. A systematic pharmacological study illustrated that QFPD exhibited immune regulation, anti-infection and anti-inflammatory properties, and multi-organ protection [3]. QFPD granules were, therefore, approved for market use by the National Medical Products Administration in China [4]. However, worldwide acceptance of QFPD granules is challenging because of the TCM complexity, and unique theory of treatment, in addition to quality and safety issues [5, 6]. Thus, comprehensive identification of the chemical components of QFPD granules is extremely critical for quality control, in addition to identification of the active ingredients and investigation of the mechanism-of-action.

Few analytical strategies have been applied to study the chemical constituents of QFPD decoctions, and no detailed analysis of the chemical composition of QFPD granules has been reported [7–9]. Hybrid quadrupole-Orbitrap mass spectrometry (MS) is a powerful tool for structure elucidation of TCMs due to its high resolution and high-quality MS² fragmentation patterns. In this study, high-performance liquid chromatography (HPLC)-Q Exactive Orbitrap-MS was used to analyze the chemical constituents of QFPD granules, with 108 compounds putatively identified, including 12 alkaloids, 49 flavonoids, 13 terpenoids, 14 phenylpropanoids, 4 phenolic acids, 5 phenols, and 11 other phytochemicals. The individual herbs within the QFPD granules were also analyzed. The aim of this study is to develop an analytical method for elucidating the chemical constituents of QFPD granules and provide valuable quality control and mechanism-of-action data.

Material and Methods

Reagents and Materials

QFPD granules were a gift from Renmin Hospital of Wuhan University. The 21 raw materials were purchased from Yifeng Pharmacy Chain Co., Ltd. (Changde, China). Acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). Formic acid was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Watsons distilled water was obtained from Jingdong Mall (Beijing, China).

Authentic standards of cytosine, sucrose, citric acid, uridine, adenosine, 2-pyrrolidinecarboxylic acid, and guanosine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nicotinic acid was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Nicotinamide and tangeretin were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Salicylic acid was acquired from Ascender Chemical Co., Ltd. (Shanghai, China). Glycyrrhizic acid, 18-β-glycyrrhetinic acid, isoliquiritigenin, baicalin, and narirutin were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Chlorogenic acid was a gift from Thermo Fisher Scientific.

Standard Solutions and Sample Preparations

The QFPD granules were ground, and the resultant powder (0.4 g) was accurately weighed, dissolved in 60% methanol (v/v; 20 mL), and sonicated for 30 min, resulting in partial precipitation of the QFPD granules. The solution was centrifuged, and the supernatant was filtered through a 0.22 μm membrane prior to HPLC-Q Exactive Orbitrap-MS.

The individual raw materials were treated using the same procedure.

The authentic standards were dissolved in 50% methanol and stored at – 80 ℃. Prior to qualitative analysis, they were mixed appropriate concentrations and filtered using a 0.22 μm membrane.

HPLC-Q Exactive Hybrid Quadrupole-Orbitrap MS

LC–MS was performed using an UltiMate 3000 UPLC system (Thermo Fisher Scientific), autosampler, a vacuum degasser, binary pump, and column compartment. A Hypersil Gold aQ C18 column (2.1 × 150 mm, 3 μm) was used at 40 ℃ for chromatography. The mobile phase consisted of acetonitrile/0.1% formic acid (A) and water/0.1% formic acid (B) at a flow rate of 0.2 mL/min. The following gradient elution program was used: 0–2 min, 0–5% (A); 2–42 min, 5–95% (A); 42–46.9 min, 95% (A); 46.9–47 min, 95–5% (A); 47–50 min, 5% (A). The total run time was 50 min, and the sample injection volume was 5 μL.

A Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) with heated electrospray ionization (ESI) was used. 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 ℃; auxiliary gas temperature, 350 ℃; sheath gas, 40 Arb; auxiliary gas, 15 Arb; sweep gas, 0 Arb; S-lens RF level, 50.

The Orbitrap mass detector was operated in full scan plus data-dependent MS² mode. The MS resolution was set at 70,000 for the full scan and 17,500 for the MS² scan. The automatic gain control target and maximum injection time were 1 × 10⁶ ions capacity and 100 ms, respectively. The top N (N: the number of most abundant ions for fragmentation) was five, while the scan range was m/z 100–1500. The normalized collision energies were 20%, 40%, and 60%, and the isolation window was 1.2 Da. The apex trigger was 5–15 s, and the loop count was 3. The dynamic exclusion was 5 s.

Data Analysis Using the Orbitrap Traditional Chinese Medicine Library (OTCML) and Manual Verification

The raw data were imported into the Compound Discoverer (CD) software, which is integrated into the OTCML. The molecular masses, retention times, fragments, and peak areas from both the positive and negative ESI modes were compared to the mzVault library, which was integrated into CD. The mzVault spectral library (Thermo Fisher Scientific) contained the retention times, precise mass ions, and MS² fragments of 1200 commercial reference standards, which were analyzed using Q Exactive Orbitrap-MS. The software identified peaks with high mass accuracy (< 10 ppm) and an isotope pattern variation within 85%. The molecular compositions adhered to the H/C ratio rules and were matched to potential compounds using ring and double-bond equivalents. The MS² profiles were compared with the reference spectra from the mzVault library. Compounds were identified only when the match score was > 85. In addition, compound identification accuracy was improved by comparing the obtained data and possible fragmentation patterns with those in the literature, and the corresponding individual herb pieces components were analyzed to determine the source of each compound and elucidate chemical compositions.

Results and discussion

Positive and negative ion modes were used to detect the chemical compounds within the QFPD granules. The base peak chromatograms (BPCs) of the QFPD granules are shown in Fig. 1. In total, 108 compounds are putatively identified (Table 1). The BPCs of the individual herb pieces are shown in Figs. S1 and S2. Compound identification is summarized below.

Fig. 1.

Base peak chromatograms of QFPD granules obtained using high performance liquid chromatography- Q Exactive hybrid quadrupole-Orbitrap mass spectrometry. A Electrospray ionization in the positive mode (ESI( +)), B electrospray ionization in the negative mode (ESI( −))

Table 1.

Identification of the chemical components of QFPD granules using high performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap mass spectrometry combined with the Orbitrap traditional Chinese medicine library

No.	RT (min)	Formula	Potential compound	Detected m/z	Characterized MS2	Compound class	Herb	Refs
1d	2.11	C12H22O11	Sucrose	341.1069 [M – H]−	89.0235 71.1031 59.0132b	Miscellaneous	PR	[10]
2d	2.12	C5H9NO2	2-Pyrrolidinecarboxylic acid	116.0709 [M + H]+	116.0708 70.0658b	Miscellaneous	DR	[11]
3d	2.12	C4H5N3O	Cytosine	112.0508 [M + H]+	112.0508b 95.0244	Alkaloids	AR
4	2.13	C5H11NO2	Betaine	118.0865 [M + H]+	118.0864b 59.0737	Alkaloids	AE
5	2.14	C7H7NO2	Trigonelline	138.0550 [M + H]+	138.0550b 110.0603 94.0656	Alkaloids	PC	[12]
6d	2.2	C6H8O7	Citric acid	191.0185 [M – H]−	111.0078b 87.0079	Miscellaneous	GR
7d	2.66	C6H5NO2	Nicotinic acid	124.0395 [M + H]+	124.0394b 96.0448 80.05	Alkaloids
8acd	2.69	C9H12N2O6	Uridine	243.0608 [M – H]−	200.0554 152.0339 122.0234 110.0238b	Miscellaneous	PR	[13]
9d	2.69	C6H6N2O	Nicotinamide	123.0555 [M + H]+	123.0554b 96.0448 80.0501	Alkaloids	FF
10ad	2.73	C10H13N5O4	Adenosine	268.1041 [M + H]+	136.0618b	Miscellaneous	PC	[14]
11acd	2.87	C10H13N5O5	Guanosine	284.0989 [M + H]+	152.0567b	Miscellaneous	PC	[14]
12	3.41	C7H6O5	Gallic acid	169.013 [M – H]−	125.0233b 97.0285 69.0337	Phenolic acids
13	3.9	C9H11NO2	l-Phenylalanine	166.0863[M + H]+	120.0809b 103.0546	Miscellaneous
14	4.22	C6H6O3	5-Hydroxymethylfurfural	127.0392 [M + H]+	109.0288b 81.0341	Miscellaneous	PC	[15]
15	4.68	C9H13NO	l-norephedrine	152.1069 [M + H]+	134.0965b 117.0701	Alkaloids	EH	[16]
16	5.12	C9H13NO	d-norpseudoephedrine	152.1069 [M + H]+	134.0965b 117.0701	Alkaloids	EH	[16]
17	5.24	C15H14O7	( −)-Gallocatechin	305.0651 [M – H]−	219.0654 137.0232 125.0232b	Phenols
18	5.32	C7H6O4	Protocatechuic acid	153.0181 [M – H]−	109.0284b	Phenolic acids	GR	[15]
19	6.09	C10H15NO	l-ephedrine	166.1226 [M + H]+	148.1120b 133.0887 117.0701 91.0547	Alkaloids	EH	[16]
20	6.47	C10H15NO	d-pseudoephedrine	166.1226 [M + H]+	148.1120b 133.0887 117.0701 91.0547	Alkaloids	EH	[16]
21	6.99	C11H17NO	Methylephedrine	180.1382 [M + H]+	162.1276b 147.1041 135.0805 117.0701	Alkaloids	EH	[16]
22	7.26	C7H6O3	Protocatechualdehyde	137.0233 [M – H]−	137.0233b 119.0126 109.0285	Phenols	CR	[15]
23c	9.16	C20H27NO11	Amygdalin	456.1492 [M – H]−	323.0963 221.0653 161.0443 59.0132b	Miscellaneous	AS	[17]
24 cd	9.43	C16H18O9	Chlorogenic acid	353.0862 [M – H]−	191.0548b 135.0441 179.0337	Phenylpropanoids	FF	[18]
25	9.46	C9H6O4	Esculetin	177.0181 [M – H]−	177.0180b 149.0236 133.0284 105.0336	Phenylpropanoids
26	9.48	C7H6O2	p-Hydroxybenzaldehyde	121.0285 [M – H]−	121.0284b 93.0336	Phenols
27	9.78	C9H8O4	Caffeic acid	179.0337 [M – H]−	135.0441b	Phenolic acids
28a	10.49	C15H14O6	Catechin hydrate	289.0703 [M – H]−	245.0805 123.044 109.0284b	Flavonoids
29a	10.63	C15H12O7	Taxifolin	303.0494 [M – H]−	177.018 125.0233b	Flavonoids	SR	[19]
30c	10.98	C27H30O15	Vicenin II	593.1482 [M – H]−	353.0648b 383.0753 473.1062 297.075	Flavonoids	GR	[8]
31	11.12	C25H24O12	1,3-Dicaffeoylquinic acid	515.1168 [M – H]−	353.0859 191.0547b 179.0336 135.044	Phenylpropanoids
32	11.87	C9H8O3	p-Coumaric acid	163.0400 [M – H]−	119.0496b 163.0394	Phenylpropanoids
33ac	11.95	C26H28O14	Isoschaftoside	563.1376 [M – H]−	353.0648b 383.0754 473.1073	Flavonoids	GR	[20]
34	11.96	C9H10O4	3,5-Dimethoxy-4-hydroxybenzaldehyde	183.0652 [M + H]+	140.0469 123.0443 95.0497b	Phenols	CR EH RE	[15]
35	12.18	C21H20O11	Orientin	447.0913 [M – H]−	357.06 327.0496b 299.0541 133.028	Alkaloids	CP	[21]
36	12.6	C10H8O4	Scopoletin	193.0497 [M + H]+	193.0496b 178.026 133.0285	Phenylpropanoids	AF	[22]
37ac	12.7	C26H30O13	Naringenin 7-O-(2-β-d-apiofuranosyl)-β-d-glucopyranoside	549.1588 [M – H]−	255.0649 135.0077 119.0492b	Flavonoids	GR	[20]
38	12.84	C10H10O4	Ferulic acid	193.0492 [M – H]−	178.0258 134.0362b	Phenylpropanoids
39	13.03	C11H10O5	Isofraxidin	223.0601 [M + H]+	223.0601b 190.0261 162.0311	Phenylpropanoids
40c	13.04	C27H32O15	Eriocitrin	595.1638 [M – H]−	459.1152 151.0025b 135.0441	Flavonoids	CP AF	[21] [23]
41c	13.04	C26H30O13	Liquiritin apioside	549.1586 [M – H]−	119.0491b 135.0077 255.0649	Flavonoids	GR	[20]
42c	13.18	C27H30O16	Rutin	609.1431[M – H]−	300.0258b 271.0234 255.0284	Flavonoids
43	13.39	C9H6O4	5,7-Dihydroxychromone	177.0180 [M – H]−	177.0180b 135.0076	Flavonoids
44d	13.75	C7H6O3	Salicylic acid	137.0233 [M – H]−	137.0233 93.0337b	Phenolic acids	AE	[15]
45	13.8	C14H12O4	Piceatannol	243.0648 [M – H]−	243.0648b 201.0544 159.0439	Phenols
46	13.84	C25H24O12	Isochlorogenic acid B	515.1165 [M – H]−	353.0856 191.0547 179.0336 135.0440b	Phenylpropanoids	FF	[18]
47 cd	13.89	C27H32O14	Narirutin	581.1863 [M + H]+	273.0755b 153.0181 85.0289 71.0498	Flavonoids	CP	[21]
48a	14.02	C29H36O15	Verbascoside	623.1945 [M – H]−	461.1639 161.0231b 133.0283	Phenylpropanoids	PH	[15]
49	14.13	C25H24O12	3,5-Dicaffeoylquinic acid	515.1165 [M – H]−	353.0878 191.0558b 179.0346 135.0448	Phenylpropanoids	FF	[18]
50	14.14	C22H22O11	Tectoridin	463.1234 [M + H]+	301.0705b 286.047	Flavonoids	BH	[24] [25]
51ac	14.27	C27H32O14	Naringin	579.1688 [M – H]−	271.0597 151.0025b 119.0491 107.0129	Flavonoids	AF CP	[23] [21]
52	14.57	C9H16O4	Azelaic acid	187.0962[M – H]−	125.0960b 97.0649	Miscellaneous
53	14.62	C28H34O15	Neohesperidin	609.1796 [M – H]−	609.1791 301.0700b 286.0466	Flavonoids	CP
54	14.95	C25H24O12	Isochlorogenic acid C	515.1166 [M – H]−	353.088 191.0558 173.0452 135.0448b	Phenylpropanoids	FF	[18]
55c	15	C28H34O15	Hesperidin	609.1796 [M – H]−	609.1791 301.0699b 286.0466	Flavonoids	CP	[26]
56	15.06	C24H26O13	Iridin	523.1445 [M + H]+	361.0915b 346.0679 331.0445	Flavonoids	BH	[25]
57	15.06	C9H6O2	Coumarin	147.0440 [M + H]+	147.0440b 103.0546 91.0547	Phenylpropanoids	CR EH	[15]
58a	15.63	C15H12O6	Eriodictyol	287.0547 [M – H]−	287.0547 161.0231 125.0233b	Flavonoids	AF	[23]
59c	15.86	C26H30O13	Isoliquiritin apioside	549.1589 [M – H]−	255.0649 153.0181 135.0077 119.0491b	Flavonoids	GR	[20]
60	16.06	C22H22O9	Ononin	431.1336 [M + H]+	269.0807b 254.0573 237.0544	Flavonoids	GR	[27]
61 cd	16.08	C21H18O11	Baicalin	445.0753 [M – H]−	269.0439b	Flavonoids	SR	[19]
62	16.15	C21H20O10	Oroxin A	433.1129 [M + H]+	271.0599b 253.0493 123.0078	Flavonoids	SR	[19]
63a	16.23	C21H22O9	Isoliquiritin	419.1334 [M + H]+	257.0806b 147.0439 137.0232	Flavonoids	GR	[20]
64c	16.51	C15H12O4	Liquiritigenin	255.0651 [M – H]−	135.0078 119.0492b 91.0181	Flavonoids	GR	[20]
65	16.87	C11H6O4	Bergaptol	201.0180 [M – H]−	201.0192b 183.1012 139.1117	Phenylpropanoids
66c	16.92	C21H18O11	Norwogonin-8-glucuronide	445.0753 [M – H]−	269.0441b	Flavonoids	SR	[19]
67	17.2	C28H34O14	Poncirin	593.1842 [M – H]−	593.184 285.0753b 151.0024	Flavonoids	AF	[23]
68	17.24	C21H18O11	Norwogonin-7-glucuronide	445.0752 [M – H]−	269.0439b	Flavonoids	SR	[19]
69c	17.51	C22H20O11	Oroxylin A-7-O-β-d-glucuronide	459.0910 [M – H]−	283.0595 268.0362b	Flavonoids	SR	[19]
70c	18.11	C21H18O11	Baicalein-6-glucuronide	445.0754 [M – H]−	269.0441b	Flavonoids	SR	[19]
71c	18.14	C22H20O11	Wogonoside	459.0909 [M – H]−	283.0595 268.0362b	Flavonoids	SR	[19]
72	18.58	C15H12O5	Naringenin chalcone	273.0756 [M + H]+	273.0757 153.0182b 147.044 119.0493	Flavonoids	GR AF CP	[20] [23] [21]
73c	19.32	C16H14O6	Hesperetin	301.0702 [M – H]−	301.0702b 286.044 164.0103 108.0207	Flavonoids	CP	[26]
74	19.38	C16H12O6	Tectorigenin	299.0546[M – H]−	284.0301b 240.0414	Flavonoids
75ac	20.01	C18H16O8	Irigenin	359.0756 [M – H]−	344.0519 329.0286b 314.0054 286.0104	Flavonoids	BH	[24]
76	20.03	C16H12O7	Isorhamnetin	315.0496 [M – H]−	315.0496 300.0260b 271.0237 151.002	Flavonoids	AE	[28]
77c	20.15	C17H14O7	Iristectorigenin B	329.0652 [M – H]−	314.0417 299.0180b 271.0235	Flavonoids	BH	[24]
78	20.39	C15H10O5	Baicalein	269.0443 [M – H]−	269.0457b 241.0507 223.0398	Flavonoids	SR	[19]
79 cd	20.97	C15H12O4	Isoliquiritigenin	255.0650 [M – H]−	135.0076 119.0491b 91.018	Flavonoids	GR	[20]
80	21.08	C16H12O4	Formononetin	269.0807 [M + H]+	269.0807b 254.0574	Flavonoids	GR	[20]
81ac	21.35	C15H16O4	Isomeranzin	261.1119 [M + H]+	189.0546b 159.0439 131.0492	Phenylpropanoids
82ac	21.65	C42H62O17	Licorice-saponin G2	837.3869 [M – H]−	837.386 351.0552 193.0341 113.0235b	Terpenoids	GR	[20]
83c	21.83	C26H30O8	Limonin	469.1846 [M – H]−	469.1831b 249.0909 229.1214	Terpenoids	AF	[22]
84	21.83	C15H22O2	Curcumenol	235.1693 [M + H]+	235.169 217.1588 199.1482b	Terpenoids
85	22.15	C20H20O7	Isosinensetin	373.1283 [M + H]+	373.1281b 343.0812	Flavonoids	CP	[26]
86 cd	22.64	C30H46O4	18 β-Glycyrrhetinic acid	471.3469 [M + H]+	417.3471b 453.3362	Terpenoids	GR	[29]
87 cd	22.65	C42H62O16	Glycyrrhizic acid	821.3926 [M – H]−	821.3915b 351.056 113.0234	Terpenoids	GR	[20]
88	22.72	C20H18O8	Irisflorentin	387.1073 [M + H]+	387.1073b 372.0843 357.0603 329.0654	Flavonoids	BH	[25]
89	22.84	C20H20O7	Sinensetin	373.1282 [M + H]+	373.1281b 343.0809	Flavonoids	CP	[26]
90	22.98	C18H14O8	Dichotomitin	359.0762 [M + H]+	359.0761b 344.0526 326.0421 299.0549	Flavonoids	BH	[24]
91a	23.16	C16H12O5	Wogonin	285.0757 [M + H]+	285.0756 270.0521b	Flavonoids	SR	[19]
92	23.34	C42H62O16	isomer of Glycyrrhizic acid	821.3922 [M – H]−	821.3919b 351.0551 113.0235	Terpenoids	GR
93c	23.57	C42H68O13	Saikosaponin A	825.4599 [M + COOH]−	779.4534b 617.40106 59.0132	Terpenoids	BR	[26]
94	23.91	C17H14O6	Pectolinarigenin	313.0703 [M – H]−	313.0701 283.0233b 255.0286	Flavonoids	SR	[19]
95	23.99	C21H22O8	Nobiletin	403.1388 [M + H]+	403.1388 373.0917b 211.0238 183.0288	Flavonoids	CP	[26]
96a	24.04	C19H18O6	6-Demethoxytangeretin	343.1174 [M + H]+	343.1173 313.0705b 285.0756	Flavonoids	CP	[26]
97c	24.09	C42H68O13	Saikosaponin B1	825.4599 [M + COOH]−	779.4542b 617.4028 59.0132	Terpenoids	BR
98a	24.13	C16H12O5	Oroxylin A	285.0758 [M + H]+	285.0757 270.0523b 168.0054	Flavonoids	SR	[19]
99	24.21	C15H20O3	Atractylenolide III	249.1486 [M + H]+	231.1379b 249.1481 213.1276 163.0752	Terpenoids	AM	[30]
100	24.49	C22H24O9	Heptamethoxyflavone	433.1493 [M + H]+	403.1021 433.1492b 165.0546	Flavonoids	CP	[26]
101c	24.7	C42H68O13	Saikosaponin D	825.4594 [M + COOH]−	779.4537b 617.4034 59.0132	Terpenoids	BR
102d	25.67	C20H20O7	Tangeretin	373.1280 [M + H]+	373.1278 358.1043 343.0808b 328.0573	Flavonoids	CP	[26]
103	26.41	C20H20O8	5-O-Demethylnobiletin	389.1230 [M + H]+	389.1227b 359.076 341.0652	Flavonoids	CP	[21]
104	26.68	C32H48O6	Alisol C 23-acetate	529.3526 [M + H]+	529.3521b 469.3314 451.3204 415.2842	Terpenoids	AR	[31]
105	27.85	C15H20O2	Atractylenolide II	233.1536 [M + H]+	233.1536b 215.1432 187.1482 151.0753	Terpenoids	AM	[30]
106	27.94	C12H16O4	Pogostone	225.1122 [M + H]+	207.1015 139.039 81.0705b	Miscellaneous	PH	[32]
107	34.7	C32H50O5	Alisol B 23-acetate	515.3733 [M + H]+	437.3412 339.2679 419.3305 97.0653b	Terpenoids	AR	[31]
108	35.79	C18H30O2	α-Linolenic acid	279.2318 [M + H]+	95.086 81.0705 67.055b	Alkaloids

RT retention time

^aRepresentative retention time, as more than one peak was identified for this compound

^bBase fragment ion

^cCompounds detected using both the positive and negative electrospray ionization modes. m/z: mass-to-charge ratio

^dCompounds identified by comparison with reference standards. Herb: Compound detected within herb experimentally and also the reference reported the source of the compound. Ref.: The references that reported the sources of the compounds. EH (Ephedrae Herba), GR (Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle), AS (Armeniacae Semen Amarum), CR (Cinnamomi Ramulus), PH (Pogostemonis Herba), AR (Alismatis Rhizoma), PP (Polyporus), AM (Atractylodis Macrocephalae Rhizoma), PR (Poria), BR (Bupleuri Radix), SR (Scutellariae Radix); PC (Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine), ZR (Zingiberis Rhizoma Recens), AE (Asteris Radix Et Rhizoma), FF (Farfarae Flos), BH (Belamcandae Rhizoma), RE (Asari Radix Et Rhizoma), DR (Dioscoreae Rhizoma), AF (Aurantii Fructus Immaturus); CP (Citri Reticulatae Pericarpium)

Alkaloids

Twelve alkaloids were detected. Compounds 19, 20, 21, 15, and 16 are observed in the positive BPC of QFPD, with no matching identification results after data processing using the OTCML. The mass spectra of compounds 15, 19, and 21 display the same fragment ions at m/z 117.0701 (Fig. S3). The mass spectra of compounds 19 and 20 exhibit the same [M + H]⁺ ions at m/z 166.1226 (C₁₀H₁₅NO), with the same fragment ions also observed at m/z 148.1120 [M + H – H₂O]⁺ and 133.0887 [M + H – H₂O – CH₃]⁺. According to the literature [16], they are identified as L-ephedrine (19) and D-pseudoephedrine (20). The mass spectrum of compound 21 (methylephedrine) reveals a peak representing the protonated molecule [M + H]⁺, at m/z 180.1382, and fragment ion peaks at m/z 162.1276 [M + H – H₂O]⁺ and 147.1041 [M + H – H₂O – CH₃]⁺. The mass spectra of compounds 15 (l-norephedrine) and 16 (D-norpseudoephedrine) reveal the same peak at m/z 152.1069, and MS² peaks at m/z 134.0965 [M + H – H₂O]⁺ and 117.0701 [M + H – H₂O – NH₃]⁺. However, they exhibit different retention times. These compounds are phytochemicals present in Ephedrae Herba.

Compounds 3 (cytosine), 7 (nicotinic acid), and 9 (nicotinamide) were identified by comparing the retention times and MS² fragmentation patterns with those of reference standards. Nicotinic acid and nicotinamide exhibit the same structural skeleton, and fragment ion peaks at m/z 96.0448 [M + H – CO]⁺ are observed in the MS² profiles. Their possible fragmentation pathways and library match results are shown in Fig. S4. The MS² profile of compound 5 reveals a peak representing a protonated molecule, [M + H]⁺, at m/z 138.0550 and peaks at m/z 110.0603 [M + H − CO]⁺ and 94.0656 [M + H − CO − O]⁺. Therefore, compound 5 is deduced to be trigonelline.

Flavonoids

Forty-nine compounds were identified as flavonoids. Compounds 47 and 51 were identified as narirutin and naringin, respectively, by comparison with the OTCML. Furthermore, compound 47 was confirmed using a reference standard. They were detected in both the positive and negative ESI modes, displaying similar MS and MS² profiles that revealed peaks representing [M − H]⁻ ions at m/z 579.1688. Fragment ions were represented by peaks at m/z 271.0615, owing to the loss of glucose (Glc) and rhamnose moieties [21]. Characterized fragment ions represented by peaks at m/z 151.0034 and 119.0499 were generated by retro-Diels–Alder cleavage. Narirutin and naringin are flavonoid O-glycoside isomers distinguished by their different retention times. Compounds 87 (isosinensetin), 89 (sinensetin), 96 (6-demethoxytangeretin), 95 (nobiletin), and 102 (tangeretin) are polymethoxyflavones, bearing numerous methoxyl and/or hydroxyl groups on the basic structure. The mass spectra of these compounds show peaks representing [M + H]⁺ ions and characterized fragment ions due to continuous CH₃ loss [26]. The MS² profiles and library match results are shown in Fig. S5. As examples, the mass spectra of compounds 85 and 89 reveal peaks representing [M + H]⁺ ions at m/z 373.1283 and characterized fragment ions at m/z 343.08 [M + H − 2CH₃]⁺. The spectra are very similar, and the compounds were identified using the OTCML by the different retention times and slight differences in the spectra. Compound 102 (tangeretin) was further confirmed using a reference standard. Compounds 30 and 33 showed similar MS² patterns, but the molecular ions were different, indicating the same basic structure. These compounds were assigned as vicenin II [8] and isoschaftoside [20, 33], respectively. For example, the mass spectrum of compound 30 revealed peaks representing the [M − H]⁻ ion at m/z 593.1482 and fragment ions at m/z 297.0750 [M − H − Glc − Glc]⁻, m/z 473.1062 [M − H − 120]⁻, m/z 383.0753 [M – H – 210]⁻, and m/z 353.0648 [M − H − 240]⁻. These are characterized fragment ions of the hexose ring-opening reaction [33]. The similarities of the MS and MS² profiles of compounds 37, 41, and 59 indicated isomers. By comparing the data in the OTCML combined with literature data [20], they were deduced as naringenin 7-O-(2-β-d-apiofuranosyl)-β-d-glucopyranoside (37), liquiritin apioside (41), and isoliquiritin apioside (59). The mass spectra of compounds 61, 68, 66, and 70 revealed peaks representing [M − H]⁻ ions at m/z 445.07 and dominant fragment ions at m/z 269.04, along with [M + H]⁺ ions at m/z 447.09 and dominant fragment ions at m/z 271.05. Individual herb pieces component mass spectra showed that these compounds, baicalin (61), norwogonin-7-glucuronide (68), norwogonin-8-glucuronide (66) and baicalein-6-glucuronide (70), were chemical components of Scutellariae Radix [19], and baicalin (61) was identified using a reference standard. Based on the literature [26], compounds 73 and 100 were assigned as hesperetin and heptamethoxyflavone, respectively. Compound 79 (isoliquiritigenin) was identified using a reference standard.

Phenylpropanoids

Fourteen compounds were identified as phenylpropanoids. Compounds 31 (1,3-dicaffeoylquinic acid), 46 (isochlorogenic acid B), 49 (3,5-dicaffeoylquinic acid) and 53 (isochlorogenic acid C) were identified using the OTCML. Compound 24 (chlorogenic acid) was identified using a reference standard. Compounds 31, 46, 49 and 53 were isomers with skeletons similar to those of quinic and caffeic acid, generating similar MS and MS² profiles and distinguished by their retention times. For example, the MS² profile of compound 46 revealed peaks representing fragment ions at m/z 191.0547 [quinic acid − H]⁻, 179.0336 [caffeic acid − H]⁻ and 135.0440 [caffeic acid − CO₂ − H]⁻. The mass spectrum of compound 38 (ferulic acid) showed peaks representing a [M – H]⁻ ion at m/z 193.0492 and the main fragment ions at m/z 134.0362 [M − H − CH₃ − CO₂]⁻ and 178.0258 [M − H − CH₃]⁻. Compounds 32 (p-coumaric acid), 57 (coumarin), 65 (bergaptol) and 25 (esculetin) were assigned using the OTCML.

Phenolic Acids and Phenols

Four phenolic acids were identified, and they exhibited the same fragmentation pattern. The MS² profile of compound 12 (gallic acid) revealed peaks representing [M − H]⁻ at m/z 169.0130 and ions at m/z 125.0233 [M − H − CO₂]⁻, 97.0285 [M − H − CO₂ − CO]⁻ and 69.0337 [M − H − CO₂ − CO − CO]⁻. The mass spectrum of compound 18 (protocatechuic acid) revealed a peak representing a base fragment ion at m/z 109.0284 [M − H − CO₂]⁻. Compound 44 (salicylic acid) was identified by comparison with a reference standard. All of these compounds exhibited successive losses of H₂O, CO and CO₂ during fragmentation [34, 35].

Five phenols were identified. Compound 26 (p-hydroxybenzaldehyde) produced several clear fragment ions at high collision energies. Compound 22 (protocatechualdehyde) was identified using the OTCML. The phenols also showed neutral losses of CO, CH₃ and H₂O in the MS² profiles.

Terpenoids

Thirteen terpenoids are identified. The mass spectra of compounds 82 and 87 reveal peaks representing [M + H]⁺ ions at m/z 839.4061 and 823.4108, respectively. The mass spectrum of compound 82 (licorice-saponin G2) reveals peaks representing fragment ions at m/z 469.3314 [Aglycone + H − H₂O]⁺, 487.3412 [Aglycone + H]⁺ and 451.3212 [Aglycone + H − 2H₂O]⁺ [36]. Compound 87 displays a similar fragmentation pattern, yet is 16 Da smaller than compound 82. Compound 87 was then confirmed as glycyrrhizic acid through a comparison between the negative ESI mode data, a reference standard, and literature data [20]. These spectra are shown in Fig.S6. The mass spectrum of compound 86, 18 β-glycyrrhetinic acid, reveals a peak representing [M + H]⁺ at m/z 471.3469. These are triterpenic acids. Compound 86 (18 β-glycyrrhetinic acid) was also identified using a reference standard.

The MS² profile of compound 104 showed peaks representing a protonated molecule, [M + H]⁺, at m/z 529.3526 and dominant fragment ions at m/z 529.3521 [M + H]⁺, 469.3314 [M + H − HAc]⁺, 451.3204 [M + H – HAc − H₂O]⁺ and 415.2842 [M + H − C₄H₈O − H₂O]⁺ [31]. This compound was identified as alisol C 23-acetate using the OTCML. The mass spectrum of compound 107, alisol B 23-acetate, revealed a peak representing [M + H]⁺ at m/z 515.3733.

The mass spectrum of compound 93 revealed peaks representing a [M + H]⁺ ion at m/z 781.4732 and fragment ions at m/z 455.3518 [M + H − H₂O − Fuc (fucose) Glc]⁺ and 437.3412 [M + H − 2H₂O − FucGlc]⁺. This compound was identified as saikosaponin A by comparison with data obtained from the OTCML. The mass spectrum of compound 83 exhibited peaks representing [M + H]⁺ at m/z 471.2016 and fragment ions at m/z 425.1957 [M + H − 46]⁺ and 161.0597. According to the literature [23] and the data in the OTCML, it was limonin.

The mass spectra of compounds 99 and 105 revealed peaks representing [M + H]⁺ ions at m/z 249.1486 and 233.1536, respectively. They were identified as atractylenolide III and atractylenolide II, respectively, using the OTCML. The MS² profile of atractylenolide III revealed peaks representing fragment ions at m/z 249.1481 [M + H]⁺, 231.1379 [M + H − H₂O]⁺, 213.1276 [M + H − 2H₂O]⁺ and 203.1430 [M + H − H₂O − CO]⁺ [30].

Other Phytochemicals

Eleven compounds were identified by comparing the obtained data to the information in the OTCML, including the hydrophilic compounds 1 (sucrose), 2 (2-pyrrolidinecarboxylic acid), 6 (citric acid), 8 (uridine), 10 (adenosine) and 11 (guanosine). These compounds were also confirmed using reference standards.

Quantification Analysis

The extracted ion chromatograms (EICs) of 17 authentic standards compared with those of their corresponding detected compounds within QFPD granules are shown in Fig. 2. The HPLC-Q Exactive hybrid quadrupole-Orbitrap MS method was also used for quantification analysis of these 17 constituents within QFPD granules. The concentration of each constituent was obtained using the respective calibration curve and their contents within the QFPD granules are listed in Table 2.

Fig. 2.

Extracted ion chromatograms (EICs) of 17 authentic standards compared with those of the corresponding compounds detected within QFPD granules. A EICs of compounds 3, 11, and 24; B EICs of compounds 7, 44, and 9; C EICs of compounds 8, 86, and 79; D EICs of compounds 1, 102, and 47; E EICs of compounds 2, 6, and 61; F EICs of compounds 10 and 87. R sample from the QFPD granules, S authentic standards

Table 2.

Contents of the constituents within QFPD granules

No	Compound	Conc.(mg/g)
1	Sucrose	0.683 ± 0.185
2	2-Pyrrolidinecarboxylic acid	0.803 ± 0.017
3	Cytosine	0.009 ± 0.0008
6	Citric acid	4.309 ± 0.352
7	Nicotinic acid	0.011 ± 0.0007
8	Uridine	0.029 ± 0.005
9	Nicotinamide	0.006 ± 0.0003
10	Adenosine	0.164 ± 0.009
11	Guanosine	0.135 ± 0.004
24	Chlorogenic acid	0.854 ± 0.015
44	Salicylic acid	0.016 ± 0.002
47	Narirutin	0.699 ± 0.119
61	Baicalin	4.383 ± 1.107
79	Isoliquiritigenin	0.007 ± 0.00005
86	18 β-Glycyrrhetinic Acid	0.0005 ± 0.000007
87	Glycyrrhizic acid	2.199 ± 0.127
102	Tangeretin	0.003 ± 0.0003

Data present the (average ± standard deviation) of three replicates. Conc. (mg/g): mg of the constituent/ g of QFPD granules

Compounds from Individual Herbs Within QFPD Granules

In total, 265 compounds were putatively identified using the OTCML combined with manual verification from 20 herbs that are components of QFPD granules (Table S1), including 33 alkaloids, 106 flavonoids, 28 terpenoids, 41 phenylpropanoids, 10 phenolic acids, 18 phenols and 29 other phytochemicals. Of these, 163 compounds were from only one herb, and 102 compounds were from more than two herbs. Within the QFPD granules, 59 compounds were from only one herb and 49 compounds were from more than two herbs.

Conclusions

In this study, HPLC-Q Exactive hybrid quadrupole-Orbitrap MS coupled with the OTCML which is an automatic data analysis platform, was used to study the chemical profile of QFPD granules, an effective TCM prescribed to treat the symptoms of SARS-CoV-2 infections. Furthermore, manual verification ensured compound identification. A total of 108 compounds were putatively identified from QFPD granules, including alkaloids, flavonoids, phenylpropanoids, phenolic acids, phenols, terpenoids and other phytochemicals. This allowed rapid chemical composition screening of QFPD granules, providing potentially valuable information for quality control and further clinical application.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Conflict of Interest

The authors declare that there are no conflicts of interest.

Ethical Statement

We certify that this manuscript is original, has not been previously published and will not be submitted elsewhere for publication while under consideration by Chromatographia. This study is not split into several parts and submitted to various journals. Results are presented clearly, honestly and without fabrication. No data, text, or theories by others are presented as our own.

Human and Animal Rights

Explicit permission to submit has been received from all co-authors. All the authors whose name appear on the submission have contributed sufficiently to this study.

This manuscript does not contain any studies involving humans or animals.