# 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

Authors: Gang Cao; Qinglin Li; Hao Cai; Sicong Tu; Baochang Cai
Journal: Evidence-Based Complementary and Alternative Medicine
 (2014)

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## Abstract

The Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair is mainly used for regulating the functions of liver and spleen, benefitingqi, 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.


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## Body

## 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, benefitingqi, 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 ofm/z 150–1500 was chosen. Resolution for higher energy collisional dissociation cell (HCD) spectra was set to 17,500 atm/z 150 on the Q Exactive.


## 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 ofm/z 150–1500 was chosen. Resolution for higher energy collisional dissociation cell (HCD) spectra was set to 17,500 atm/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 Figure1. 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.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
PaeoniaeRadix Alba
Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair
(Measured area)
(Measured area)
Crude
Processed
Crude
Processed
1
0.84
6-O-galloylsucrose
C19H26O15
1.8570
E
+
08
1.9012
E
+
08
4.2870
E
+
07
4.4158
E
+
07
2
0.84
Glucogallin
C13H16O10
2.9739
E
+
08
2.5698
E
+
08
1.0931
E
+
08
—
3
1.05
Desbenzoylpaeoniflorin
C16H24O10
1.6682
E
+
08
1.6263
E
+
08
9.8500
E
+
07
—
4
1.06
1′-O-galloylsucrose
C19H26O15
3.2574
E
+
08
2.9123
E
+
08
—
—
5
1.07
1-O-glucopyranosyl paeonisuffrone
C16H24O9
2.8667
E
+
08
2.3654
E
+
08
1.1532
E
+
08
—
6
1.13
Gallic acid
C7H6O5
4.1152
E
+
09
4.0736
E
+
09
2.7186
E
+
09
3.1711
E
+
09
7
1.18
Oxypaeoniflorin sulfonate
C23H28O14S
4.9527
E
+
07
3.5407
E
+
07
6.1568
E
+
06
8.5010
E
+
07
8
1.22
Ethyl gallate
C9H10O5
5.1351
E
+
07
6.7200
E
+
07
1.5592
E
+
07
4.8337
E
+
07
9
1.22
6-O-galloyl desbenzoylpaeoniflorin
C23H28O14
9.9020
E
+
07
9.5040
E
+
07
5.3798
E
+
07
—
10
1.26
6-O-glucopyranosyl-lactinolide
C16H26O9
1.0875
E
+
08
1.1180
E
+
08
—
3.7130
E
+
07
11
1.30
Paeoniflorin sulfonate I
C23H28O13S
5.3777
E
+
07
3.6391
E
+
07
7.3077
E
+
06
7.7185
E
+
07
12
1.30
Mudanpioside E sulfonate
C24H30O15S
5.3777
E
+
07
3.6391
E
+
07
7.3077
E
+
06
7.7185
E
+
07
13
1.43
6-O-glucopyranosyl-lactinolide
C16H26O9
7.4342
E
+
08
6.5904
E
+
08
4.0712
E
+
08
3.1407
E
+
08
14
1.64
Mudanpioside F
C16H24O8
6.4178
E
+
08
6.0980
E
+
08
4.0130
E
+
08
6.6680
E
+
07
15
1.76
Isomaltopaeoniflorin sulfonate
C29H38O18S
1.8858
E
+
09
1.1382
E
+
09
2.6277
E
+
08
5.8622
E
+
07
16
1.81
Pedunculagin
C34H24O22
4.8098
E
+
07
—
5.6076
E
+
07
1.2660
E
+
09
17
1.97
Paeoniflorin sulfonate I
C23H28O13S
3.1881
E
+
10
2.3387
E
+
10
6.6202
E
+
09
5.5241
E
+
10
18
2.25
Oxypaeoniflorin
C23H28O12
2.3173
E
+
09
2.4115
E
+
09
1.6734
E
+
09
1.4513
E
+
09
19
2.36
Gallotannin
C27H24O18
2.2850
E
+
08
2.2458
E
+
08
1.6284
E
+
08
1.6703
E
+
08
20
2.37
1-O-benzoylsucrose
C19H26O12
1.3761
E
+
08
1.3161
E
+
08
1.1673
E
+
08
8.2986
E
+
07
21
2.41
d-catechin
C15H14O6
3.7822
E
+
09
4.2278
E
+
09
2.6339
E
+
09
2.5982
E
+
09
22
2.63
Methyl gallate
C8H8O5
2.3823
E
+
10
2.4116
E
+
10
2.3388
E
+
10
—
23
2.63
Salicylic acid
C7H6O3
2.3823
E
+
10
2.4116
E
+
10
2.3388
E
+
10
1.7399
E
+
10
24
2.72
Albiflorin R1
C23H28O11
5.2469
E
+
08
5.6725
E
+
08
5.6329
E
+
08
4.5647
E
+
08
25
3.00
Kaempferol-3,7-di-O-glucoside
C27H30O16
3.8065
E
+
07
2.1896
E
+
07
3.5719
E
+
07
1.5513
E
+
07
26
3.00
Paeonoside
C27H30O16
3.8065
E
+
07
2.1896
E
+
07
3.5719
E
+
07
1.5513
E
+
07
27
3.46
Galloylpaeoniflorin
C30H32O15
1.3912
E
+
08
1.5200
E
+
08
1.1501
E
+
08
1.0863
E
+
08
28
3.47
Paeonolide
C20H28O12
1.0622
E
+
07
1.1936
E
+
07
9.1812
E
+
06
—
29
3.58
6-O-glucopyranosyl-lactinolide
C16H26O9
2.5249
E
+
08
2.3834
E
+
08
2.2675
E
+
08
2.1241
E
+
08
30
3.68
Oxypaeoniflorin
C23H28O12
1.5407
E
+
08
—
1.4345
E
+
08
1.3307
E
+
08
31
3.76
6-O-glucopyranosyl-lactinolide
C16H26O9
3.2664
E
+
08
—
3.0588
E
+
08
3.1627
E
+
08
32
3.88
Paeonilactone B
C10H12O4
9.0325
E
+
07
9.3539
E
+
07
5.1257
E
+
07
8.9597
E
+
07
33
3.93
Isomaltopaeoniflorin
C29H38O16
1.1545
E
+
10
1.1941
E
+
10
1.2282
E
+
10
9.4600
E
+
09
34
4.07
Albiflorin
C23H28O11
2.9587
E
+
10
2.9296
E
+
10
2.8430
E
+
10
2.8684
E
+
10
35
4.32
Glucopyranosylalbiorin
C29H38O16
2.2813
E
+
09
2.4109
E
+
08
2.0383
E
+
08
1.6844
E
+
08
36
4.34
Galloylpaeoniflorin sulfonate
C30H32O17S
7.6943
E
+
08
5.6793
E
+
08
1.5501
E
+
08
1.4886
E
+
09
37
4.34
Galloylpaeoniflorin isomer
C30H32O15
6.7592
E
+
08
7.4322
E
+
08
6.0470
E
+
08
5.4051
E
+
08
38
4.38
1,2,3,6-tetra-O-galloylglucose
C34H28O22
4.6602
E
+
08
3.4977
E
+
08
4.0697
E
+
08
3.6901
E
+
08
39
4.38
Tetragalloyl glucose A
C34H28O22
4.6602
E
+
08
3.4977
E
+
08
4.0697
E
+
08
3.6901
E
+
08
40
4.56
Mudanpioside F
C16H24O8
8.4156
E
+
07
8.2734
E
+
07
7.2666
E
+
07
7.9227
E
+
07
41
4.60
Oxypaeoniflorin isomer
C23H28O12
9.6610
E
+
08
9.8706
E
+
08
9.2464
E
+
08
8.7359
E
+
08
42
4.65
Gallotannin
C27H24O18
6.7737
E
+
07
—
—
—
43
4.77
Paeoniflorin
C23H28O11
5.9556
E
+
10
6.1356
E
+
10
5.9929
E
+
10
5.8832
E
+
10
44
4.89
Paeoniflorin sulfonate II
C23H28O13S
1.1095
E
+
08
1.4567
E
+
08
5.5052
E
+
07
2.7813
E
+
08
45
4.98
Isogalloylpaeoniflorin sulfonate
C30H32O17S
3.6742
E
+
07
—
—
—
46
5.05
Ethyl gallate
C9H10O5
6.0669
E
+
07
5.4850
E
+
07
1.6681
E
+
07
2.6790
E
+
07
47
5.05
Methyl salicylate
C8H8O3
6.0669
E
+
07
5.4850
E
+
07
1.6681
E
+
07
2.6790
E
+
07
48
5.15
Benzoic acid
C7H6O2
4.0163
E
+
07
4.5493
E
+
07
2.9695
E
+
07
2.9727
E
+
07
49
5.25
Paeonol
C9H10O3
6.8567
E
+
07
7.4129
E
+
07
9.9992
E
+
07
6.5619
E
+
07
50
5.25
4-hydroxy-3-methoxy acetophenone
C9H10O3
6.8567
E
+
07
7.4129
E
+
07
9.9992
E
+
07
6.5619
E
+
07
51
5.31
ortho-oxypaeoniflorin
C23H28O12
1.9080
E
+
09
1.9263
E
+
09
1.8723
E
+
09
1.6842
E
+
09
52
5.63
Ethyl gallate
C9H10O5
1.4627
E
+
08
1.2812
E
+
08
1.0365
E
+
08
8.8155
E
+
07
53
5.63
Methyl salicylate
C8H8O3
1.4627
E
+
08
1.2812
E
+
08
1.0365
E
+
08
8.8155
E
+
07
54
5.66
Kaempferol-3-O-glucoside
C21H20O11
1.6012
E
+
07
1.7385
E
+
07
—
—
55
5.66
Astragalin
C21H20O11
1.6012
E
+
07
1.7385
E
+
07
—
—
56
6.01
Eugeniin
C41H30O26
2.7483
E
+
08
3.0279
E
+
08
2.8080
E
+
08
3.0479
E
+
08
57
6.01
Dihydroxymethyl benzoyl tetragalloyl glucose
C41H30O26
2.7483
E
+
08
3.0279
E
+
08
2.8080
E
+
08
3.0479
E
+
08
58
6.03
1,2,3,6-tetra-O-galloylglucose isomer A
C34H28O22
1.3555
E
+
09
—
1.1980
E
+
09
1.1039
E
+
09
59
6.03
Tetragalloyl glucose B
C34H28O22
1.3555
E
+
09
—
—
—
60
6.08
Astragalin
C21H20O11
1.5009
E
+
07
1.8552
E
+
07
1.5922
E
+
07
1.4002
E
+
07
61
6.09
Isomaltopaeoniflorin isomer
C29H38O16
7.5172
E
+
07
—
—
—
62
6.47
1,2,3,6-tetra-O-galloylglucose isomer B
C34H28O22
1.5882
E
+
09
—
—
1.2570
E
+
09
63
6.47
Tetragalloyl glucose C
C34H28O22
1.5882
E
+
09
—
—
1.2570
E
+
09
64
6.85
3,6-di-O-galloyl paeoniorin
C37H36O19
7.6512
E
+
07
—
—
—
65
6.96
1,2,3,6-tetra-O-galloylglucose
C34H28O22
4.4729
E
+
08
4.5825
E
+
08
4.0642
E
+
08
4.2393
E
+
08
66
6.96
Tetragalloyl glucose D
C34H28O22
4.4729
E
+
08
4.5825
E
+
08
4.0642
E
+
08
4.2393
E
+
08
67
7.35
Galloylpaeoniflorin isomer I
C30H32O15
1.2156
E
+
10
1.2451
E
+
10
1.1484
E
+
10
1.0962
E
+
10
68
7.60
1-O-glucopyranosyl-8-O-benzoyl paeonisuffrone
C23H28O10
4.3983
E
+
07
4.5347
E
+
07
4.4927
E
+
07
3.9869
E
+
07
69
7.71
Glucopyranosylalbiorin isomer I
C29H38O16
7.2982
E
+
07
7.9341
E
+
07
—
1.8872
E
+
07
70
8.18
1-O-glucopyranosyl-8-O-benzoyl paeonisuffrone
C23H28O10
7.4648
E
+
07
8.3204
E
+
07
6.5957
E
+
07
5.9832
E
+
07
71
8.31
Ortho-oxypaeoniflorin
C23H28O12
2.4469
E
+
07
2.4504
E
+
07
2.3932
E
+
07
2.2796
E
+
07
72
8.45
1,2,3,4,6-Penta-O-galloyl–D-glucopyranose
C41H32O26
1.1843
E
+
10
1.0905
E
+
10
1.0518
E
+
10
1.0489
E
+
10
73
8.45
Pentagalloyl glucose
C41H32O26
1.1843
E
+
10
1.0905
E
+
10
1.0518
E
+
10
1.0489
E
+
10
74
8.64
Lactiflorin
C23H26O10
1.0818
E
+
08
1.8628
E
+
08
1.3689
E
+
08
—
75
8.80
Galloylalbiroin
C30H32O15
3.2696
E
+
09
—
—
—
76
9.17
Astragalin
C21H20O11
1.0717
E
+
07
1.3960
E
+
07
1.2843
E
+
07
1.0582
E
+
07
77
9.25
Lactinolide
C10H16O4
2.7251
E
+
07
2.6105
E
+
07
2.1735
E
+
07
3.2770
E
+
07
78
9.29
Galloylpaeoniflorin isomer II
C30H32O15
2.8831
E
+
09
—
2.6829
E
+
09
2.2850
E
+
09
79
9.68
Glucopyranosylalbiorin isomer II
C29H38O16
2.4321
E
+
07
2.6804
E
+
07
2.2576
E
+
07
2.4950
E
+
07
80
9.84
Hexagalloyl glucose
C48H36O30
4.9153
E
+
07
—
6.8676
E
+
08
5.7793
E
+
08
81
9.95
Oxybenzoyl-oxypaeoniflorin
C30H32O14
1.4385
E
+
07
1.6654
E
+
07
1.1051
E
+
07
1.1345
E
+
07
82
10.07
1-O-glucopyranosyl-8-O-benzoylpaeonisuffrone
C23H28O10
3.6916
E
+
09
3.5634
E
+
09
3.1333
E
+
09
3.2106
E
+
09
83
10.29
Albiflorin R1 isomer I
C23H28O11
6.3346
E
+
09
6.6205
E
+
09
5.9528
E
+
09
5.8736
E
+
09
84
10.74
Hexagalloyl glucose
C48H36O30
4.9225
E
+
08
2.5582
E
+
08
1.9395
E
+
09
1.5439
E
+
09
85
10.76
Lactiflorin
C23H26O10
1.2785
E
+
09
3.5174
E
+
09
9.9713
E
+
08
3.4524
E
+
09
86
10.84
Benzoylpaeoniflorin Sulfonate
C30H32O14S
9.0616
E
+
08
6.4075
E
+
08
1.5946
E
+
08
2.1931
E
+
09
87
10.88
3,6-di-O-galloyl paeoniorin
C37H36O19
1.6123
E
+
08
—
—
—
88
10.95
Ortho-oxypaeoniflorin isomer
C23H28O12
5.5563
E
+
07
5.8774
E
+
07
5.7147
E
+
07
5.6640
E
+
07
89
11.52
3,6-di-O-galloyl paeoniorin
C37H36O19
3.6509
E
+
08
3.9290
E
+
08
5.2162
E
+
08
5.3781
E
+
08
90
11.72
3,6-di-O-galloyl paeoniorin isomer
C37H36O19
9.7356
E
+
08
—
1.2523
E
+
09
9.5929
E
+
08
91
11.75
Galloylalbiroin isomer I
C30H32O15
2.3457
E
+
08
—
—
—
92
11.84
Oxypaeoniflorin sulfonate isomer
C23H28O14S
2.1063
E
+
07
1.9747
E
+
07
1.3875
E
+
07
1.0840
E
+
07
93
12.15
1-O-glucopyranosyl-8-O-benzoylpaeonisuffrone
C23H28O10
7.2104
E
+
07
7.1468
E
+
07
6.7309
E
+
07
6.5917
E
+
07
94
12.15
Oxybenzoyl-oxypaeoniflorin
C30H32O14
1.9982
E
+
08
—
—
1.6891
E
+
08
95
12.18
Benzoyloxypaeoniflorin
C30H32O13
2.0822
E
+
08
—
2.0163
E
+
08
1.9074
E
+
08
96
13.42
Benzoyloxypaeoniflorin isomer
C30H32O13
8.6458
E
+
07
6.2282
E
+
07
7.6048
E
+
07
7.2791
E
+
07
97
13.44
Oxybenzoyl-oxypaeoniflorin isomer I
C30H32O14
1.4728
E
+
07
1.7389
E
+
07
1.5360
E
+
07
1.6008
E
+
07
98
13.85
Galloylalbiroin isomer II
C30H32O15
9.6403
E
+
07
1.2196
E
+
08
1.0506
E
+
08
1.0272
E
+
08
99
14.05
Oxybenzoyl-oxypaeoniflorin isomer II
C30H32O14
2.5323
E
+
07
2.9603
E
+
07
2.3556
E
+
07
2.8526
E
+
07
100
14.13
Benzoyloxypaeoniflorin
C30H32O13
3.8096
E
+
07
3.8557
E
+
07
3.7499
E
+
07
3.5800
E
+
07
101
15.07
Benzoyloxypaeoniflorin isomer I
C30H32O13
1.9827
E
+
07
2.3616
E
+
07
—
—
102
15.38
Benzoyloxypaeoniflorin isomer II
C30H32O13
1.1841
E
+
07
1.3730
E
+
07
—
—
103
16.01
Oxybenzoyl-paeoniflorin
C30H32O12
1.8152
E
+
07
—
1.8435
E
+
07
—
104
16.95
Isobenzoylpaeoniflorin
C30H32O12
1.2225
E
+
10
1.3228
E
+
10
1.2158
E
+
10
1.2391
E
+
10
105
16.95
Oxybenzoyl-paeoniflorin isomer I
C30H32O12
1.2225
E
+
10
1.3228
E
+
10
1.2158
E
+
10
1.2391
E
+
10
106
17.23
Benzoylpaeoniflorin Sulfonate
C30H32O14S
1.5680
E
+
07
1.2235
E
+
07
5.6573
E
+
06
3.5831
E
+
07
107
17.48
Isobenzoylpaeoniflorin isomer I
C30H32O12
5.4138
E
+
09
5.4432
E
+
09
5.2522
E
+
09
5.3238
E
+
09
108
17.48
Oxybenzoyl-paeoniflorin isomer II
C30H32O12
5.4138
E
+
09
5.4432
E
+
09
5.2522
E
+
09
5.3238
E
+
09
109
17.86
Benzoyloxypaeoniflorin
C30H32O13
3.4347
E
+
07
3.4852
E
+
07
3.5980
E
+
07
3.8814
E
+
07
110
18.55
Benzoyloxypaeoniflorin isomer
C30H32O13
1.5397
E
+
07
1.7656
E
+
07
1.7246
E
+
07
1.8012
E
+
07
111
18.69
Albiflorin R1 isomer II
C23H28O11
2.0046
E
+
07
1.9851
E
+
07
2.3462
E
+
07
—
112
19.30
Albiflorin R1 isomer III
C23H28O11
2.9827
E
+
06
—
—
5.6105
E
+
06
113
21.79
Palbinone
C22H30O4
8.9687
E
+
07
1.3174
E
+
08
1.2834
E
+
08
5.7610
E
+
07
114
21.93
Isobenzoylpaeoniflorin isomer II
C30H32O12
4.5356
E
+
08
4.2874
E
+
07
3.4016
E
+
08
2.7347
E
+
08
115
21.93
Oxybenzoyl-paeoniflorin isomer III
C30H32O12
4.5356
E
+
08
4.2874
E
+
07
3.4016
E
+
08
2.7347
E
+
08
116
22.15
Paeonilactinone
C10H16O2
7.0423
E
+
06
3.7108
E
+
06
8.0036
E
+
06
6.6886
E
+
06
117
36.46
Hederagenin
C30H48O4
7.6725
E
+
07
8.1456
E
+
07
9.7498
E
+
07
4.7332
E
+
07
118
37.31
23-hydroxybetulinic acid
C30H48O4
3.9836
E
+
07
4.0995
E
+
07
3.9906
E
+
07
2.2611
E
+
07
119
38.14
Astrantiagenin D
C30H46O4
7.8714
E
+
06
7.9560
E
+
06
1.1904
E
+
07
3.8958
E
+
06
120
43.00
Astrantiagenin D isomer
C30H46O4
4.0450
E
+
06
—
3.1585
E
+
06
—
121
45.65
Oleanolic acid
C30H48O3
1.1266
E
+
08
9.4258
E
+
07
7.6434
E
+
07
4.3295
E
+
07
122
46.10
Betulinic acid
C30H48O3
6.2494
E
+
06
2.3289
E
+
07
4.0543
E
+
07
2.3912
E
+
07
123
52.48
Daucosterol
C35H60O6
1.4060
E
+
07
1.9624
E
+
07
8.5440
E
+
06
6.3156
E
+
06Total ion chromatograms of crude (a) and processed (b) Paeoniae Radix Alba obtained from both positive and negative ion modes.
(a)
(b)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 atm/z 319.11731, 197.08075, 133.06473, and 105.03342 in positive ion mode, andm/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.Mass spectra and proposed fragmentations of albiflorin (a), paeoniflorin (b), and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose (c).
(a)
(b)
(c)


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

Figure3 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.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
2.0389
E
+
07
1.4454
E
+
07
2.0881
E
+
07
2.4383
E
+
07
2
2.67
Protocatechuic acid isomer I
C7H6O4
9.6661
E
+
07
—
—
—
3
3.24
Caffeic acid
C9H8O4
3.6818
E
+
08
1.7393
E
+
08
2.8796
E
+
08
1.2882
E
+
08
4
3.73
Protocatechuic acid isomer II
C7H6O4
2.0846
E
+
07
—
—
1.2022
E
+
07
5
4.21
Dictamnoside A isomer I
C21H36O9
1.8843
E
+
07
2.4981
E
+
07
1.0636
E
+
07
1.3140
E
+
07
6
4.70
Dictamnoside A isomer II
C21H36O9
2.8770
E
+
07
3.4768
E
+
07
1.0395
E
+
07
1.4208
E
+
07
7
5.63
Scopoletin
C10H8O4
6.1458
E
+
07
4.1494
E
+
07
6.1562
E
+
07
5.3342
E
+
07
8
5.82
Dictamnoside A
C21H36O9
9.6195
E
+
07
1.1991
E
+
08
7.5446
E
+
07
9.4190
E
+
07
9
8.77
Atracetylentriol
C14H16O3
1.2538
E
+
07
5.4052
E
+
06
—
—
10
9.33
Ferulic acid
C10H10O4
1.3958
E
+
07
9.1214
E
+
06
1.1912
E
+
07
9.6849
E
+
06
11
25.81
Atractylenolide I isomer
C15H18O2
4.5224
E
+
09
4.2401
E
+
09
5.9401
E
+
09
6.5277
E
+
09
12
25.83
Atractylenolide III
C15H20O3
2.5549
E
+
09
1.8023
E
+
09
2.8280
E
+
09
3.1632
E
+
09
13
26.17
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol
C21H26O5
2.4755
E
+
07
—
—
—
14
26.95
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol isomer
C21H26O5
7.5991
E
+
07
—
—
—
15
31.10
Atractylenolide II isomer
C15H20O2
6.7883
E
+
09
4.5794
E
+
09
7.6246
E
+
09
7.8814
E
+
09
16
31.66
Atractylenolide II
C15H20O2
2.8279
E
+
10
1.9902
E
+
10
3.0285
E
+
10
3.1294
E
+
10
17
33.44
Atractylodin
C13H10O
6.4157
E
+
06
—
7.0452
E
+
07
—
18
35.07
Atractylenolide I isomer
C15H18O2
8.2226
E
+
08
1.4781
E
+
09
1.0831
E
+
09
3.2083
E
+
09
19
35.94
Atractylenolide I
C15H18O2
8.8877
E
+
09
7.2520
E
+
09
8.3857
E
+
09
1.2742
E
+
10
20
39.03
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol isomer I
C21H26O5
3.0978
E
+
07
3.7863
E
+
07
2.9171
E
+
07
—
21
39.81
Dibutyl phthalate
C16H22O4
1.1372
E
+
08
9.8325
E
+
07
1.2659
E
+
08
1.4865
E
+
08
22
40.00
12-methylbutyryl-14-acetyl 2E,8EZ,10E-atractylentriol isomer II
C21H26O5
3.8810
E
+
07
7.7498
E
+
07
3.3885
E
+
07
7.0522
E
+
07
23
40.26
Dibutyl phthalate isomer
C16H22O4
1.0631
E
+
08
5.4902
E
+
07
6.1958
E
+
07
4.6227
E
+
07
24
41.50
14-methylbutyryl-2E,8EZ,10Es-atractylentriol
C19H24O4
4.9587
E
+
07
2.8423
E
+
07
5.1146
E
+
07
4.7855
E
+
07
25
46.43
Spinasteryl
C29H48O
8.6778
E
+
06
7.9096
E
+
06
1.0609
E
+
07
7.7832
E
+
06
26
47.32
Atractylon
C15H20O
7.4433
E
+
07
5.4063
E
+
07
6.6146
E
+
07
—
27
47.37
Biatractylolide
C30H38O4
1.0949
E
+
09
9.5665
E
+
08
1.2797
E
+
09
—
28
47.96
Linoleic acid
C18H32O2
1.8499
E
+
08
1.5041
E
+
08
1.8777
E
+
08
2.3743
E
+
08
29
48.25
Linoleic acid isomer
C18H32O2
2.1059
E
+
07
—
—
—
30
48.59
Biepiasterolid isomer
C30H38O4
9.0255
E
+
08
7.0863
E
+
08
7.4011
E
+
08
—
31
48.90
Atractylon isomer
C15H20O
9.5308
E
+
07
8.7683
E
+
07
8.2967
E
+
07
1.0132
E
+
08
32
49.42
Palmitic acid
C16H32O2
2.2356
E
+
07
2.2942
E
+
07
2.5949
E
+
07
2.0153
E
+
07Total ion chromatograms of crude (a) and processed (b) Atractylodis Macrocephalae Rhizoma obtained from both positive and negative ion modes.
(a)
(b)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 atm/z 231.13799, which could lead to four MS2 ions atm/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 atm/z 215.14310, 187.14818, 159.08055, 151.07541, 133.10117, and 95.08547. Meanwhile, the MS2 spectrum ofm/z 249.14836 from atractylenolide III contained six major fragment ions atm/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.Mass spectra of atractylenolide I (a), atractylenolide II (b), and atractylenolide III (c).
(a)
(b)
(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 Figure5 and Table 1.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.
(a)
(b)


### 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 Figure5 and Table 2.


## 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 Figure1. 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.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
PaeoniaeRadix Alba
Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair
(Measured area)
(Measured area)
Crude
Processed
Crude
Processed
1
0.84
6-O-galloylsucrose
C19H26O15
1.8570
E
+
08
1.9012
E
+
08
4.2870
E
+
07
4.4158
E
+
07
2
0.84
Glucogallin
C13H16O10
2.9739
E
+
08
2.5698
E
+
08
1.0931
E
+
08
—
3
1.05
Desbenzoylpaeoniflorin
C16H24O10
1.6682
E
+
08
1.6263
E
+
08
9.8500
E
+
07
—
4
1.06
1′-O-galloylsucrose
C19H26O15
3.2574
E
+
08
2.9123
E
+
08
—
—
5
1.07
1-O-glucopyranosyl paeonisuffrone
C16H24O9
2.8667
E
+
08
2.3654
E
+
08
1.1532
E
+
08
—
6
1.13
Gallic acid
C7H6O5
4.1152
E
+
09
4.0736
E
+
09
2.7186
E
+
09
3.1711
E
+
09
7
1.18
Oxypaeoniflorin sulfonate
C23H28O14S
4.9527
E
+
07
3.5407
E
+
07
6.1568
E
+
06
8.5010
E
+
07
8
1.22
Ethyl gallate
C9H10O5
5.1351
E
+
07
6.7200
E
+
07
1.5592
E
+
07
4.8337
E
+
07
9
1.22
6-O-galloyl desbenzoylpaeoniflorin
C23H28O14
9.9020
E
+
07
9.5040
E
+
07
5.3798
E
+
07
—
10
1.26
6-O-glucopyranosyl-lactinolide
C16H26O9
1.0875
E
+
08
1.1180
E
+
08
—
3.7130
E
+
07
11
1.30
Paeoniflorin sulfonate I
C23H28O13S
5.3777
E
+
07
3.6391
E
+
07
7.3077
E
+
06
7.7185
E
+
07
12
1.30
Mudanpioside E sulfonate
C24H30O15S
5.3777
E
+
07
3.6391
E
+
07
7.3077
E
+
06
7.7185
E
+
07
13
1.43
6-O-glucopyranosyl-lactinolide
C16H26O9
7.4342
E
+
08
6.5904
E
+
08
4.0712
E
+
08
3.1407
E
+
08
14
1.64
Mudanpioside F
C16H24O8
6.4178
E
+
08
6.0980
E
+
08
4.0130
E
+
08
6.6680
E
+
07
15
1.76
Isomaltopaeoniflorin sulfonate
C29H38O18S
1.8858
E
+
09
1.1382
E
+
09
2.6277
E
+
08
5.8622
E
+
07
16
1.81
Pedunculagin
C34H24O22
4.8098
E
+
07
—
5.6076
E
+
07
1.2660
E
+
09
17
1.97
Paeoniflorin sulfonate I
C23H28O13S
3.1881
E
+
10
2.3387
E
+
10
6.6202
E
+
09
5.5241
E
+
10
18
2.25
Oxypaeoniflorin
C23H28O12
2.3173
E
+
09
2.4115
E
+
09
1.6734
E
+
09
1.4513
E
+
09
19
2.36
Gallotannin
C27H24O18
2.2850
E
+
08
2.2458
E
+
08
1.6284
E
+
08
1.6703
E
+
08
20
2.37
1-O-benzoylsucrose
C19H26O12
1.3761
E
+
08
1.3161
E
+
08
1.1673
E
+
08
8.2986
E
+
07
21
2.41
d-catechin
C15H14O6
3.7822
E
+
09
4.2278
E
+
09
2.6339
E
+
09
2.5982
E
+
09
22
2.63
Methyl gallate
C8H8O5
2.3823
E
+
10
2.4116
E
+
10
2.3388
E
+
10
—
23
2.63
Salicylic acid
C7H6O3
2.3823
E
+
10
2.4116
E
+
10
2.3388
E
+
10
1.7399
E
+
10
24
2.72
Albiflorin R1
C23H28O11
5.2469
E
+
08
5.6725
E
+
08
5.6329
E
+
08
4.5647
E
+
08
25
3.00
Kaempferol-3,7-di-O-glucoside
C27H30O16
3.8065
E
+
07
2.1896
E
+
07
3.5719
E
+
07
1.5513
E
+
07
26
3.00
Paeonoside
C27H30O16
3.8065
E
+
07
2.1896
E
+
07
3.5719
E
+
07
1.5513
E
+
07
27
3.46
Galloylpaeoniflorin
C30H32O15
1.3912
E
+
08
1.5200
E
+
08
1.1501
E
+
08
1.0863
E
+
08
28
3.47
Paeonolide
C20H28O12
1.0622
E
+
07
1.1936
E
+
07
9.1812
E
+
06
—
29
3.58
6-O-glucopyranosyl-lactinolide
C16H26O9
2.5249
E
+
08
2.3834
E
+
08
2.2675
E
+
08
2.1241
E
+
08
30
3.68
Oxypaeoniflorin
C23H28O12
1.5407
E
+
08
—
1.4345
E
+
08
1.3307
E
+
08
31
3.76
6-O-glucopyranosyl-lactinolide
C16H26O9
3.2664
E
+
08
—
3.0588
E
+
08
3.1627
E
+
08
32
3.88
Paeonilactone B
C10H12O4
9.0325
E
+
07
9.3539
E
+
07
5.1257
E
+
07
8.9597
E
+
07
33
3.93
Isomaltopaeoniflorin
C29H38O16
1.1545
E
+
10
1.1941
E
+
10
1.2282
E
+
10
9.4600
E
+
09
34
4.07
Albiflorin
C23H28O11
2.9587
E
+
10
2.9296
E
+
10
2.8430
E
+
10
2.8684
E
+
10
35
4.32
Glucopyranosylalbiorin
C29H38O16
2.2813
E
+
09
2.4109
E
+
08
2.0383
E
+
08
1.6844
E
+
08
36
4.34
Galloylpaeoniflorin sulfonate
C30H32O17S
7.6943
E
+
08
5.6793
E
+
08
1.5501
E
+
08
1.4886
E
+
09
37
4.34
Galloylpaeoniflorin isomer
C30H32O15
6.7592
E
+
08
7.4322
E
+
08
6.0470
E
+
08
5.4051
E
+
08
38
4.38
1,2,3,6-tetra-O-galloylglucose
C34H28O22
4.6602
E
+
08
3.4977
E
+
08
4.0697
E
+
08
3.6901
E
+
08
39
4.38
Tetragalloyl glucose A
C34H28O22
4.6602
E
+
08
3.4977
E
+
08
4.0697
E
+
08
3.6901
E
+
08
40
4.56
Mudanpioside F
C16H24O8
8.4156
E
+
07
8.2734
E
+
07
7.2666
E
+
07
7.9227
E
+
07
41
4.60
Oxypaeoniflorin isomer
C23H28O12
9.6610
E
+
08
9.8706
E
+
08
9.2464
E
+
08
8.7359
E
+
08
42
4.65
Gallotannin
C27H24O18
6.7737
E
+
07
—
—
—
43
4.77
Paeoniflorin
C23H28O11
5.9556
E
+
10
6.1356
E
+
10
5.9929
E
+
10
5.8832
E
+
10
44
4.89
Paeoniflorin sulfonate II
C23H28O13S
1.1095
E
+
08
1.4567
E
+
08
5.5052
E
+
07
2.7813
E
+
08
45
4.98
Isogalloylpaeoniflorin sulfonate
C30H32O17S
3.6742
E
+
07
—
—
—
46
5.05
Ethyl gallate
C9H10O5
6.0669
E
+
07
5.4850
E
+
07
1.6681
E
+
07
2.6790
E
+
07
47
5.05
Methyl salicylate
C8H8O3
6.0669
E
+
07
5.4850
E
+
07
1.6681
E
+
07
2.6790
E
+
07
48
5.15
Benzoic acid
C7H6O2
4.0163
E
+
07
4.5493
E
+
07
2.9695
E
+
07
2.9727
E
+
07
49
5.25
Paeonol
C9H10O3
6.8567
E
+
07
7.4129
E
+
07
9.9992
E
+
07
6.5619
E
+
07
50
5.25
4-hydroxy-3-methoxy acetophenone
C9H10O3
6.8567
E
+
07
7.4129
E
+
07
9.9992
E
+
07
6.5619
E
+
07
51
5.31
ortho-oxypaeoniflorin
C23H28O12
1.9080
E
+
09
1.9263
E
+
09
1.8723
E
+
09
1.6842
E
+
09
52
5.63
Ethyl gallate
C9H10O5
1.4627
E
+
08
1.2812
E
+
08
1.0365
E
+
08
8.8155
E
+
07
53
5.63
Methyl salicylate
C8H8O3
1.4627
E
+
08
1.2812
E
+
08
1.0365
E
+
08
8.8155
E
+
07
54
5.66
Kaempferol-3-O-glucoside
C21H20O11
1.6012
E
+
07
1.7385
E
+
07
—
—
55
5.66
Astragalin
C21H20O11
1.6012
E
+
07
1.7385
E
+
07
—
—
56
6.01
Eugeniin
C41H30O26
2.7483
E
+
08
3.0279
E
+
08
2.8080
E
+
08
3.0479
E
+
08
57
6.01
Dihydroxymethyl benzoyl tetragalloyl glucose
C41H30O26
2.7483
E
+
08
3.0279
E
+
08
2.8080
E
+
08
3.0479
E
+
08
58
6.03
1,2,3,6-tetra-O-galloylglucose isomer A
C34H28O22
1.3555
E
+
09
—
1.1980
E
+
09
1.1039
E
+
09
59
6.03
Tetragalloyl glucose B
C34H28O22
1.3555
E
+
09
—
—
—
60
6.08
Astragalin
C21H20O11
1.5009
E
+
07
1.8552
E
+
07
1.5922
E
+
07
1.4002
E
+
07
61
6.09
Isomaltopaeoniflorin isomer
C29H38O16
7.5172
E
+
07
—
—
—
62
6.47
1,2,3,6-tetra-O-galloylglucose isomer B
C34H28O22
1.5882
E
+
09
—
—
1.2570
E
+
09
63
6.47
Tetragalloyl glucose C
C34H28O22
1.5882
E
+
09
—
—
1.2570
E
+
09
64
6.85
3,6-di-O-galloyl paeoniorin
C37H36O19
7.6512
E
+
07
—
—
—
65
6.96
1,2,3,6-tetra-O-galloylglucose
C34H28O22
4.4729
E
+
08
4.5825
E
+
08
4.0642
E
+
08
4.2393
E
+
08
66
6.96
Tetragalloyl glucose D
C34H28O22
4.4729
E
+
08
4.5825
E
+
08
4.0642
E
+
08
4.2393
E
+
08
67
7.35
Galloylpaeoniflorin isomer I
C30H32O15
1.2156
E
+
10
1.2451
E
+
10
1.1484
E
+
10
1.0962
E
+
10
68
7.60
1-O-glucopyranosyl-8-O-benzoyl paeonisuffrone
C23H28O10
4.3983
E
+
07
4.5347
E
+
07
4.4927
E
+
07
3.9869
E
+
07
69
7.71
Glucopyranosylalbiorin isomer I
C29H38O16
7.2982
E
+
07
7.9341
E
+
07
—
1.8872
E
+
07
70
8.18
1-O-glucopyranosyl-8-O-benzoyl paeonisuffrone
C23H28O10
7.4648
E
+
07
8.3204
E
+
07
6.5957
E
+
07
5.9832
E
+
07
71
8.31
Ortho-oxypaeoniflorin
C23H28O12
2.4469
E
+
07
2.4504
E
+
07
2.3932
E
+
07
2.2796
E
+
07
72
8.45
1,2,3,4,6-Penta-O-galloyl–D-glucopyranose
C41H32O26
1.1843
E
+
10
1.0905
E
+
10
1.0518
E
+
10
1.0489
E
+
10
73
8.45
Pentagalloyl glucose
C41H32O26
1.1843
E
+
10
1.0905
E
+
10
1.0518
E
+
10
1.0489
E
+
10
74
8.64
Lactiflorin
C23H26O10
1.0818
E
+
08
1.8628
E
+
08
1.3689
E
+
08
—
75
8.80
Galloylalbiroin
C30H32O15
3.2696
E
+
09
—
—
—
76
9.17
Astragalin
C21H20O11
1.0717
E
+
07
1.3960
E
+
07
1.2843
E
+
07
1.0582
E
+
07
77
9.25
Lactinolide
C10H16O4
2.7251
E
+
07
2.6105
E
+
07
2.1735
E
+
07
3.2770
E
+
07
78
9.29
Galloylpaeoniflorin isomer II
C30H32O15
2.8831
E
+
09
—
2.6829
E
+
09
2.2850
E
+
09
79
9.68
Glucopyranosylalbiorin isomer II
C29H38O16
2.4321
E
+
07
2.6804
E
+
07
2.2576
E
+
07
2.4950
E
+
07
80
9.84
Hexagalloyl glucose
C48H36O30
4.9153
E
+
07
—
6.8676
E
+
08
5.7793
E
+
08
81
9.95
Oxybenzoyl-oxypaeoniflorin
C30H32O14
1.4385
E
+
07
1.6654
E
+
07
1.1051
E
+
07
1.1345
E
+
07
82
10.07
1-O-glucopyranosyl-8-O-benzoylpaeonisuffrone
C23H28O10
3.6916
E
+
09
3.5634
E
+
09
3.1333
E
+
09
3.2106
E
+
09
83
10.29
Albiflorin R1 isomer I
C23H28O11
6.3346
E
+
09
6.6205
E
+
09
5.9528
E
+
09
5.8736
E
+
09
84
10.74
Hexagalloyl glucose
C48H36O30
4.9225
E
+
08
2.5582
E
+
08
1.9395
E
+
09
1.5439
E
+
09
85
10.76
Lactiflorin
C23H26O10
1.2785
E
+
09
3.5174
E
+
09
9.9713
E
+
08
3.4524
E
+
09
86
10.84
Benzoylpaeoniflorin Sulfonate
C30H32O14S
9.0616
E
+
08
6.4075
E
+
08
1.5946
E
+
08
2.1931
E
+
09
87
10.88
3,6-di-O-galloyl paeoniorin
C37H36O19
1.6123
E
+
08
—
—
—
88
10.95
Ortho-oxypaeoniflorin isomer
C23H28O12
5.5563
E
+
07
5.8774
E
+
07
5.7147
E
+
07
5.6640
E
+
07
89
11.52
3,6-di-O-galloyl paeoniorin
C37H36O19
3.6509
E
+
08
3.9290
E
+
08
5.2162
E
+
08
5.3781
E
+
08
90
11.72
3,6-di-O-galloyl paeoniorin isomer
C37H36O19
9.7356
E
+
08
—
1.2523
E
+
09
9.5929
E
+
08
91
11.75
Galloylalbiroin isomer I
C30H32O15
2.3457
E
+
08
—
—
—
92
11.84
Oxypaeoniflorin sulfonate isomer
C23H28O14S
2.1063
E
+
07
1.9747
E
+
07
1.3875
E
+
07
1.0840
E
+
07
93
12.15
1-O-glucopyranosyl-8-O-benzoylpaeonisuffrone
C23H28O10
7.2104
E
+
07
7.1468
E
+
07
6.7309
E
+
07
6.5917
E
+
07
94
12.15
Oxybenzoyl-oxypaeoniflorin
C30H32O14
1.9982
E
+
08
—
—
1.6891
E
+
08
95
12.18
Benzoyloxypaeoniflorin
C30H32O13
2.0822
E
+
08
—
2.0163
E
+
08
1.9074
E
+
08
96
13.42
Benzoyloxypaeoniflorin isomer
C30H32O13
8.6458
E
+
07
6.2282
E
+
07
7.6048
E
+
07
7.2791
E
+
07
97
13.44
Oxybenzoyl-oxypaeoniflorin isomer I
C30H32O14
1.4728
E
+
07
1.7389
E
+
07
1.5360
E
+
07
1.6008
E
+
07
98
13.85
Galloylalbiroin isomer II
C30H32O15
9.6403
E
+
07
1.2196
E
+
08
1.0506
E
+
08
1.0272
E
+
08
99
14.05
Oxybenzoyl-oxypaeoniflorin isomer II
C30H32O14
2.5323
E
+
07
2.9603
E
+
07
2.3556
E
+
07
2.8526
E
+
07
100
14.13
Benzoyloxypaeoniflorin
C30H32O13
3.8096
E
+
07
3.8557
E
+
07
3.7499
E
+
07
3.5800
E
+
07
101
15.07
Benzoyloxypaeoniflorin isomer I
C30H32O13
1.9827
E
+
07
2.3616
E
+
07
—
—
102
15.38
Benzoyloxypaeoniflorin isomer II
C30H32O13
1.1841
E
+
07
1.3730
E
+
07
—
—
103
16.01
Oxybenzoyl-paeoniflorin
C30H32O12
1.8152
E
+
07
—
1.8435
E
+
07
—
104
16.95
Isobenzoylpaeoniflorin
C30H32O12
1.2225
E
+
10
1.3228
E
+
10
1.2158
E
+
10
1.2391
E
+
10
105
16.95
Oxybenzoyl-paeoniflorin isomer I
C30H32O12
1.2225
E
+
10
1.3228
E
+
10
1.2158
E
+
10
1.2391
E
+
10
106
17.23
Benzoylpaeoniflorin Sulfonate
C30H32O14S
1.5680
E
+
07
1.2235
E
+
07
5.6573
E
+
06
3.5831
E
+
07
107
17.48
Isobenzoylpaeoniflorin isomer I
C30H32O12
5.4138
E
+
09
5.4432
E
+
09
5.2522
E
+
09
5.3238
E
+
09
108
17.48
Oxybenzoyl-paeoniflorin isomer II
C30H32O12
5.4138
E
+
09
5.4432
E
+
09
5.2522
E
+
09
5.3238
E
+
09
109
17.86
Benzoyloxypaeoniflorin
C30H32O13
3.4347
E
+
07
3.4852
E
+
07
3.5980
E
+
07
3.8814
E
+
07
110
18.55
Benzoyloxypaeoniflorin isomer
C30H32O13
1.5397
E
+
07
1.7656
E
+
07
1.7246
E
+
07
1.8012
E
+
07
111
18.69
Albiflorin R1 isomer II
C23H28O11
2.0046
E
+
07
1.9851
E
+
07
2.3462
E
+
07
—
112
19.30
Albiflorin R1 isomer III
C23H28O11
2.9827
E
+
06
—
—
5.6105
E
+
06
113
21.79
Palbinone
C22H30O4
8.9687
E
+
07
1.3174
E
+
08
1.2834
E
+
08
5.7610
E
+
07
114
21.93
Isobenzoylpaeoniflorin isomer II
C30H32O12
4.5356
E
+
08
4.2874
E
+
07
3.4016
E
+
08
2.7347
E
+
08
115
21.93
Oxybenzoyl-paeoniflorin isomer III
C30H32O12
4.5356
E
+
08
4.2874
E
+
07
3.4016
E
+
08
2.7347
E
+
08
116
22.15
Paeonilactinone
C10H16O2
7.0423
E
+
06
3.7108
E
+
06
8.0036
E
+
06
6.6886
E
+
06
117
36.46
Hederagenin
C30H48O4
7.6725
E
+
07
8.1456
E
+
07
9.7498
E
+
07
4.7332
E
+
07
118
37.31
23-hydroxybetulinic acid
C30H48O4
3.9836
E
+
07
4.0995
E
+
07
3.9906
E
+
07
2.2611
E
+
07
119
38.14
Astrantiagenin D
C30H46O4
7.8714
E
+
06
7.9560
E
+
06
1.1904
E
+
07
3.8958
E
+
06
120
43.00
Astrantiagenin D isomer
C30H46O4
4.0450
E
+
06
—
3.1585
E
+
06
—
121
45.65
Oleanolic acid
C30H48O3
1.1266
E
+
08
9.4258
E
+
07
7.6434
E
+
07
4.3295
E
+
07
122
46.10
Betulinic acid
C30H48O3
6.2494
E
+
06
2.3289
E
+
07
4.0543
E
+
07
2.3912
E
+
07
123
52.48
Daucosterol
C35H60O6
1.4060
E
+
07
1.9624
E
+
07
8.5440
E
+
06
6.3156
E
+
06Total ion chromatograms of crude (a) and processed (b) Paeoniae Radix Alba obtained from both positive and negative ion modes.
(a)
(b)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 atm/z 319.11731, 197.08075, 133.06473, and 105.03342 in positive ion mode, andm/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.Mass spectra and proposed fragmentations of albiflorin (a), paeoniflorin (b), and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose (c).
(a)
(b)
(c)


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

Figure3 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.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
2.0389
E
+
07
1.4454
E
+
07
2.0881
E
+
07
2.4383
E
+
07
2
2.67
Protocatechuic acid isomer I
C7H6O4
9.6661
E
+
07
—
—
—
3
3.24
Caffeic acid
C9H8O4
3.6818
E
+
08
1.7393
E
+
08
2.8796
E
+
08
1.2882
E
+
08
4
3.73
Protocatechuic acid isomer II
C7H6O4
2.0846
E
+
07
—
—
1.2022
E
+
07
5
4.21
Dictamnoside A isomer I
C21H36O9
1.8843
E
+
07
2.4981
E
+
07
1.0636
E
+
07
1.3140
E
+
07
6
4.70
Dictamnoside A isomer II
C21H36O9
2.8770
E
+
07
3.4768
E
+
07
1.0395
E
+
07
1.4208
E
+
07
7
5.63
Scopoletin
C10H8O4
6.1458
E
+
07
4.1494
E
+
07
6.1562
E
+
07
5.3342
E
+
07
8
5.82
Dictamnoside A
C21H36O9
9.6195
E
+
07
1.1991
E
+
08
7.5446
E
+
07
9.4190
E
+
07
9
8.77
Atracetylentriol
C14H16O3
1.2538
E
+
07
5.4052
E
+
06
—
—
10
9.33
Ferulic acid
C10H10O4
1.3958
E
+
07
9.1214
E
+
06
1.1912
E
+
07
9.6849
E
+
06
11
25.81
Atractylenolide I isomer
C15H18O2
4.5224
E
+
09
4.2401
E
+
09
5.9401
E
+
09
6.5277
E
+
09
12
25.83
Atractylenolide III
C15H20O3
2.5549
E
+
09
1.8023
E
+
09
2.8280
E
+
09
3.1632
E
+
09
13
26.17
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol
C21H26O5
2.4755
E
+
07
—
—
—
14
26.95
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol isomer
C21H26O5
7.5991
E
+
07
—
—
—
15
31.10
Atractylenolide II isomer
C15H20O2
6.7883
E
+
09
4.5794
E
+
09
7.6246
E
+
09
7.8814
E
+
09
16
31.66
Atractylenolide II
C15H20O2
2.8279
E
+
10
1.9902
E
+
10
3.0285
E
+
10
3.1294
E
+
10
17
33.44
Atractylodin
C13H10O
6.4157
E
+
06
—
7.0452
E
+
07
—
18
35.07
Atractylenolide I isomer
C15H18O2
8.2226
E
+
08
1.4781
E
+
09
1.0831
E
+
09
3.2083
E
+
09
19
35.94
Atractylenolide I
C15H18O2
8.8877
E
+
09
7.2520
E
+
09
8.3857
E
+
09
1.2742
E
+
10
20
39.03
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol isomer I
C21H26O5
3.0978
E
+
07
3.7863
E
+
07
2.9171
E
+
07
—
21
39.81
Dibutyl phthalate
C16H22O4
1.1372
E
+
08
9.8325
E
+
07
1.2659
E
+
08
1.4865
E
+
08
22
40.00
12-methylbutyryl-14-acetyl 2E,8EZ,10E-atractylentriol isomer II
C21H26O5
3.8810
E
+
07
7.7498
E
+
07
3.3885
E
+
07
7.0522
E
+
07
23
40.26
Dibutyl phthalate isomer
C16H22O4
1.0631
E
+
08
5.4902
E
+
07
6.1958
E
+
07
4.6227
E
+
07
24
41.50
14-methylbutyryl-2E,8EZ,10Es-atractylentriol
C19H24O4
4.9587
E
+
07
2.8423
E
+
07
5.1146
E
+
07
4.7855
E
+
07
25
46.43
Spinasteryl
C29H48O
8.6778
E
+
06
7.9096
E
+
06
1.0609
E
+
07
7.7832
E
+
06
26
47.32
Atractylon
C15H20O
7.4433
E
+
07
5.4063
E
+
07
6.6146
E
+
07
—
27
47.37
Biatractylolide
C30H38O4
1.0949
E
+
09
9.5665
E
+
08
1.2797
E
+
09
—
28
47.96
Linoleic acid
C18H32O2
1.8499
E
+
08
1.5041
E
+
08
1.8777
E
+
08
2.3743
E
+
08
29
48.25
Linoleic acid isomer
C18H32O2
2.1059
E
+
07
—
—
—
30
48.59
Biepiasterolid isomer
C30H38O4
9.0255
E
+
08
7.0863
E
+
08
7.4011
E
+
08
—
31
48.90
Atractylon isomer
C15H20O
9.5308
E
+
07
8.7683
E
+
07
8.2967
E
+
07
1.0132
E
+
08
32
49.42
Palmitic acid
C16H32O2
2.2356
E
+
07
2.2942
E
+
07
2.5949
E
+
07
2.0153
E
+
07Total ion chromatograms of crude (a) and processed (b) Atractylodis Macrocephalae Rhizoma obtained from both positive and negative ion modes.
(a)
(b)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 atm/z 231.13799, which could lead to four MS2 ions atm/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 atm/z 215.14310, 187.14818, 159.08055, 151.07541, 133.10117, and 95.08547. Meanwhile, the MS2 spectrum ofm/z 249.14836 from atractylenolide III contained six major fragment ions atm/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.Mass spectra of atractylenolide I (a), atractylenolide II (b), and atractylenolide III (c).
(a)
(b)
(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 Figure5 and Table 1.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.
(a)
(b)


## 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 Figure5 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.



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*Source: 170959-2014-05-04.xml*