Investigation of the Chemical Changes from Crude and Processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma Herbal Pair Extracts by Using Q Exactive High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS.

The Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair is mainly used for regulating the functions of liver and spleen, benefiting qi, and nourishing blood. However, the bioactive compounds for the pharmacological activities of the crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair extracts are still unclear to date. In the present study, Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS was applied to identify the complicated components from crude and processed Paeoniae Radix Alba, crude and processed Atractylodis Macrocephalae Rhizoma, and their crude and processed herbal pair extracts. 123 and 101 compounds were identified in crude and processed Paeoniae Radix Alba samples, respectively. Meanwhile, 32 and 26 compounds were identified in crude and processed Atractylodis Macrocephalae Rhizoma samples, respectively. In the crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair extracts, co-decoction could significantly change the chemical composition of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma in solution. The developed method may provide a scientific foundation for deeply elucidating the processing and compatibility mechanism of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma.

1. Introduction

Traditional Chinese medicine (TCM) processing is regarded as a pharmaceutical technology based on TCM theory, the requirements of different syndrome treatment, the quality nature of medicine, and different demands of clinical dispensing and preparations [1]. It is one of the characteristics in application of TCM. The compatible components of prescription are composed of prepared Chinese crude drugs after TCM processing.

The prescription compatibility and TCM processing are not only two major features of clinical medication in TCM, but are also critical to distinguish TCM from natural medicine. The research on structural features, compatible effect, and material basis of the herbal pair is the important support in the study of the prescription compatibility since the herbal pair is the minimum unit in prescription of TCM [2, 3]. They play a guidance and significant role in reveal of the compatibility rule and the scientific connotation. The herbal pair compatibility theory can explain the relationship of the prescription compatibility to some extent. The research on the relationship between the herbal pair compatibility and the prescription compatibility contributes to the elucidation of the prescription compatibility mechanism and the action mechanism of treatment. There are many herbal pairs commonly used in the clinical practice of TCM, such as the herbal pairs of Paeonia Lactiflora-Liquorice, Ginseng-Aconite, and Aconite-Rhizome Zingiberis [4, 5] besides the Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair frequently used in all China dynasties [6, 7]. Paeoniae Radix Alba nourishes blood and liver, and Atractylodis Macrocephalae Rhizoma helps invigorate spleen and eliminate dampness [8-12]. Thus, the compatibility of these two medicines could help achieve the goal of purging wood from the earth, regulating the functions of liver and spleen, benefiting qi, and nourishing blood [13-15]. Although the compositions of these two medicines have been extensively studied, the appropriate processing method of them, such as frying, which is believed by the practitioners of traditional medicine to have the effects for enhancing the efficacy of the medicine, and their underlying compatibility mechanism are still under investigation.

The objective of this study is to investigate the qualitative, preprocessing, and postprocessing changes in the composition and compatibility of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma by using Q Exactive hybrid quadrupole-Orbitrap mass spectrometer combined with high-performance quadrupole precursor selection with high-resolution and accurate-mass Orbitrap detection. The work could serve as a theoretical basis for the development of medicines from Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma, and the reasonable clinical medication. Furthermore, it provides new insights into the investigation of the herbal pair and for the study of the appropriate processing method for Chinese herbal medicines and their underlying compatibility mechanism.

2. Experimental

2.1. Chemicals, Solvents, and Herbal Materials

Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma samples were acquired from Zhejiang suppliers. All of these herbal samples were authenticated by Professor Jianwei Chen (College of Pharmacy, Nanjing University of Chinese Medicine). HPLC-grade acetonitrile and formic acid were obtained from Merck (Darmstadt, Germany). Deionized water was purified using the Milli-Q system (Millipore, Bedford, MA, USA). All other reagents and chemicals were analytical grade.

2.2. Preparation of the Sample Solutions

The dried and powdered samples of crude and processed Paeoniae Radix Alba, crude and processed Atractylodis Macrocephalae Rhizoma, and their crude and processed herbal pair extracts (1 : 1, g/g) were prepared. A total of 2.0 g of each sample powder was accurately weighed and transferred into a 50 mL round bottom flask with 20 mL of 70% methanol aqueous solution (v/v) and refluxed in a 80°C water bath for 1 h. The filtrate was collected after filtration and the residue was then refluxed with 20 mL of 70% methanol aqueous solution in a 80°C water bath for 1 h, the filtrate was collected again after filtration and the residue was removed. Finally, the combined filtrates were treated by rotary evaporation concentration and the resultant residue was dissolved and transferred into a 25 mL volumetric flask with 70% methanol aqueous solution to make it up to a final concentration of 0.08 g·mL−1. All solutions were stored at 4°C and filtered through a 0.22 μm filter membrane before injection into the HPLC system.

2.3. Liquid Chromatography and Mass Spectrometry

Analyses were performed by using Dionex UltiMate 3000 HPLC system (Dionex, Sunnyvale, CA, USA) with a diode array detector. Detection wavelengths were set at 255 nm. A Thermo Scientific Hypersil Gold C18 column (100 mm × 2.1 mm, 1.9 μm) was used with a flow rate of 0.35 mL·min−1. The injection volume was 5 μL, and the column temperature was maintained at 30°C. The sample separation was performed according to the previous reports with minor modification [16-18]. The mobile phase was composed of (a) aqueous formic acid (0.1%, v/v) and (b) acetonitrile under following gradient elution: 10–55% B from 0 to 40 min, 55–90% B from 40 to 51 min, 90% B from 51 to 56 min, 90–10% B from 56 to 56.1 min, and 10% B from 56.1 to 60 min. Mass spectrometry was performed on a Q Exactive high-resolution benchtop quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, USA) using a heated electrospray ionization (HESI-II) source for ionization of the target compounds in positive and negative ion modes. The key parameters were as follows: ionization voltage, +3.0 kV/−2.8 kV; sheath gas pressure, 35 arbitrary units; auxiliary gas, 10 arbitrary units; heat temperature, 300°C; and capillary temperature, 300°C. For the compounds of interest, a scan range of m/z 150–1500 was chosen. Resolution for higher energy collisional dissociation cell (HCD) spectra was set to 17,500 at m/z 150 on the Q Exactive.

3. Results and Discussion

3.1. Identification of the Main Components in Crude and Processed Paeoniae Radix Alba

Tentative identification of the main compounds in crude and processed Paeoniae Radix Alba samples was generated based on elemental composition data determined from accurate mass measurements and comparison with the literature data. The total ion chromatograms of crude and processed Paeoniae Radix Alba samples obtained from both positive and negative ion modes were shown in Figure 1. In the preliminary study, the Q Exactive mass spectrometer was confirmed to be highly selective and sensitive. Under the present chromatographic and MS conditions, 123 and 101 compounds were identified in crude and processed Paeoniae Radix Alba samples, respectively. Compounds 16, 30, 31, 42, 45, 58, 59, 61, 62, 63, 64, 75, 78, 80, 87, 90, 91, 94, 95, 103, 112, and 120 were not detected in processed Paeoniae Radix Alba sample. Meanwhile, the ESI-MS data of crude and processed samples demonstrated that the peak areas of components 8, 113, and 122 varied significantly, and their amounts were dramatically increased in processed sample. The results were shown in Table 1.

Figure 1

Total ion chromatograms of crude (a) and processed (b) Paeoniae Radix Alba obtained from both positive and negative ion modes.

Table 1

Major chemical constituents identified in crude and processed Paeoniae Radix Alba and in crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair.

No.	t R (min)	Compound name	Formula	Paeoniae Radix Alba		Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair
				(Measured area)		(Measured area)
				Crude	Processed	Crude	Processed
1	0.84	6-O-galloylsucrose	C19H26O15	1.8570E + 08	1.9012E + 08	4.2870E + 07	4.4158E + 07
2	0.84	Glucogallin	C13H16O10	2.9739E + 08	2.5698E + 08	1.0931E + 08	—
3	1.05	Desbenzoylpaeoniflorin	C16H24O10	1.6682E + 08	1.6263E + 08	9.8500E + 07	—
4	1.06	1′-O-galloylsucrose	C19H26O15	3.2574E + 08	2.9123E + 08	—	—
5	1.07	1-O-glucopyranosyl paeonisuffrone	C16H24O9	2.8667E + 08	2.3654E + 08	1.1532E + 08	—
6	1.13	Gallic acid	C7H6O5	4.1152E + 09	4.0736E + 09	2.7186E + 09	3.1711E + 09
7	1.18	Oxypaeoniflorin sulfonate	C23H28O14S	4.9527E + 07	3.5407E + 07	6.1568E + 06	8.5010E + 07
8	1.22	Ethyl gallate	C9H10O5	5.1351E + 07	6.7200E + 07	1.5592E + 07	4.8337E + 07
9	1.22	6-O-galloyl desbenzoylpaeoniflorin	C23H28O14	9.9020E + 07	9.5040E + 07	5.3798E + 07	—
10	1.26	6-O-glucopyranosyl-lactinolide	C16H26O9	1.0875E + 08	1.1180E + 08	—	3.7130E + 07
11	1.30	Paeoniflorin sulfonate I	C23H28O13S	5.3777E + 07	3.6391E + 07	7.3077E + 06	7.7185E + 07
12	1.30	Mudanpioside E sulfonate	C24H30O15S	5.3777E + 07	3.6391E + 07	7.3077E + 06	7.7185E + 07
13	1.43	6-O-glucopyranosyl-lactinolide	C16H26O9	7.4342E + 08	6.5904E + 08	4.0712E + 08	3.1407E + 08
14	1.64	Mudanpioside F	C16H24O8	6.4178E + 08	6.0980E + 08	4.0130E + 08	6.6680E + 07
15	1.76	Isomaltopaeoniflorin sulfonate	C29H38O18S	1.8858E + 09	1.1382E + 09	2.6277E + 08	5.8622E + 07
16	1.81	Pedunculagin	C34H24O22	4.8098E + 07	—	5.6076E + 07	1.2660E + 09
17	1.97	Paeoniflorin sulfonate I	C23H28O13S	3.1881E + 10	2.3387E + 10	6.6202E + 09	5.5241E + 10
18	2.25	Oxypaeoniflorin	C23H28O12	2.3173E + 09	2.4115E + 09	1.6734E + 09	1.4513E + 09
19	2.36	Gallotannin	C27H24O18	2.2850E + 08	2.2458E + 08	1.6284E + 08	1.6703E + 08
20	2.37	1-O-benzoylsucrose	C19H26O12	1.3761E + 08	1.3161E + 08	1.1673E + 08	8.2986E + 07
21	2.41	d-catechin	C15H14O6	3.7822E + 09	4.2278E + 09	2.6339E + 09	2.5982E + 09
22	2.63	Methyl gallate	C8H8O5	2.3823E + 10	2.4116E + 10	2.3388E + 10	—
23	2.63	Salicylic acid	C7H6O3	2.3823E + 10	2.4116E + 10	2.3388E + 10	1.7399E + 10
24	2.72	Albiflorin R1	C23H28O11	5.2469E + 08	5.6725E + 08	5.6329E + 08	4.5647E + 08
25	3.00	Kaempferol-3,7-di-O-glucoside	C27H30O16	3.8065E + 07	2.1896E + 07	3.5719E + 07	1.5513E + 07
26	3.00	Paeonoside	C27H30O16	3.8065E + 07	2.1896E + 07	3.5719E + 07	1.5513E + 07
27	3.46	Galloylpaeoniflorin	C30H32O15	1.3912E + 08	1.5200E + 08	1.1501E + 08	1.0863E + 08
28	3.47	Paeonolide	C20H28O12	1.0622E + 07	1.1936E + 07	9.1812E + 06	—
29	3.58	6-O-glucopyranosyl-lactinolide	C16H26O9	2.5249E + 08	2.3834E + 08	2.2675E + 08	2.1241E + 08
30	3.68	Oxypaeoniflorin	C23H28O12	1.5407E + 08	—	1.4345E + 08	1.3307E + 08
31	3.76	6-O-glucopyranosyl-lactinolide	C16H26O9	3.2664E + 08	—	3.0588E + 08	3.1627E + 08
32	3.88	Paeonilactone B	C10H12O4	9.0325E + 07	9.3539E + 07	5.1257E + 07	8.9597E + 07
33	3.93	Isomaltopaeoniflorin	C29H38O16	1.1545E + 10	1.1941E + 10	1.2282E + 10	9.4600E + 09
34	4.07	Albiflorin	C23H28O11	2.9587E + 10	2.9296E + 10	2.8430E + 10	2.8684E + 10
35	4.32	Glucopyranosylalbiorin	C29H38O16	2.2813E + 09	2.4109E + 08	2.0383E + 08	1.6844E + 08
36	4.34	Galloylpaeoniflorin sulfonate	C30H32O17S	7.6943E + 08	5.6793E + 08	1.5501E + 08	1.4886E + 09
37	4.34	Galloylpaeoniflorin isomer	C30H32O15	6.7592E + 08	7.4322E + 08	6.0470E + 08	5.4051E + 08
38	4.38	1,2,3,6-tetra-O-galloylglucose	C34H28O22	4.6602E + 08	3.4977E + 08	4.0697E + 08	3.6901E + 08
39	4.38	Tetragalloyl glucose A	C34H28O22	4.6602E + 08	3.4977E + 08	4.0697E + 08	3.6901E + 08
40	4.56	Mudanpioside F	C16H24O8	8.4156E + 07	8.2734E + 07	7.2666E + 07	7.9227E + 07
41	4.60	Oxypaeoniflorin isomer	C23H28O12	9.6610E + 08	9.8706E + 08	9.2464E + 08	8.7359E + 08
42	4.65	Gallotannin	C27H24O18	6.7737E + 07	—	—	—
43	4.77	Paeoniflorin	C23H28O11	5.9556E + 10	6.1356E + 10	5.9929E + 10	5.8832E + 10
44	4.89	Paeoniflorin sulfonate II	C23H28O13S	1.1095E + 08	1.4567E + 08	5.5052E + 07	2.7813E + 08
45	4.98	Isogalloylpaeoniflorin sulfonate	C30H32O17S	3.6742E + 07	—	—	—
46	5.05	Ethyl gallate	C9H10O5	6.0669E + 07	5.4850E + 07	1.6681E + 07	2.6790E + 07
47	5.05	Methyl salicylate	C8H8O3	6.0669E + 07	5.4850E + 07	1.6681E + 07	2.6790E + 07
48	5.15	Benzoic acid	C7H6O2	4.0163E + 07	4.5493E + 07	2.9695E + 07	2.9727E + 07
49	5.25	Paeonol	C9H10O3	6.8567E + 07	7.4129E + 07	9.9992E + 07	6.5619E + 07
50	5.25	4-hydroxy-3-methoxy acetophenone	C9H10O3	6.8567E + 07	7.4129E + 07	9.9992E + 07	6.5619E + 07
51	5.31	ortho-oxypaeoniflorin	C23H28O12	1.9080E + 09	1.9263E + 09	1.8723E + 09	1.6842E + 09
52	5.63	Ethyl gallate	C9H10O5	1.4627E + 08	1.2812E + 08	1.0365E + 08	8.8155E + 07
53	5.63	Methyl salicylate	C8H8O3	1.4627E + 08	1.2812E + 08	1.0365E + 08	8.8155E + 07
54	5.66	Kaempferol-3-O-glucoside	C21H20O11	1.6012E + 07	1.7385E + 07	—	—
55	5.66	Astragalin	C21H20O11	1.6012E + 07	1.7385E + 07	—	—
56	6.01	Eugeniin	C41H30O26	2.7483E + 08	3.0279E + 08	2.8080E + 08	3.0479E + 08
57	6.01	Dihydroxymethyl benzoyl tetragalloyl glucose	C41H30O26	2.7483E + 08	3.0279E + 08	2.8080E + 08	3.0479E + 08
58	6.03	1,2,3,6-tetra-O-galloylglucose isomer A	C34H28O22	1.3555E + 09	—	1.1980E + 09	1.1039E + 09
59	6.03	Tetragalloyl glucose B	C34H28O22	1.3555E + 09	—	—	—
60	6.08	Astragalin	C21H20O11	1.5009E + 07	1.8552E + 07	1.5922E + 07	1.4002E + 07
61	6.09	Isomaltopaeoniflorin isomer	C29H38O16	7.5172E + 07	—	—	—
62	6.47	1,2,3,6-tetra-O-galloylglucose isomer B	C34H28O22	1.5882E + 09	—	—	1.2570E + 09
63	6.47	Tetragalloyl glucose C	C34H28O22	1.5882E + 09	—	—	1.2570E + 09
64	6.85	3,6-di-O-galloyl paeoniorin	C37H36O19	7.6512E + 07	—	—	—
65	6.96	1,2,3,6-tetra-O-galloylglucose	C34H28O22	4.4729E + 08	4.5825E + 08	4.0642E + 08	4.2393E + 08
66	6.96	Tetragalloyl glucose D	C34H28O22	4.4729E + 08	4.5825E + 08	4.0642E + 08	4.2393E + 08
67	7.35	Galloylpaeoniflorin isomer I	C30H32O15	1.2156E + 10	1.2451E + 10	1.1484E + 10	1.0962E + 10
68	7.60	1-O-glucopyranosyl-8-O-benzoyl paeonisuffrone	C23H28O10	4.3983E + 07	4.5347E + 07	4.4927E + 07	3.9869E + 07
69	7.71	Glucopyranosylalbiorin isomer I	C29H38O16	7.2982E + 07	7.9341E + 07	—	1.8872E + 07
70	8.18	1-O-glucopyranosyl-8-O-benzoyl paeonisuffrone	C23H28O10	7.4648E + 07	8.3204E + 07	6.5957E + 07	5.9832E + 07
71	8.31	Ortho-oxypaeoniflorin	C23H28O12	2.4469E + 07	2.4504E + 07	2.3932E + 07	2.2796E + 07
72	8.45	1,2,3,4,6-Penta-O-galloyl–D-glucopyranose	C41H32O26	1.1843E + 10	1.0905E + 10	1.0518E + 10	1.0489E + 10
73	8.45	Pentagalloyl glucose	C41H32O26	1.1843E + 10	1.0905E + 10	1.0518E + 10	1.0489E + 10
74	8.64	Lactiflorin	C23H26O10	1.0818E + 08	1.8628E + 08	1.3689E + 08	—
75	8.80	Galloylalbiroin	C30H32O15	3.2696E + 09	—	—	—
76	9.17	Astragalin	C21H20O11	1.0717E + 07	1.3960E + 07	1.2843E + 07	1.0582E + 07
77	9.25	Lactinolide	C10H16O4	2.7251E + 07	2.6105E + 07	2.1735E + 07	3.2770E + 07
78	9.29	Galloylpaeoniflorin isomer II	C30H32O15	2.8831E + 09	—	2.6829E + 09	2.2850E + 09
79	9.68	Glucopyranosylalbiorin isomer II	C29H38O16	2.4321E + 07	2.6804E + 07	2.2576E + 07	2.4950E + 07
80	9.84	Hexagalloyl glucose	C48H36O30	4.9153E + 07	—	6.8676E + 08	5.7793E + 08
81	9.95	Oxybenzoyl-oxypaeoniflorin	C30H32O14	1.4385E + 07	1.6654E + 07	1.1051E + 07	1.1345E + 07
82	10.07	1-O-glucopyranosyl-8-O-benzoylpaeonisuffrone	C23H28O10	3.6916E + 09	3.5634E + 09	3.1333E + 09	3.2106E + 09
83	10.29	Albiflorin R1 isomer I	C23H28O11	6.3346E + 09	6.6205E + 09	5.9528E + 09	5.8736E + 09
84	10.74	Hexagalloyl glucose	C48H36O30	4.9225E + 08	2.5582E + 08	1.9395E + 09	1.5439E + 09
85	10.76	Lactiflorin	C23H26O10	1.2785E + 09	3.5174E + 09	9.9713E + 08	3.4524E + 09
86	10.84	Benzoylpaeoniflorin Sulfonate	C30H32O14S	9.0616E + 08	6.4075E + 08	1.5946E + 08	2.1931E + 09
87	10.88	3,6-di-O-galloyl paeoniorin	C37H36O19	1.6123E + 08	—	—	—
88	10.95	Ortho-oxypaeoniflorin isomer	C23H28O12	5.5563E + 07	5.8774E + 07	5.7147E + 07	5.6640E + 07
89	11.52	3,6-di-O-galloyl paeoniorin	C37H36O19	3.6509E + 08	3.9290E + 08	5.2162E + 08	5.3781E + 08
90	11.72	3,6-di-O-galloyl paeoniorin isomer	C37H36O19	9.7356E + 08	—	1.2523E + 09	9.5929E + 08
91	11.75	Galloylalbiroin isomer I	C30H32O15	2.3457E + 08	—	—	—
92	11.84	Oxypaeoniflorin sulfonate isomer	C23H28O14S	2.1063E + 07	1.9747E + 07	1.3875E + 07	1.0840E + 07
93	12.15	1-O-glucopyranosyl-8-O-benzoylpaeonisuffrone	C23H28O10	7.2104E + 07	7.1468E + 07	6.7309E + 07	6.5917E + 07
94	12.15	Oxybenzoyl-oxypaeoniflorin	C30H32O14	1.9982E + 08	—	—	1.6891E + 08
95	12.18	Benzoyloxypaeoniflorin	C30H32O13	2.0822E + 08	—	2.0163E + 08	1.9074E + 08
96	13.42	Benzoyloxypaeoniflorin isomer	C30H32O13	8.6458E + 07	6.2282E + 07	7.6048E + 07	7.2791E + 07
97	13.44	Oxybenzoyl-oxypaeoniflorin isomer I	C30H32O14	1.4728E + 07	1.7389E + 07	1.5360E + 07	1.6008E + 07
98	13.85	Galloylalbiroin isomer II	C30H32O15	9.6403E + 07	1.2196E + 08	1.0506E + 08	1.0272E + 08
99	14.05	Oxybenzoyl-oxypaeoniflorin isomer II	C30H32O14	2.5323E + 07	2.9603E + 07	2.3556E + 07	2.8526E + 07
100	14.13	Benzoyloxypaeoniflorin	C30H32O13	3.8096E + 07	3.8557E + 07	3.7499E + 07	3.5800E + 07
101	15.07	Benzoyloxypaeoniflorin isomer I	C30H32O13	1.9827E + 07	2.3616E + 07	—	—
102	15.38	Benzoyloxypaeoniflorin isomer II	C30H32O13	1.1841E + 07	1.3730E + 07	—	—
103	16.01	Oxybenzoyl-paeoniflorin	C30H32O12	1.8152E + 07	—	1.8435E + 07	—
104	16.95	Isobenzoylpaeoniflorin	C30H32O12	1.2225E + 10	1.3228E + 10	1.2158E + 10	1.2391E + 10
105	16.95	Oxybenzoyl-paeoniflorin isomer I	C30H32O12	1.2225E + 10	1.3228E + 10	1.2158E + 10	1.2391E + 10
106	17.23	Benzoylpaeoniflorin Sulfonate	C30H32O14S	1.5680E + 07	1.2235E + 07	5.6573E + 06	3.5831E + 07
107	17.48	Isobenzoylpaeoniflorin isomer I	C30H32O12	5.4138E + 09	5.4432E + 09	5.2522E + 09	5.3238E + 09
108	17.48	Oxybenzoyl-paeoniflorin isomer II	C30H32O12	5.4138E + 09	5.4432E + 09	5.2522E + 09	5.3238E + 09
109	17.86	Benzoyloxypaeoniflorin	C30H32O13	3.4347E + 07	3.4852E + 07	3.5980E + 07	3.8814E + 07
110	18.55	Benzoyloxypaeoniflorin isomer	C30H32O13	1.5397E + 07	1.7656E + 07	1.7246E + 07	1.8012E + 07
111	18.69	Albiflorin R1 isomer II	C23H28O11	2.0046E + 07	1.9851E + 07	2.3462E + 07	—
112	19.30	Albiflorin R1 isomer III	C23H28O11	2.9827E + 06	—	—	5.6105E + 06
113	21.79	Palbinone	C22H30O4	8.9687E + 07	1.3174E + 08	1.2834E + 08	5.7610E + 07
114	21.93	Isobenzoylpaeoniflorin isomer II	C30H32O12	4.5356E + 08	4.2874E + 07	3.4016E + 08	2.7347E + 08
115	21.93	Oxybenzoyl-paeoniflorin isomer III	C30H32O12	4.5356E + 08	4.2874E + 07	3.4016E + 08	2.7347E + 08
116	22.15	Paeonilactinone	C10H16O2	7.0423E + 06	3.7108E + 06	8.0036E + 06	6.6886E + 06
117	36.46	Hederagenin	C30H48O4	7.6725E + 07	8.1456E + 07	9.7498E + 07	4.7332E + 07
118	37.31	23-hydroxybetulinic acid	C30H48O4	3.9836E + 07	4.0995E + 07	3.9906E + 07	2.2611E + 07
119	38.14	Astrantiagenin D	C30H46O4	7.8714E + 06	7.9560E + 06	1.1904E + 07	3.8958E + 06
120	43.00	Astrantiagenin D isomer	C30H46O4	4.0450E + 06	—	3.1585E + 06	—
121	45.65	Oleanolic acid	C30H48O3	1.1266E + 08	9.4258E + 07	7.6434E + 07	4.3295E + 07
122	46.10	Betulinic acid	C30H48O3	6.2494E + 06	2.3289E + 07	4.0543E + 07	2.3912E + 07
123	52.48	Daucosterol	C35H60O6	1.4060E + 07	1.9624E + 07	8.5440E + 06	6.3156E + 06

From ESI-MS information, it was found that the sensitivities for all kinds of components in Paeoniae Radix Alba were high in both positive and negative ion modes. In present study, we chose peaks 1, 2, and 3 to explain the identification process using Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS. Peaks 1, 2, and 3 were eluted at retention times of 4.08, 4.79, and 8.47 min, respectively. Peak 1 showed the [M+H]+ m/z 481.16986, [2 M+NH4]+ m/z 978.35950, [M–H]− m/z 479.15591, [M–H+HCOOH]− m/z 525.16101, and [2 M−H+HCOOH]− m/z 1005.32404 and the corresponding elemental compositions were C23H29O11, C46H60O22N, C23H27O11, C24H29O13, and C47H57O24, respectively. On the basis of above data we deduced that the elemental composition of peak 1 was C23H28O11. The molecular ion of peak 1 could lead to seven main MS2 ions at m/z 319.11731, 197.08075, 133.06473, and 105.03342 in positive ion mode, and m/z 479.15594, 283.08231, and 121.02956 in negative ion mode. On the basis of the elemental compositions of fragment ions, peak 1 was assigned as albiflorin. Peaks 2 and 3 were therefore identified as paeoniflorin, and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose with above mentioned method. The mass spectra and proposed fragmentations of albiflorin, paeoniflorin, and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose were shown in Figure 2.

Figure 2

Mass spectra and proposed fragmentations of albiflorin (a), paeoniflorin (b), and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose (c).

3.2. Identification of the Main Components in Crude and Processed Atractylodis Macrocephalae Rhizoma

Figure 3 showed the total ion chromatograms of crude and processed Atractylodis Macrocephalae Rhizoma samples obtained from both positive and negative ion modes. 32 and 26 compounds were identified in crude and processed Atractylodis Macrocephalae Rhizoma samples, respectively. Compounds 2, 4, 13, 14, 17, and 29 were not detected in processed Atractylodis Macrocephalae Rhizoma sample. Moreover, the amounts of compounds 3, 7, 9, 10, 21, 23, and 27 were substantially decreased, and the amounts of compounds 8, 18, and 22 were increased in processed sample compared with crude one. The results were shown in Table 2.

Figure 3

Total ion chromatograms of crude (a) and processed (b) Atractylodis Macrocephalae Rhizoma obtained from both positive and negative ion modes.

Table 2

Major chemical constituents identified in crude and processed Atractylodis Macrocephalae Rhizoma and in crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair.

No.	t R (min)	Compound name	Formula	Atractylodis Macrocephalae Rhizoma		Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair
				(Measured area)		(Measured area)
				Crude	Processed	Crude	Processed
1	1.72	Protocatechuic acid	C7H6O4	2.0389E + 07	1.4454E + 07	2.0881E + 07	2.4383E + 07
2	2.67	Protocatechuic acid isomer I	C7H6O4	9.6661E + 07	—	—	—
3	3.24	Caffeic acid	C9H8O4	3.6818E + 08	1.7393E + 08	2.8796E + 08	1.2882E + 08
4	3.73	Protocatechuic acid isomer II	C7H6O4	2.0846E + 07	—	—	1.2022E + 07
5	4.21	Dictamnoside A isomer I	C21H36O9	1.8843E + 07	2.4981E + 07	1.0636E + 07	1.3140E + 07
6	4.70	Dictamnoside A isomer II	C21H36O9	2.8770E + 07	3.4768E + 07	1.0395E + 07	1.4208E + 07
7	5.63	Scopoletin	C10H8O4	6.1458E + 07	4.1494E + 07	6.1562E + 07	5.3342E + 07
8	5.82	Dictamnoside A	C21H36O9	9.6195E + 07	1.1991E + 08	7.5446E + 07	9.4190E + 07
9	8.77	Atracetylentriol	C14H16O3	1.2538E + 07	5.4052E + 06	—	—
10	9.33	Ferulic acid	C10H10O4	1.3958E + 07	9.1214E + 06	1.1912E + 07	9.6849E + 06
11	25.81	Atractylenolide I isomer	C15H18O2	4.5224E + 09	4.2401E + 09	5.9401E + 09	6.5277E + 09
12	25.83	Atractylenolide III	C15H20O3	2.5549E + 09	1.8023E + 09	2.8280E + 09	3.1632E + 09
13	26.17	12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol	C21H26O5	2.4755E + 07	—	—	—
14	26.95	12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol isomer	C21H26O5	7.5991E + 07	—	—	—
15	31.10	Atractylenolide II isomer	C15H20O2	6.7883E + 09	4.5794E + 09	7.6246E + 09	7.8814E + 09
16	31.66	Atractylenolide II	C15H20O2	2.8279E + 10	1.9902E + 10	3.0285E + 10	3.1294E + 10
17	33.44	Atractylodin	C13H10O	6.4157E + 06	—	7.0452E + 07	—
18	35.07	Atractylenolide I isomer	C15H18O2	8.2226E + 08	1.4781E + 09	1.0831E + 09	3.2083E + 09
19	35.94	Atractylenolide I	C15H18O2	8.8877E + 09	7.2520E + 09	8.3857E + 09	1.2742E + 10
20	39.03	12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol isomer I	C21H26O5	3.0978E + 07	3.7863E + 07	2.9171E + 07	—
21	39.81	Dibutyl phthalate	C16H22O4	1.1372E + 08	9.8325E + 07	1.2659E + 08	1.4865E + 08
22	40.00	12-methylbutyryl-14-acetyl 2E,8EZ,10E-atractylentriol isomer II	C21H26O5	3.8810E + 07	7.7498E + 07	3.3885E + 07	7.0522E + 07
23	40.26	Dibutyl phthalate isomer	C16H22O4	1.0631E + 08	5.4902E + 07	6.1958E + 07	4.6227E + 07
24	41.50	14-methylbutyryl-2E,8EZ,10Es-atractylentriol	C19H24O4	4.9587E + 07	2.8423E + 07	5.1146E + 07	4.7855E + 07
25	46.43	Spinasteryl	C29H48O	8.6778E + 06	7.9096E + 06	1.0609E + 07	7.7832E + 06
26	47.32	Atractylon	C15H20O	7.4433E + 07	5.4063E + 07	6.6146E + 07	—
27	47.37	Biatractylolide	C30H38O4	1.0949E + 09	9.5665E + 08	1.2797E + 09	—
28	47.96	Linoleic acid	C18H32O2	1.8499E + 08	1.5041E + 08	1.8777E + 08	2.3743E + 08
29	48.25	Linoleic acid isomer	C18H32O2	2.1059E + 07	—	—	—
30	48.59	Biepiasterolid isomer	C30H38O4	9.0255E + 08	7.0863E + 08	7.4011E + 08	—
31	48.90	Atractylon isomer	C15H20O	9.5308E + 07	8.7683E + 07	8.2967E + 07	1.0132E + 08
32	49.42	Palmitic acid	C16H32O2	2.2356E + 07	2.2942E + 07	2.5949E + 07	2.0153E + 07

Atractylenolide I, atractylenolide II, and atractylenolide III are the main active compounds that belong to the sesquiterpenes in Atractylodis Macrocephalae Rhizoma. The mass spectra of atractylenolide I showed a [M+H]+ ion at m/z 231.13799, which could lead to four MS2 ions at m/z 213.12740, 185.13251, 157.10127, and 143.08569. The molecular ion of atractylenolide II ([M+H]+ m/z 233.15358) could lead to six MS2 ions at m/z 215.14310, 187.14818, 159.08055, 151.07541, 133.10117, and 95.08547. Meanwhile, the MS2 spectrum of m/z 249.14836 from atractylenolide III contained six major fragment ions at m/z 231.13802, 213.12758, 189.09108, 163.07541, 135.04411, and 105.06989. The mass spectra of the above three compounds were shown in Figure 4.

Figure 4

Mass spectra of atractylenolide I (a), atractylenolide II (b), and atractylenolide III (c).

3.3. Analysis of Chemical Changes of Paeoniae Radix Alba after Compatibility with Atractylodis Macrocephalae Rhizoma

In the present study, the Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS based on chemical profiling approach was used to evaluate chemical constitution between co-decoction and single decoction of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma. For crude Paeoniae Radix Alba, the relative contents of most compounds were dramatically decreased except those of compounds 80, 90, 98, 113, 119, and 122 were significantly increased and 19 compounds were not detected after its compatibility with crude Atractylodis Macrocephalae Rhizoma. For processed Paeoniae Radix Alba, the relative contents of compounds 12, 36, 84, and 86 were remarkably increased except 12 compounds including pedunculagin, oxypaeoniflorin, 6-O-glucopyranosyl-lactinolide, 1, 2, 3, 6-tetra-O-galloylglucose isomer A, 1, 2, 3, 6-tetra-O-galloylglucose isomer B, tetragalloyl glucose C, galloylpaeoniflorin isomer II, hexagalloyl glucose, 3, 6-di-O-galloyl paeoniorin isomer, oxybenzoyl-oxypaeoniflorin, benzoyloxypaeoniflorin, and albiflorin R1 isomer III were newly generated and 13 compounds were not found after its compatibility with processed Atractylodis Macrocephalae Rhizoma. The results were presented in Figure 5 and Table 1.

Figure 5

Total ion chromatograms of crude (a) and processed (b) Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair obtained from both positive and negative ion modes.

3.4. Analysis of the Chemical Changes of Atractylodis Macrocephalae Rhizoma after Compatibility with Paeoniae Radix Alba

For crude Atractylodis Macrocephalae Rhizoma, the relative contents of compounds 17, 18, and 25 were increased clearly except those of compounds 6, 23, and 30 decreased considerably and six compounds including protocatechuic acid isomer I, protocatechuic acid isomer II, atracetylentriol, 12-methylbutyryl-14-acetyl-2E, 8EZ, 10E-atractylentriol, 12-methylbutyryl-14-acetyl-2E, 8EZ, 10E-atractylentriol isomer, and linoleic acid isomer were lost after its compatibility with crude Paeoniae Radix Alba. For processed Atractylodis Macrocephalae Rhizoma, compounds 9, 20, 26, 27, and 30 were not found except the relative contents of compounds 5, 6, and 8 were decreased while those of compounds 15, 19, 21, and 31 were increased after its compatibility with processed Paeoniae Radix Alba. Furthermore, compound 4 (protocatechuic acid isomer II) was not found in processed Atractylodis Macrocephalae Rhizoma but could be detected in processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair by using Exact Finder and MassFrontier softwares. The above results illustrated that Paeoniae Radix Alba significantly changed the components of Atractylodis Macrocephalae Rhizoma in solution when they decocted together. The corresponding results were presented in Figure 5 and Table 2.

4. Conclusions

Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS is a powerful tool for discriminating the chemical changes between single herbal and co-decocting medicines. In our present study, the Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS based on chemical profiling approach to investigate and evaluate chemical changes from crude and processed Paeoniae Radix Alba, crude and processed Atractylodis Macrocephalae Rhizoma, and their crude and processed herbal pair extracts was proposed. The results showed that processing and compatibility of TCM could significantly change the chemical composition of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma. The developed method is considered to provide a scientific foundation for deeply elucidating the processing and compatibility mechanism of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma.