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. 2015 Jan 19;20(1):1594–1609. doi: 10.3390/molecules20011594

Identification and Quantitative Characterization of PSORI-CM01, a Chinese Medicine Formula for Psoriasis Therapy, by Liquid Chromatography Coupled with an LTQ Orbitrap Mass Spectrometer

Shao-Dan Chen 1,2,3, Chuan-Jian Lu 1,2,*, Rui-Zhi Zhao 1,2
Editor: Derek J McPhee
PMCID: PMC6272627  PMID: 25608042

Abstract

PSORI-CM01 is a Chinese medicine formula prepared from medicinal herbs and used in China for the treatment of psoriasis. However, the chemical constituents in PSORI-CM01 have not been clarified yet. In order to quickly define the chemical profiles and control the quality of PSORI-CM01 preparations, ultra-high liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole Orbitrap mass spectrometry (UHPLC-ESI-LTQ/Orbitrap-MS) was applied for simultaneous identification and quantification of multiple constituents. A total of 108 compounds, including organic acids, phenolic acids, flavonoids, and terpenoids, were identified or tentatively deduced on the base of their retention behaviors, MS and MSn data, or by comparing with reference substances and literature data. In addition, an optimized UHPLC-ESI-MS method was established for the quantitative determination of 14 marker compounds in different dosage forms of PSORI-CM01 preparations. The validation of the method, including spike recoveries, linearity, sensitivity (LOQ), precision, and repeatability, was carried out and demonstrated to be satisfied the requirements of quantitative analysis. This is the first report on the comprehensive determination of chemical constituents in PSORI-CM01 preparations by UHPLC-ESI-LTQ/Orbitrap mass spectrometry. The results suggested that the established methods would be a powerful and reliable analytical tool for the characterization of multi-constituents in complex chemical system and quality control of TCM preparations.

Keywords: PSORI-CM01, UHPLC-ESI-LTQ/Orbitrap-MS, identification, quantification, TCM

1. Introduction

As known, the Chinese nation, with a history of more than 5000 years of civilization, largely relies on Traditional Chinese Medicine (TCM). TCM still plays a huge role in health care, disease prevention and treatment in China today. This is the reason why TCM has been deeply rooted in China and other Chinese cultural circles around the world for thousands of years. Traditional medicine, especially TCM, is not only an important and indispensable “alternative medicine” or “complementary medicine”, but has also been entrusted with hopes for disease prevention and treatment in the future.

Traditional Chinese medicine prescriptions (TCMPs), or TCM formulae, which are applied according to certain compatibility rules, are the main and important clinical applications of TCM. However, TCMPs are facing difficulties because their effective constituents are unclear and sometimes inexplicable, which seriously restricts their development in the international market [1]. The chemical constituents of TCMPs are the key object of the study of TCM [2]. TCM is commonly considered to operate due to the synergistic effects of all the major and minor components in the medicines. Hence sensitive and comprehensive analytical techniques are needed to acquire a better understanding of the substance basis of TCM and to enhance the product quality control [3].

PSORI-CM01 was a novel formulated Chinese medicine used for psoriasis therapy [4]. It was optimized on the basis of a Chinese medicine formula Yin-Xie Ling, which was originated by Guo-Wei Xuan, the State Medical Master of China [5,6]. PSORI-CM01 was composed of seven herbs including Rhizoma Curcumae (E Zhu), Radix Paeoniae Rubra (Chi Shao), Sarcandra glabra (Zhong-Jie Feng), Radix Arnebiae (Zi Cao), Rhizoma Smilacis Glabrae (Tu-Fu Ling), Fructus Mume (Wu Mei), and Radix Glycyrrhizae (Gan Cao). Guo-Wei Xuan believed that one of the main causes of psoriasis is blood stasis. In this TCM formula PSORI-CM01, Curcumae bearing blood-activating and stasis-dissolving efficacy acts as the monarch drug, Paeoniae Rubra, Sarcandra glabra and Arnebiae, as the minister drugs, that together help Curcumae in activating blood and removing blood stasis. Smilacis Glabrae and Mume produce the effect of reducing the itch and together act as assistant drugs. Licorice, as a guide drug can mediate the other drugs’ properties. When combined, the seven drugs have great therapeutic effects. Although the chemical constituents of the individual herbs have previously been well studied [7,8,9,10,11,12,13,14,15], little is known about the integrated chemical composition of PSORI-CM01. Unlike chemical drugs, botanical products contain a complex mixture of compounds. The contents of these compounds may be significantly affected by plant species, geographical sources, harvesting, processing and storage [16]. In addition, three dosage forms of PSORI-CM01 preparations (tablet, granules and decoction) are produced and used clinically in the Guangdong Provincial Hospital of Chinese Medicine. In order to guarantee drug safety and batch-to-batch consistency, quality control is therefore critically important for preparations such as PSORI-CM01. In recent years, the combination of Orbitrap technology with a linear ion trap, known as LTQ Orbitrap mass spectrometer were introduced, which could provide all the traditional MS and MSn scan functions using a linear IT and high mass accuracy measurements (errors within 5 ppm) [17]. Our previous study indicated that this analytical technique has the potential capability of simple, sensitive and reliable detection and identification of complex samples such as TCMs [9].

In the present study, a sensitive LC-ESI-MSn method was established for rapid separating, reliable identifying and quantifying the multiple components in PSORI-CM01 preparations, by using a hybrid LTQ-Orbitrap mass spectrometer coupled with an UHPLC system. The qualitative analysis was carried out in negative ionization mode to acquire accurate mass data in full scan mode and MS/MS in a data dependent product ion spectrum. Further, 14 reference compounds were quantitatively determined in negative ionization mode and eight samples of PSORI-CM01 preparations were analyzed for assessment of quality consistence.

2. Results and Discussion

2.1. Optimization of Chromatographic Conditions

To improve the resolution and sensitivity of the analysis but reduce the analytical time, the mobile phase system was optimized. To inhibit ionization of the acidic ingredients in PSORI-CM01, formic acid was added to the mobile phase. Two mobile phase systems, methanol-aqueous solution and acetonitrile-aqueous solution were compared. Both negative and positive modes were examined. Generally, in positive mode, low abundance of [M+H]+, [M+NH4]+ ions and few product ions were observed, while, in negative ion mode, a series of [M−H] ions and/or adduct ions ([M+HCOOH−H]) appeared with sufficient abundance. Thus the negative ion mode was chosen and the [M−H]/([M+HCOOH−H]) ions were further subjected to LC-MSn analysis. For the extracted target ions in full scan mode, an accurate mass limit of 5 ppm accurate mass filter limit was used to characterize “real” compounds signals from the background peaks, as well as to increase the signal-to-noise ratio for each analyte.

2.2. Identification of Chemical Constituents in PSORI-CM01 Preparations

The reference substances and PSORI-CM01 sample were analyzed by using the optimized LC-ESI-MSn method. The TIC chromatogram of PSORI-CM01 sample in negative ESI mode was shown in Figure 1, and 108 peaks were observed in PSORI-CM01 sample. The MS data showed high precision with all the mass accuracy within 5 ppm. For most of the constituents, [M−H] ions were observed. Due to the use of formic acid in mobile phase, there were adduct ions of [M+46−H] corresponding to [M+HCOOH−H] in negative ion mode. These results provided valuable information for confirming accurate molecular weight and composition of the constituents.

Figure 1.

Figure 1

UHPLC-(-) ESI-MS total ion chromatograms of a mixture of fourteen standards (A) and PSORI-CM01 (B).

One hundred and eight compounds were tentatively identified on the basis of their retention behaviors, accurate molecular weights and MSn fragment data, or by comparison with reference substances or literature data. Corresponding quasimolecular ions and their fragment ions in the MSn spectra were listed in Table 1. The identified compounds can be classified into four classes including organic acids, phenolic acids, flavonoids, and terpenoids, which were mainly from Radix Paeoniae Rubra, Rhizoma Smilacis Glabrae, Sarcandra glabra, and Radix Glycyrrhizae. Monoterpene glucosides and phenolic acids are the main constituents of Paeoniae Rubra, caffeic acid derivatives and flavonoids are abundant in Sarcandra glabra, flavonoids and phenolic compounds are mainly from Rhizoma Smilacis Glabrae, while flavonoids and triterpene glucoside are from Radix Glycyrrhizae. To some extent, the UHPLC-ESI-MS chromatogram reflected the rationale of PSORI-CM01. However, the characteristic constituents (sesquiterpenoids and curcuminoids) from the monarch drug Rhizoma Curcumae were not detected.

Table 1.

Identification of the chemical constituents of PSORI-CM01 by UHPLC-ESI-MSn analysis.

Peak No. tR (min) SelectedIon ObservedMass (m/z) CalculatedMass (m/z) Formula MS/MS PatternsFragmentation Identifieation Source a Reference
1 0.41 [M−H] 191.0559 191.0556 C7H12O6 191→173, 127, 109 quinic acid Sa [8]
2 0.51 [M−H] 133.0140 133.0137 C4H6O5 133→115, 87, 71 malic acid M
3 0.60 [M−H] 173.0451 173.0450 C7H10O5 173→155, 145, 129 shikimic acid Sm [9]
4 0.88 [M−H] 191.0198 191.0192 C6H8O7 191→173, 155→111 citric acid M
5 0.98 [M−H] 115.0037 115.0031 C4H4O4 115→97, 71 fumaric acid Sa [8]
6 2.16 [M−H] 169.0142 169.0137 C7H6O5 169→125 gallic acid P [24,25]
7 2.60 [M−H] 331.0663 331.0665 C13H16O10 331→169 gallic acid-1-O-glucoside P
8 3.28 [M−H] 315.0716 315.0716 C13H16O9 315→297, 247,153 protocatechuic acid-4-O-glucoside P, C
9 3.64 [M−H] 197.0453 197.0450 C9H10O5 197→153, 123 syringic acid Sa, Sm [9]
10 3.69 [M−H] 315.0716 315.0716 C13H16O9 315→169 gallic acid-1-O-rhamnoside P
11 3.78 [M−H] 153.0192 153.0188 C7H6O4 153→109 protocatechuic acid Sa,Sm,C [8,9]
12 3.82 [M−H] 493.1208 493.1193 C19H26O15 493→313, 169 gallic acid-1-O-glucosyl-(6→1)-glucoside P
13 4.02 [M−H] 359.0977 359.0978 C15H20O10 359→197, 179 syringic acid-4-O-glucoside Sa, Sm [9]
14 4.83 [M−H] 181.0495 181.0450 C9H10O5 197→169, 133 3,5-dimethoxy-4-hydroxy-benzaldehyde Sa
15 4.93 [M−H] 137.0243 137.0239 C7H6O3 137→109, 93 4-hydroxybenzoic acid C, M, Sm, Sa,
16 5.01 [M−H] 353.0873 353.0873 C16H18O9 353→191, 179→135 3-O-caffeoylquinic acid Sa [8]
17 5.24 [M−H] 165.0556 165.0552 C9H10O3 165→151, 135 paeonal P [24,25]
18 5.31 [M−H] 345.1182 345.1186 C15H22O9 345→183 (3,5-dimethoxy-4-hydroxyphenyl)methy-O-glucoside Sa
19 5.59 [M−H] 183.0296 183.0293 C8H8O5 183→124 methyl gallate P
20 6.20 [M−H] 609.1445 609.1456 C27H30O16 609→445, 301 rutin M
21 6.20 [M−H] 495.1498 495.1503 C23H28O12 495→465,333,281 oxypaeoniflorin P
22 6.12 [M−H] 353.0872 353.0873 C16H18O9 353→191, 179, 135 5-O-caffeoylquinic acid Sa [8]
23 6.17 [M−H] 289.0713 289.0712 C15H14O6 289→245, 205, 179 (+)-catechin P, Sm [9]
24 6.18 [M−H] 335.0767 335.0767 C16H16O8 335→289, 179, 135, 111 3-O-caffeoylshikimic acid Sa, Sm [9]
25 6.21 [M−H] 321.0246 321.0247 C14H10O9 321→169 p-digallic acid P
26 6.41 [M−H] 353.0873 353.0873 C16H18O9 353→191, 179, 135 4-O-caffeoylquinic acid Sa [8]
27 6.43 [M−H] 469.1129 469.1135 C24H22O10 469→423, 371, 315, 289 8-[β-(3,4-dihydroxyphenyl)-α-carboxyl-3-xoxpropyl]-substituted catechin Sm [9]
28 6.45 [M−H] 179.0345 179.0344 C9H8O4 179→135, 85 caffeic acid Sa, Sm, A [8,9]
29 6.59 [M+COOH] 447.1500 447.1503 C18H26O10 447→401, 349, 317, 191 phenylmethyl-glucoside-(6→1)-apiose M
30 6.62 [M−H] 221.0452 221.0450 C11H10O5 221→206 fraxidin Sa [8]
31 6.67 [M−H] 369.0821 369.0822 C16H18O10 369→207 fraxin Sa [8]
32 6.87 [M−H] 435.1289 435.1291 C21H24O10 435→273 isoliquiritigenin-7-O-glucoside Sa, P [8]
33 6.87 [M−H] 431.1914 431.1917 C20H32O10 431→385, 223, 205, 153 drovomifoliol-O-glucopyranoside Sa [8]
34 6.96 [M−H] 433.2070 433.2074 C20H34O10 433→387, 369 , 225 dihydrovomifoliol-O-glucoside Sa [8]
35 7.13 [M−H] 335.0767 335.0767 C16H16O8 335→289, 179, 135, 111 4-O-caffeoylshikimic acid Sa, Sm [9]
36 7.20 [M−H] 289.0712 289.0712 C15H14O6 289→245, 205, 179 epi-catechin P, Sm [9]
37 7.23 [M+COOH] 525.1604 481.1710 C23H30O11 525→479, 465, 121 albiflorin P [24]
38 7.28 [M−H] 207.0296 207.0293 C10H8O5 207→192 fraxetin Sa [8]
39 7.38 [M−H] 335.0767 335.0767 C16H16O8 335→289, 179, 135, 111 5-O-caffeoylshikimic acid Sa, Sm [9]
40 7.61 [M+COOH] 525.1604 481.1710 C23H30O11 525→479, 327, 283,161 paeoniflorin P [24,25]
41 7.72 [M−H] 471.1863 471.1866 C22H32O11 471→425, 263 sarcaglaboside G Sa [8]
42 7.81 [M−H] 339.0715 339.0716 C15H16O9 339→193, 165,137 6,7,8-trihydroxycoumarin-7-rhamnoside Sa, Sm [9]
43 7.94 [M−H] 629.1514 629.1506 C30H30O15 629→483, 475, 449, 303, 285 8-[β-(3,4-dihydroxyphenyl)-α-carboxyl-3-oxopropyl]-substituted neoastilbin Sm [9]
44 8.05 [M−H] 473.2022 473.2023 C22H34O11 473.202 sarcaglaboside H Sa [8]
45 8.16 [M−H] 629.1514 629.1506 C30H30O15 629→483, 475, 449, 303, 285 8-[β-(3,4-dihydroxyphenyl)-α-carboxyl-3-oxopropyl]-substituted astilbin Sm [9]
46 8.18 [M−H] 565.1552 565.1557 C26H30O14 565→313, 193 (2R/2S)-naringenin-6-C-β-d-glucopy ranoside-(6-1)-apiose Sa [8]
47 8.27 [M−H] 537.1025 537.1033 C27H22O12 537→493, 295, 159, 109 lithospermic acid A
48 8.33 [M−H] 565.1550 565.1557 C26H30O14 565→313, 193 (2R/2S)-naringenin-6-C-glucopyranoside-(6-1)-apiose Sa [9]
49 8.52 [M−H] 629.1514 629.1506 C30H30O15 629→483, 475, 449, 303, 285 8-[β-(3,4-dihydroxyphenyl)-α-carboxyl-3-oxopropyl]-substituted Sm [9]
50 8.57 [M−H] 221.0452 221.0450 C11H10O5 221→206, 191, 163 isofraxidin Sa [8]
51 8.62 [M−H] 417.1186 417.1186 C21H22O9 417→255 liquiritin G [26]
52 8.62 [M−H] 549.1607 549.1608 C26H30O13 549→429, 255 liquiritin apioside G [26]
53 8.77 [M−H] 451.1025 451.1029 C24H20O9 451→341, 299 cinchonain Ia Sm
54 8.80 [M−H] 303.0505 303.0505 C15H12O7 303→285 taxifolin Sm [9]
55 8.82 [M−H] 300.9986 300.9984 C14H6O8 301→257 gallogen P
56 8.84 [M−H] 449.1088 449.1084 C21H22O11 449→303, 285 neoastilbin Sa, Sm [8,9]
57 8.98 [M−H] 629.1514 629.1506 C30H30O15 629→483, 475, 449, 303, 285 8-[β-(3,4-dihydroxyphenyl)-α-carboxyl-3-oxopropyl]-substituted isoastilbin Sm [9]
58 8.96 [M−H] 631.1656 631.1663 C30H32O15 631→613, 491, 399, 169 galloyl paeoniflorin P
59 9.02 [M−H] 477.0668 477.0669 C21H18O13 477→301 quercetin-3-O-glucruronide Sa [8]
60 9.02 [M−H] 449.1088 449.1084 C21H22O11 449→303, 285 astilbin Sa, Sm [8,9]
61 9.05 [M−H] 521.1295 521.1295 C24H26O13 521→359, 197 rosmarinic acid-4-O-glucoside Sa
62 9.38 [M−H] 939.1089 939.1104 C41H32O26 939→769, 617, 393, 317 penta-O-galloyl-glucose P
63 9.49 [M−H] 515.1184 515.1190 C25H24O12 515→353 dicaffeoylquinic acid Sa
64 9.64 [M−H] 449.1088 449.1084 C21H22O11 449→303, 285 neoisoastilbin Sa, Sm [8,9]
65 9.68 [M−H] 717.1442 717.1456 C36H30O16 717→519, 475, 321 caffeic acid tetramer A
66 9.78 [M−H] 187.0974 187.0970 C9H16O4 187→142, 125 nonandioic acid P, G
67 9.84 [M−H] 449.1088 449.1084 C21H22O11 449→303, 285 isoastilbin Sa, Sm [8,9]
68 9.87 [M−H] 451.1025 451.1029 C24H20O9 451→341, 299 cinchonain Ib Sm
69 9.95 [M−H] 717.1442 717.1456 C36H30O16 717→519, 475, 321 caffeic acid tetramer isomer A
70 9.96 [M−H] 597.1605 597.1608 C30H30O13 597→451, 341, 217 glabraoside C Sa [8]
71 9.95 [M−H] 719.1599 719.1512 C36H32O16 719→539, 359 dirosmarinic acid Sa
72 10.08 [M−H] 433.1131 433.1135 C21H22O10 433→343, 313, 271, 179 (2R/2S)-naringenin-6-C-glucopyranoside Sa [8]
73 10.17 [M−H] 433.1131 433.1135 C21H22O10 433→343, 313, 271, 179 (2R/2S)-naringenin-6-C-glucopyranoside Sa [8]
74 10.36 [M−H] 587.2327 587.2340 C27H40O14 587→451, 341, 217 sarcaglaboside D Sa [8]
75 10.61 [M−H] 515.1184 515.1190 C25H24O12 515→353 dicaffeoylquinic acid Sa
76 10.64 [M−H] 359.0768 359.0767 C18H16O8 359→197, 161 rosmarinic acid Sa, Sm [8,9]
77 10.73 [M−H] 279.1234 279.1232 C15H20O5 279→235, 139 zedoalactone D Sa [8]
78 10.97 [M+COOH] 507.1497 461.1448 C23H26O10 461→417, 295 lactiflorin P
79 11.03 [M−H] 433.1132 433.1135 C21H22O10 433→287, 269 engeletin Sm [9]
80 11.41 [M−H] 423.1653 423.1655 C21H28O9 423→261, 243 chloranoside A Sa [8]
81 11.80 [M−H] 549.1603 549.1608 C26H30O13 549→417, 255 isoliquiritin apioside G [26]
82 11.94 [M−H] 433.1132 433.1135 C21H22O10 433→287, 269 isoengeletin Sa, Sm [8,9]
83 11.82 [M−H] 549.1607 549.1608 C26H30O13 549→417, 255 liquiritin apioside G [26]
84 12.40 [M−H] 417.1183 417.1186 C21H22O9 417→255 isoliquiritin G [26]
85 12.97 [M−H] 599.1756 599.1765 C30H32O13 599→569 benzoyloxypaeoniflorin P [24]
86 12.98 [M−H] 451.1027 451.1029 C24H20O9 451→341, 299 cinchonain Ic Sm [8]
87 13.06 [M−H] 255.0658 255.0657 C15H12O4 255→135 liquiritigenin G [26]
88 13.14 [M−H] 451.1025 451.1029 C24H20O9 451→341, 299 cinchonain Id Sm [8]
89 13.33 [M−H] 373.0918 373.0923 C19H18O8 373→211, 161 methyl rosmarina Sa, Sm [8]
90 13.52 [M−H] 823.4102 823.4116 C42H64O16 823→647, 351 uralsaponin C G [27]
91 13.86 [M−H] 835.3742 835.3752 C42H60O17 823→661, 351 uralsaponin D G [27]
92 13.52 [M−H] 999.4421 999.4433 C48H72O22 999→837, 645, 351 24-hydroxyl-licorice-saponin A3 G [27]
93 13.64 [M−H] 895.3950 895.3964 C44H64O19 895→719, 501,351 uralsaponin F G [27]
94 13.63 [M−H] 853.3845 853.3858 C42H62O18 853→809, 791, 677, 351 22-hydroxyl-licorice-saponin G2 G [27]
95 13.61 [M−H] 983.4470 983.4488 C48H72O21 983→821, 645, 351 licorice saponin A3 G [27]
96 13.68 [M−H] 1025.4579 1025.4593 C50H74O22 1025→993, 833, 497 22-acetoxyl-rhaoglycyrrhizin G [27]
97 13.70 [M−H] 849.3538 849.3545 C42H58O18 849→673, 479 uralsaponin E G [27]
98 13.76 [M−H] 879.3996 879.4014 C44H64O18 879→861, 643, 351 22-acetoxyl-glycyrrhizin G [27]
99 13.77 [M−H] 837.3891 837.3909 C42H62O17 837→819, 661, 351 24-hydroxyl-glycyrrhizin G [27]
100 13.78 [M−H] 271.0607 271.0606 C15H12O5 271→254, 177 naringenin Sm [27]
101 13.86 [M−H] 835.3742 835.3752 C42H60O17 823→661, 351 24-hydroxyl-licorice-saponin E2 G [27]
102 14.00 [M−H] 837.3891 837.3909 C42H62O17 837→819, 775, 661, 351 licorice saponin G2 G [27]
103 14.00 [M−H] 967.4523 967.4539 C48H72O20 967→805,497, 407, 321 rhaoglycyrrhizin G [27]
104 14.01 [M−H] 819.3787 819.3803 C42H60O16 819→777, 643, 351 licorice saponin E2 G [27]
105 14.06 [M−H] 863.4049 863.4065 C44H64O17 863→819, 729, 687, 351, 289 22-acetoxyl-glycyrrhaldehyde G [27]
106 14.13 [M−H] 255.0660 255.0657 C15H12O4 255→135 isoliquiritigenin G [27]
107 14.14 [M−H] 821.3945 821.3945 C42H62O16 821→803, 759, 645, 351 glycyrrhizin G [27]
108 14.33 [M−H] 821.3945 821.3960 C42H62O16 821→803, 759, 645, 351 18α-glycyrrhizin G [27]

Note: a: A, Arnebiae radix; C, Curcumae rhizome; G, Glycyrrhizae radix et rhizome; M, Mume fructus; P, Paeoniae radix rubra; Sa, Sarcandrae Herba; Sm, Smilacis glabrae rhizome.

Among the 108 compounds, 3-O-caffeoylquinic acid (16), 5-O-caffeoylquinic acid (22), 4-O-caffeoylquinic acid (24), 5-O-caffeoylskimic acid (39), paeoniflorin (40), liquiritin (51), neoastilbin (56), astilbin (60), neoisoastilbin (64), isoastilbin (67), engeletin (79), isoengeletin (82), liquiritigenin (87) and glycyrrhizic acid (107) were the main components in PSORI-CM01. Besides, 3-O-caffeoylquinic acid (16), paeoniflorin (40), liquiritin (51), astilbin (60), engeletin (79), liquiritigenin (87) and glycyrrhizic acid (107) have been reported to have multiple biological activities such as anti-inflammation, immunoregulation and anti-tumor properties [18,19,20,21,22,23], which are related to psoriasis. Thus, these 14 ingredients were selected as markers for quality control.

2.3. Method Validation of the Quantitative Analysis

The calibration curves, linear ranges, LOQ, and repeatability of the 14 analytes were established using the developed UHPLC-MS method (Table 2). Reasonable correlation coefficient values (r2 > 0.9981) indicated good correlations between the investigated concentrations of the standards and their peak areas within the ranges tested. The ranges of LOQ for all the analytes were from 0.013 to 0.065 μg/mL.

Table 2.

Calibration curves, linear range, LOQ and repeatability for fourteen compounds analyzed with the UHPLC-MS system.

Analyte Linear Range (μg/mL) Calibration Curve (n = 7) r2 LOQ (μg/mL) Repeatability RSD (%)
3-O-Caffeoylquinic acid (16) 0.16–6.37 y = 301,311 x− 46,304 0.9984 0.064 2.3
5-O-Caffeoylquinic acid (22) 0.23–9.24 y = 341,667 x− 39,368 0.9982 0.046 3.2
4-O-Caffeoylquinic acid (24) 0.41–16.34 y = 267,929 x− 80,795 0.9990 0.065 3.8
5-O-Caffeoylskimic acid (39) 0.57–22.92 y = 396,547 x + 152,855 0.9985 0.057 3.5
Paeoniflorin (40) 2.54–101.62 y = 207,558 x + 1,315,316 0.9988 0.040 1.7
Liquiritin (51) 0.46–18.24 y = 616,184 x+ 109,090 0.9987 0.036 1.8
Neoastilbin (56) 0.47–18.84 y = 572,723 x + 139,006 0.9983 0.016 1.7
Astilbin (60) 0.55–22.16 y = 500,903 x + 165,817 0.9990 0.055 1.5
Neoisoastilbin (64) 0.39–15.77 y = 339,480 x + 93,661 0.9986 0.067 2.0
Isoastilbin (67) 0.32–12.84 y = 531,497 x− 76,292 0.9984 0.018 1.5
Engeletin (79) 0.31–12.50 y = 743,986 x− 120,215 0.9983 0.013 2.1
Isoengeletin (82) 0.67–26.66 y = 355,937 x+ 110,394 0.9987 0.027 3.8
Liquiritigenin (87) 0.07–2.96 y = 426,840 x− 24,980 0.9981 0.030 2.0
glycyrrhizic acid (107) 0.99–39.56 y = 422,502 x+ 468,934 0.9986 0.040 1.6

The repeatability presented as RSD (n = 5) was between 1.5% and 3.8% for the 14 compounds. The overall intra- and inter-day variations (RSD) of the 14 analytes were in the range from 0.6% to 3.8%, and 1.5 to 4.4% (Table 3), respectively. The developed method had good accuracy with the recoveries were between 86.3% and 109.5% (Table 3). Therefore, the results demonstrated that the UHPLC-ESI-MS method was sensitive, precise, and accurate enough for quantitative evaluation of multi-compounds in PSORI-CM01 preparations.

Table 3.

Intra-day and inter-day precisions and recoveries for fourteen compounds analyzed with the UHPLC-MS system.

Analyte Intra-Day (RSD, %) (n = 6) Inter-Day (RSD, %) (n = 3) Recoveries (n = 6)
Initial (μg) Spiked (μg) Detected (μg) Recoveries (%) RSD (%)
3-O-Caffeoylquinic acid (16) 2.9 2.8 1.71 1.75 3.23 92.6 4.8
5-O-Caffeoylquinic acid (22) 1.3 4.4 1.38 1.27 2.87 108.4 3.8
4-O-Caffeoylquinic acid (24) 1.3 3.4 2.07 2.25 4.51 104.1 3.4
5-O-Caffeoylskimic acid (39) 0.6 3.7 2.99 3.15 5.54 90.2 1.4
Paeoniflorin (40) 3.2 2.7 27.34 27.95 57.69 104.5 3.8
Liquiritin (51) 2.9 2.5 4.95 5.02 10.05 101.3 2.5
Neoastilbin (56) 1.9 3.1 5.25 5.18 11.38 109.5 2.3
Astilbin (60) 3.4 3.0 6.02 6.10 11.86 97.8 3.7
Neoisoastilbin (64) 3.0 3.2 4.24 4.34 8.21 95.6 3.9
Isoastilbin (67) 3.2 3.1 3.60 3.53 7.49 105.1 3.2
Engeletin (79) 2.5 2.8 3.31 3.44 6.99 104.1 4.1
Isoengeletin (82) 3.8 2.3 2.51 2.62 4.83 94.4 3.0
Liquiritigenin (87) 2.3 2.6 0.82 0.81 1.52 93.2 2.4
glycyrrhizic acid (107) 0.8 1.5 10.75 10.88 22.83 106.0 2.1

2.4. Quantitative Determination of PSORI-CM01 Preparations

A total of eight different batches of PSORI-CM01 preparations were tested using the developed LC-ESI-MS method. The contents (n = 3) of 14 investigated compounds are summarized in Table 4. It was recognized that 3-O-caffeoylquinic acid (16), 5-O-caffeoylquinic acid (22), 4-O-caffeoylquinic acid (24), 5-O-caffeoylskimic acid (39), paeoniflorin (40), liquiritin (51), neoastilbin (56), astilbin (60), neoisoastilbin (64), isoastilbin (67), and glycyrrhizic acid (107) were the dominant compounds in all examined samples. However, the contents of each compound or the total content of certain type of constituents varied in different PSORI-CM01 preparation.

Table 4.

The contents of the 14 compounds in different batches of PSORI-CM01 prepations (n = 3).

Analyte a KLJ-1 KLJ-2 PJ-3 PJ-4 PJ-5 TJ-6 TJ-7 TJ-8
3-O-Caffeoylquinic acid (16) 520.94 531.48 391.56 132.01 429.07 122.89 30.68 121.18
5-O-Caffeoylquinic acid (22) 514.55 503.94 391.56 146.51 488.10 123.45 27.30 183.64
4-O-Caffeoylquinic acid (24) 601.74 545.00 558.82 211.85 683.57 129.75 40.53 160.64
5-O-Caffeoylskimic acid (39) 540.55 520.10 789.39 324.27 397.06 333.60 153.44 429.46
Paeoniflorin (40) 5855.02 6030.52 7218.12 5177.23 5368.14 2326.21 445.51 3511.46
Liquiritin (51) 1654.18 1650.73 892.15 360.64 1115.65 145.56 71.55 248.20
Neoastilbin (56) 2545.86 2767.29 2442.62 423.15 1680.52 369.88 124.41 588.89
Astilbin (60) 3819.23 4061.92 3743.95 844.53 2575.83 661.96 174.14 879.05
Neoisoastilbin (64) 2459.06 2359.70 1872.94 736.75 2211.36 605.51 154.36 802.79
Isoastilbin (67) 879.60 916.61 650.53 260.87 902.06 244.03 74.96 418.97
Engeletin (79) 678.32 740.00 503.10 196.27 621.69 165.13 84.25 274.81
Isoengeletin (82) 543.19 1089.55 348.12 132.29 331.67 119.27 16.30 116.39
Liquiritigenin (87) 224.07 246.07 477.28 62.14 202.73 75.50 24.55 130.18
glycyrrhizic acid (107) 2225.13 2359.89 2610.50 933.51 2770.51 306.01 68.44 257.72

Notes: a The content unit of granule (KLJ-1, KLJ-2) and pills (PJ-3, PJ-4, PJ-5) was expressed as μg/g; The content unit of decoction was ug/mL.

In order to evaluate the variations in detail, hierarchical cluster analysis was performed based on the contents of 14 analytes of the eight investigated batches. Between-groups linkages method was applied, and Squared Euclidean distance was selected as measurement. Figure 2 shows the results on the investigated batches of PSORI-CM01 preparations, which were divided into two main clusters. The results suggested that the contents of 14 analytes were relatively more stable and higher in granule preparations (the batches of K1 and K2) but varied in tablet and decoction preparations. This may be related to the origin of raw medicinal plants origin and extraction technology and so on. As mentioned above, 3-O-caffeoylquinic acid (16), paeoniflorin (40), liquiritin (51), astilbin (60), engeletin (79), liquiritigenin (87) and glycyrrhizic acid (107) may be the main active components of PSORI-CM01, The content of the six ingredients should be stressed on in establishing quality control methods for PSORI-CM01 preparations.

Figure 2.

Figure 2

Dendrogram of hierarchical cluster analysis for the eight investigated batches of PSORI-CM01 preparations.

3. Experimental Section

3.1. Chemicals and Materials

Methanol and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ, USA); Formic acid (HPLC grade) was purchased from the Sigma-Aldrich (Seelze, Germany); Ultra-pure water was prepared using a Millipore Milli-Q purification system (Bedford, MA, USA).

5-O-caffeoylskimic acid (39), paeoniflorin (40), neoastilbin (56), astilbin (60), neoisoastilbin (64), isoastilbin (67), engeletin (79) and isoengeletin (82) were isolated in our lab and identified by the authors. 3-O-caffeoylquinic acid (16), 5-O-caffeoylquinic acid (22), 4-O-caffeoylquinic acid (24), liquiritin (51), liquiritigenin (87), and glycyrrhizic acid (107) were purchase from the National Institutes for Food and Drug Control (Beijing, China). All of the purities were above 98% by HPLC analysis. Eight batches of PSORI-CM01 preparations were supplied by Guangdong Provincial Hospital of Chinese Medicine, and voucher samples were deposited in the laboratory of Materia Medica Preparation, Guangdong Province Academy of Chinese Medicine Science.

3.2. Standard Solutions and Sample Preparation

Stock solutions of the 14 reference substances were prepared in concentrations ranging from 0.420 to 5.083 mg/mL in 60% methanol and stored at 4 °C until use. A standard working solution of the mixtures was obtained by diluting stock solutions to desired concentrations. Aliquots of this solution were further diluted with initial mobile phase to a series of concentrations for quantification.

PSORI-CM01 preparations (granules and tablets) were pulverized into a fine powder. The powder (0.5 g) was accurately weighed, immersed in 60% MeOH (v/v, 10 mL) for 1 h at room temperature, then extracted in an ultrasonic water bath for 30 min. After recording the weight, the extract was filtered through filter paper. Aliquots of 500 μL of filtrate was transferred into a 5 mL volumetric flask which was made up to its volume with initial mobile phase. PSORI-CM01 decoction was diluted 10 times in 60% MeOH (v/v) for quantitative analysis. All of the samples were filtered through a 0.22 μm syringe filter before use, and 10 μL was injected into the LC instrument for LC-MS analysis.

3.3. UHPLC-ESI-MS/MS System

Chromatographic separation was performed on an Accela™ ultra high pressure liquid chromatography (UHPLC) system (Thermo Fisher Scientific, San Jose, CA, USA) comprising a UHPLC pump, a PDA detector, scanning from 200 to 400 nm, and an autosampler settled to 30 °C. The LC conditions were as follows: column: Thermo Scientific Syncronis C18 (50 mm × 2.1 mm, 1.7 μm); mobile phase: acetonitrile (A) and water containing 0.1% (v/v) formic acid (B); gradient: 0 min, 5:95; 10 min, 27:83; 15 min, 50:50; 18–20 min, 100:0 (A:B, v/v); flow rate: 0.4 mL/min; injection volume: 10 μL.

The above UHPLC system was connected with a LTQ/Orbitrap mass spectrometry system (Thermo-Fisher Scientific, Bremen, Germany) via an ESI interface. High purity nitrogen (N2) was used as the sheath gas and helium (He) as the auxiliary gas with a flow rate of 40 and 10 arbitrary units, respectively.

3.4. Qualitative Characteristic of Chemical Constituents

Identification of chemical constituents in PSORI-CM01 preparations was performed by LC-ESI-MSn analysis. The ESI-MS spectra of samples and reference compounds were acquired in negative ionization mode. The parameters were as follows: capillary temperature at 320 °C, capillary voltage at −28 V, ion spray voltage at −4.0 kV, and tube lens voltage at −90 V. For full scan MS analysis, the spectra were recorded in the range of m/z 100–1500 with a resolution of 30,000. Data-dependant acquisition was applied and the most intense ions detected in each MS scan were selected for MSn data records with a resolution of 15,000. The activation time was 30 ms and the collision energy was adjusted to 35%. Data were processed by Xcalibur software (Thermo-Fisher Scientific, Bremen, Germany). An external calibration for mass accuracy was carried out the day before the analysis according to the manufacturer’s guidelines.

3.5. Validation of the Quantitative Analysis

The stock solution containing 14 reference compounds was prepared and diluted to seven-point calibration levels for the construction of calibration curves. Each concentration of the mixed standard solution was injected in triplicate. Calibration curves were established by plotting the peak area versus concentration of each analyte. Intra- and inter-day variations were utilized to assess the precision of the method. The intra-day variation was determined by analyzing six replicates within 1 day and the inter-day variation was examined in three consecutive days. Recovery was used to evaluate the accuracy of the method. Recovery test was carried out to investigate accuracy of this method by adding certain amounts of the 14 standard solutions to 0.25 g powder of sample in sextuplicate. Samples were processed as described in Section 2.2. To confirm the repeatability, five replicates of the same sample were extracted and analyzed. Variations were expressed by relative standard deviation (RSD) in all three tests above.

4. Conclusions

An efficient and sensitive method employing ultra-high liquid chromatography coupled with linear trap quadrupole and high resolution mass analyzer-orbitrap (UHPLC-LTQ/Orbitrap) was developed for the qualitative and quantitative analysis of chemical constituents of PSORI-CM01 preparations. One hundred and eight compounds, including organic acids, phenolic acids, flavonoids, and terpenoids, were characterized on the basis of retention behaviors, abundant MS and MSn data, or by comparing with reference substances and literatures. All compounds identified were found to be existed in individual traditional Chinese medicines of PSORI-CM01 preparation. However, the characteristic constituents from Rhizoma Curcumae were not detected. Further investigation focused on those lipophilic constituents in PSORI-CM01 preparation is required.

An optimized LC-ESI-MS method was then established for assay of the 14 marker compounds in PSORI-CM01 preparations. The validation of the method, represent a good accuracy, sensitivity and repeatability. The quantification results indicated an obvious difference of marker compounds contents among various samples. This is the first report on the comprehensive determination of chemical constituents in PSORI-CM01 preparations by LC-ESI-MSn. The results would provide the chemical support for the further pharmacokinetic studies and for the improvement of quality control of PSORI-CM01 preparations. The study also suggested that LC-LTQ/Orbitrap-MS would be a powerful and reliable analytical tool for the characterization of chemical profile in complex chemical system, such as TCM preparations.

Acknowledgments

This research was financially supported by Guangdong Natural Science Fund (S2013030011515), Guangdong Financial Industry Technology Research Development Fund [2011(285)05], Guangdong Science and Technology Department-Guangdong Province Academy of Chinese Medicine Science Joint Special Fund (2011B032200009) and Guangdong Provincial Hospital of Chinese Medicine Special Fund (YK2013B1N11).

Author Contributions

C.-J. Lu designed the experiments and provided critical advice on operation of the analytical equipment. S.-D. Chen was responsible for performing most of the experiment and analysis, and preparing the draft of the manuscript. R.-Z. Zhao helped revising the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of the compounds 16, 22, 24, 39, 40, 51, 56, 60, 64, 67, 79, 82, 87 and 107 are available from the authors.

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