Journal of Analytical Methods in Chemistry

Volume 2014 (2014), Article ID 241505, 12 pages

http://dx.doi.org/10.1155/2014/241505

Research Article

A Metabolomic Strategy to Screen the Prototype Components and Metabolites of Shuang-Huang-Lian Injection in Human Serum by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

Mingxing Guo,¹ Baosheng Zhao,¹ Haiyu Liu,¹ Li Zhang,¹ Long Peng,¹ Lingling Qin,¹ Zhixin Zhang,¹ Jian Li,² Chengke Cai,³ and Xiaoyan Gao¹

¹Science Experiment Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, No. 11, North Third Ring Road, Chaoyang District, Beijing 100029, China

²School of Basic Medical Sciences, Beijing University of Chinese Medicine, No. 11, North Third Ring Road, Chaoyang District, Beijing 100029, China

³School of Chinese Material Medica, Beijing University of Chinese Medicine, South of Wangjing Middle Ring Road, Chaoyang District, Beijing, 100102, China

Received 22 December 2013; Accepted 19 January 2014; Published 26 February 2014

Academic Editor: Ying-Yong Zhao

Copyright © 2014 Mingxing Guo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Shuang-huang-lian injection (SHLI) is a famous Chinese patent medicine, which has been wildly used in clinic to treat acute respiratory tract infection, pneumonia, influenza, and so forth. Despite the widespread clinical application, the prototype components and metabolites of SHLI have not been fully elucidated, especially in human body. To discover and screen the constituents or metabolites of Chinese medicine in biofluids tends to be more and more difficult due to the complexity of chemical compositions, metabolic reactions and matrix effects. In this work, a metabolomic strategy to comprehensively elucidate the prototype components and metabolites of SHLI in human serum conducted by UPLC-Q-TOF/MS was developed. Orthogonal partial least squared discriminant analysis (OPLS-DA) was applied to distinguish the exogenous, namely, drug-induced constituents, from endogenous in human serum. In the S-plot, 35 drug-induced constituents were found, including 23 prototype compounds and 12 metabolites which indicated that SHLI in human body mainly caused phase II metabolite reactions. It was concluded that the metabolomic strategy for identification of herbal constituents and metabolites in biological samples was successfully developed. This identification and structural elucidation of the chemical compounds provided essential data for further pharmacological and pharmacokinetics study of SHLI.

1. Introduction

Shuang-huang-lian injection (SHLI) is a typical Chinese herbal injection that is made from the extracts of Flos Lonicerae Japonicae, Radix Scutellariae, and Fructus Forsythiae. It has been widely used for the treatment of acute upper respiratory tract infections [1, 2]. Baicalin, chlorogenic acid, and forsythin are the marker compounds representing Radix Scutellariae, Flos Lonicerae Japonicae,   and Fructus Forsythiae, respectively, for the quality control of this medicine [3]. Though several published papers have reported the determination of major active components and metabolites in Shuang-huang-lian (SHL) preparations [4–6], there is no substantial evidence to confirm the holistic existing form of SHLI in vivo, especially in human body. Therefore, systematically, screening the constituents and metabolites of SHLI in human blood is of great significance for interpreting its material basis for pharmacological effects. Currently, the ingredients of SHL formula have been detected in rat blood [7]. However, the recent study suggests that species differences in key hepatic efflux transporters are sufficiently profound to warrant careful re-examination of conclusions and to design future studies with caution [8]. Some data have revealed that rat liver contains much more (~10-fold) apical multidrug resistance-associated protein 2 (Mrp2) resulting in a much higher capacity for the biliary excretion of organic anions in rats than human or other preclinical species [9]. Therefore, to reveal the pharmacological mechanism of SHLI, comprehensive analysis of the constituents and metabolites in human body is more scientific and rational.

The process of metabolite detection and identification is typically a labor-intensive and time-consuming process. This process has been simplified by the use of radiolabeled compounds and/or spectroscopic techniques such as mass spectrometry and NMR spectroscopy [10–13]. Of these analysis techniques, liquid chromatography coupled with electrospray ionization mass spectrometer has been widely used to detect and identify trace levels of drugs and metabolites in various biological samples due to its high sensitivity and selectivity [14–16]. Ultra performance liquid chromatography (UPLC) applied for short run times combined with a quadrupole/time of flight-mass spectrometer (Q/TOF-MS) which offers high mass accuracy has become a major tool that provides a significant source of global constituent and metabolite profiling data [17–19]. Given the chemical complexity of SHLI in vivo, UPLC-Q-TOF/MS provides faster separations for complex blood samples and valuable structural insights into the characterization of SHLI metabolites.

A straightforward approach for identifying exogenous metabolites in vivo is to compare the LC-MS chromatograms of biological samples collected before and after xenobiotic treatment. However, without using effective analysis method, it is difficult to identify exogenous metabolites through visual examination of LC-MS chromatograms that contain information from thousands of chemical species [20]. A metabolomic strategy has been developed to handle the acquired data and to search for the discriminating features from biosample sets. A xenobiotic and its metabolites only appear in the samples after xenobiotic treatment, and so when using metabolomic strategy, the differences between the control group and the xenobiotic-treated group are mainly defined by the presence of the xenobiotic and its metabolites. With appropriate data processing, the separation of the control group and the xenobiotic-treated group can be achieved in the score plot of a multivariate model, and exogenous metabolites can be conveniently identified by analyzing ions contributing to the separation of the two groups. Employing this approach, the present study aims to develop a metabolomic strategy to comprehensively elucidate the prototype components and metabolites of SHLI in human serum conducted by UPLC-Q-TOF/MS.

2. Experiment

2.1. Materials

SHLI was achieved from the Second Chinese Medicine Factory of Harbin Pharm. Group CO., Ltd. (No. 1204014). HPLC grade formic acid was obtained from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). Methanol (HPLC grade) was acquired from Fisher Corporation (Michigan, USA). Water was purified with a Milli-Q system (Millipore, Bedford, USA).

2.2. Subjects and Clinical Trial Design

The study was approved by an independent ethics committee at Beijing University of Chinese Medicine, before recruitment commenced. Before the initiation of study procedures, all volunteers gave their written informed consent for participation in the study. Thirteen healthy volunteers, without taking any medication, participated in the study. They were aged between 25 and 40 years and with weight between 50 and 80 kg. After overnight fasting, early-morning blood samples (20 mL each) were collected from the medial cubital vein into evacuated tubes and marked as the control group (C group). Then participants were intravenous infusion of 60 mg/kg of SHLI (dissolved with 500 mL saline solution). The blood samples were collected at 0.5 h after SHLI administration and marked as SHLI dosed group (SHLI group). The blood supernatant was allowed to clot overnight at room temperature, and the clotted material was removed by centrifugation (3000 rpm, 15 min). The serum was collected and stored at −80°C.

2.3. Pretreatment Procedure for SHLI

The Shuang-huang-lian lyophilized powder for injection (0.1 g) was weighed and dissolved with 100 mL water. Then, it was filtered by a 0.22 μm filter before UPLC-Q-TOF/MS analysis.

2.4. Pretreatment Procedure for Serum Samples

All serum samples were thawed at room temperature followed by methanol protein precipitation. Serum (200 μL) was added with 600 μL methanol, vortexed for 30 s, and centrifuged at 14000 g for 10 min at 4°C. Then, supernatant (400 μL) was transferred to a clean tube and evaporated to dryness under a gentle stream of nitrogen. The residue was redissolved with 100 μL ultra high purity water and transferred to an autosampler vial.

2.5. UPLC-Q-TOF/MS Analysis

Separation and detection of the components and metabolites of SHLI were performed on a Waters Acquit UPLC chromatographic system (Waters Corp., Milford, USA) equipped with a Evoe G2 Q/TOF (Waters MS Technologies, Manchester, UK). An electrospray ionization source (ESI) interface was used in both positive and negative ion modes. Acquit UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters, UK) was applied for all analyses. The mobile phase was composed of A (0.1% formic acid in water) and B (methanol) with a linear gradient elution: 0–1 min, maintained at 0% B; 1–5 min, from 0% B to 40% B; 5–8 min, from 40% B to 100% B; 8–13 min, maintained at 0% B; 13.0–13.1 min, isocratic of 0% B; 13.1–15 min, maintained at 0% B. The flow rate was 0.30 L/min. The analytic column and autosampler were maintained at temperatures of 45°C and 4°C, respectively. Then, 1 μL of sample solution was injected for each run. Data were collected from m/z 50 to m/z 1200. For positive ion mode, the capillary and cone voltage were set at 3 kV and 35 V. For negative ion mode, the capillary and cone voltage were set at 2.5 kV and 35 V. The conservation gas was set at 700 L/h at a temperature of 350°C. The source temperature was set at 100°C. The cone gas was set at 50 L/h. Leucine-enkephalin was used as the lock mass solution to ensure the accuracy and reproducibility.

2.6. Data Processing and Statistical Analysis

The ES+ and ES− raw data was analyzed by MarkerLynx XS software (Waters Corp., Milford, USA). For extracting data from the raw file and detecting potential markers, the retention time range was set at 0–13 min, the mass range at 50–1000 amu, and the mass tolerance as 0.01. For detecting chromatographic peaks in the Apex Track Peak, peak width at 5% height was set at 1.00, and the peak-to-peak baseline noise was 0.00. For collecting parameters, the marker intensity threshold was set at 1000 cps, the mass window was 0.02 amu, and retention time window was 0.20 min. The noise elimination level was 6. This process provided alignment of drift (retention time and accurate mass) in data and ensured that a chromatographic peak was identified with the same parameters in each sample. Subsequently, a list of intensities or peak areas of the peaks was then generated for the first chromatogram, using the ER-m/z pairs as identifiers. The procedure was applied for each UPLC/MS analysis. The ion intensities or peak area for each peak detected was also normalized within each sample to the sum of the peak intensities in that sample. The three-dimensional data were introduced into the EZinfo 2.0 software (Waters Corporation, Milford, MA, USA) for orthogonal partial least-squares-discriminate analysis (OPLS-DA).

3. Results and Discussion

3.1. Identification and Analysis of Chemical Components in SHLI

Global profiling of both positive and negative ion modes was analyzed by UPLC-Q-TOF/MS. The typical base peak intensity (BPI) chromatograms (positive ion mode and negative ion mode) of SHLI were shown in Figure 1. In total, 38 constituents were detected and tentatively characterized in SHLI (Table 1). technique, a new technique used in deducing the splitting disciplinary of MS, was applied to data collection. technique could provide parallel alternating scans for acquisition at low collision energy to obtain precursor ion information or at a ramping of high collision energy to obtain a full-scan accurate mass of fragments, precursor ions, and neutral loss information [21, 22]. Here, the high precision MS/MS fragments information obtained from the technique were also listed in Table 1 to explain the structure information of the chemical constituents. All the constituents and the fragmentation information were consistent with previous reports [23, 24].

Table 1: UPLC-Q-TOF/MS identification of the constituents in SHLI.

NO.
(min)
Positive ion MS
Negative ion MS
Formula
Identification
Positive ion

MS/MS
Negative ion

MS/MS
Class

1
0.88
193.0722
191.0557
C₇H₁₂O₆
Quinic acid
112.0521

127.0400

85.0288
Quinic acid

2
3.77
355.1033
353.0873
C₁₆H₁₈O₉
Chlorogenic acid
163.0395

145.0279

135.0454
191.0549

179.0341

135.0449
Quinic acid

3
3.87
—
375.1287
C₁₆H₂₄O₁₀
Isomer of loganic acid
—
213.0765

169.0867

151.0759
Iridoid

4
3.90
623.2080
—
C₂₉H₃₄O₁₅
Isomer of suspensaside A
191.0568

149.0232
461.1674

443.1554

205.0319
Phenylethanoid glycoside

5
3.97
—
461.1659
C₂₀H₃₀O₁₂
Forsythoside E
—
315.1076

205.0718

135.0448
Phenylethanoid glycoside

6
4.20
—
375.1287
C₁₆H₂₄O₁₀
Loganic acid

—
213.0778

169.0853

151.0773
Iridoid

7
4.24
355.1023
353.0866
C₁₆H₁₈O₉
3-O-Caffeoylquinic acid
163.0393

145.0286
191.0569

179.0365
Quinic acid

8
4.29
—
353.0873
C₁₆H₁₈O₉
4-O-Caffeoylquinic acid
—
173.0450

135.0453
Quinic acid

9
4.35
—
639.1925
C₂₉H₃₆O₁₆
Suspensaside
—
621.1841

469.1273
Phenylethanoid glycoside

10
4.43
375.1288
373.1129
C₁₆H₂₂O₁₀
Secologanic acid
213.0749

195.0638
193.0494

149.0605
Iridoid

11
4.45
391.1255
389.1074
C₁₆H₂₂O₁₁
Monotropein
211.0586

177.0546

151.0395
209.0455

165.0554

149.0605
Iridoid

12
4.58
—
639.1918
C₂₉H₃₆O₁₆
Isomer of suspensaside
—
445.1318

205.0318

179.0346
Phenylethanoid glycoside

13
4.72
—
403.1239
C₁₇H₂₄O₁₁
Isomer of secoxyloganin
—
241.1177
Iridoid

14
4.73
359.1348
—
C₁₆H₂₂O₉
Sweroside
197.0812

151.0400
—
Iridoid

15
4.80
625.2124
623.1982
C₂₉H₃₆O₁₅
Acteoside
471.1504

325.0927

163.0398
461.1671

443.1567

203.0428
Phenylethanoid glycoside

16
4.89
—
755.2399
C₃₄H₄₄O₁₉
Forsythoside B
—
593.2103

447.1500

315.1137
Phenylethanoid glycoside

17
4.93
623.1986
621.1816
C₂₉H₃₄O₁₅
Suspensaside A
191.0571

149.0234
487.1371

469.1180
Phenylethanoid glycoside

18
5.03
625.2133
623.1970
C₂₉H₃₆O₁₅
Forsythoside A
471.1512

325.0919

163.0398
461.1671

443.1567

205.0321
Phenylethanoid glycoside

19
5.06
405.1387
403.1236
C₁₇H₂₄O₁₁
Secoxyloganin
243.0880

211.0612
371.0979

223.0611
Iridoid

20
5.09
—
515.1174
C₂₅H₂₄O₁₂
3,4-Dicaffeoylquinic

acid
—
353.0906

191.0561

135.0446
Quinic acid

21
5.13
—
515.1174
C₂₅H₂₄O12
3,5-Dicaffeoylquinic

acid
—
353.0906

173.0355

135.0446
Quinic acid

22
5.21
—
519.1863
C₂₆H₃₂O₁₁
Pinoresinol 4-O-glucoside
—
357.1336

151.0398

136.0164
Lignan

23
5.30
463.0876
461.0730
C₂₁H₁₈O₁₂
Luteolin 7-galacturonide
287.0552

269.0462

241.0493
285.0399

211.0400

113.0238
Flavonoid

24
5.32
—
447.0927
C₂₁H₂₀O11
5,6-Dihydroxy flavanone-7-O-glucuronide
—
285.0399

267.0309

239.0356
Flavonoid

25
5.35
—
621.1788
C₂₉H₃₄O₁₅
Suspensaside A
—
487.1510

179.0351
Phenylethanoid glycoside

26
5.37
611.1599
609.1453
C₂₇H₃₀O₁₆
Rutin
465.1016

303.1489

300.0253

271.0236

255.0290
Flavonoid

27
5.38
517.1344
515.1186
C₂₅H₂₄O₁₂
4,5-Dicaffeoylquinic

acid
499.1206

355.1702

337.0856
353.0866

191.0553

173.0451
Quinic acid

28
5.54
—
757.2550
C₃₄H₄₆O₁₉
Centauroside
—
525.1569

493.1695

179.0511
Iridoid

29
5.78
—
533.2020
C₂₇H₃₄O₁₁
Phillyrin
—
371.1484

356.1257

121.0295
Lignan

30
5.88
447.0925
445.0771
C₂₁H₁₈O₁₁
Baicalin
271.0603
269.0455

241.0503
Flavonoid

31
6.01
477.1027
475.0876
C₂₂H₁₉O₁₂
5,2′-Dihydroxy-6′-methoxyflavone-7-O-glucuronide
301.0713

443.0556

299.0546
Flavonoid

32
6.17
431.0978
429.0815
C₂₁H₁₈O₁₀
Chrysin 7-glucuronide
255.0658
253.0505
Flavonoid

33
6.23
461.1079
459.0927
C₂₂H₂₀O₁₁
Wogonoside
285.0767

270.0534
283.0611

268.0375

239.0345
Flavonoid

34
6.38
—
445.0779
C₂₁H₁₈O₁₁
Norwogonin-7-O-glucuronide
—
269.0422
Flavonoid

35
6.81
271.0608
269.0446
C₁₅H₁₀O₅
Baicalein
271.0623

253.0498

123.0083
251.0362

223.0379

195.0447
Flavonoid

36
7.26
285.0761
283.0602
C₁₆H₁₂O₅
Wogonin
270.0489

268.0377

162.9845
Flavonoid

37
7.35
255.0654
—
C₁₅H₁₀O₄
Chrysin
153.0173
—
Flavonoid

38
7.40
285.0762
283.0601
C₁₆H₁₂O₅
Isomer of wogonin
270.0489
268.0409
Flavonoid

Figure 1: UPLC-Q-TOF/MS BPI chromatograms of SHLI in positive ion mode and negative ion mode.

3.2. Analysis of Human Serum by Metabolomic Strategy

Figure 2 represented the typical BPI chromatograms (positive ion mode and negative ion mode) of human serum samples before and after SHLI administration. The prototype components and metabolites of SHLI in human serum were almost submerged by the endogenous metabolites due to the high level of endogenous signals. Interferences from biological matrices remain a major challenge to detection of metabolites in vivo. Without the presence of a radiolabeled isotope or a data-mining tool, it would be almost impossible to identify low level exogenous metabolites. In our work, a metabolomic strategy was employed to phenotype the differences between C group and SHLI group. The LC/MS data were processed using MarkerLynx XS to detect peaks and generate a three-dimensional data with -m/z pairs and the corresponding intensities. Statistical analysis by OPLS-DA was subsequently performed on the entire dataset. Figure 3 showed the OPLS-DA score plots of human serum samples before and after SHLI injection. Clear separation was observed between the two groups, which indicated that the drug-induced constituents were contributed to the clustering.

(a)

(b)

(c)

(d)

Figure 2: UPLC-Q-TOF/MS BPI chromatograms of human serum samples (a) before SHLI administration in positive ion mode, (b) after SHLI administration in positive ion mode, (c) before SHLI administration in negative ion mode, and (d) after SHLI administration in negative ion mode.

(a)

(b)

Figure 3: Score plots of OPLS-DA in human serum samples between C group (■) and SHLI group (▲) in (a) positive ion mode and (b) negative ion mode.

3.3. Identification and Analysis of Prototype Components and Metabolites

In order to discover the multiple prototype components and metabolites of SHLI in human serum, S-plot, a tool for visualization and interpretation of multivariate classification models, was used. In the S-plot, each point represented an ion detected by UPLC-Q-TOF/MS. Variables that were the farthest from the origin in the S-plot were representative of the most significant changes between the two groups. Based on this, even subtle differences in the two groups could be easily extracted. Figure 4 showed the ions in S-plot that were most responsible for distinguishing the C and SHLI groups and had a higher level in SHLI group.

(a)

(b)

Figure 4: S-plots of human serum samples between C and SHLI groups in (a) positive ion mode and (b) negative ion mode. The ions marked with box were at the higher level in SHLI group.

The S-plot responsible for the variances in the data was a combination of metabolites derived from the SHLI administration and endogenous molecules which were ubiquitous to serum and were interfered by SHLI. From a drug metabolite identification perspective, it was important that the disturbance endogenous molecules could be eliminated, and the prototype components and metabolites could be easily screened between SHLI-treated group and the control group. This comparison was achieved by using the trend plot. From the trend plots, the variables that only existed in the dosed serums were marked as the prototype components or the metabolites of SHLI. Figure 5 showed the visualized trend plot of 7.41-285.0762 in positive mode between C group and SHLI group. The ion only appeared in the SHLI group. Therefore, 7.41-285.0762 might be a prototype component or a metabolite of SHLI.

Figure 5: The trend plot of 7.41-285.0762 in positive mode between C group (■) and SHLI group (▲).

Based on the metabolomic strategy, 35 exogenous components in human serum were found, among them, 23 prototype components of SHLI and 12 metabolites were identified and their information was shown in Table 2.

Table 2: The prototype components and metabolites in human serum after SHLI dosed in both positive and negative mode.

NO.
(min)
Positive ion MS
Negative ion MS
Formula
Identification
Positive ion

MS/MS
Negative ion

MS/MS
Relegation

1
0.88
193.0722
191.0557
C₇H₁₂O₆
Quinic acid
112.0521

127.0400

85.0288
Prototype component

2
3.77
355.1033
353.0873
C₁₆H₁₈O₉
Chlorogenic acid
163.0395

145.0279

135.0454
191.0549

179.0341

135.0449
Prototype component

3
3.87
—
375.1287
C₁₆H₂₄O₁₀
Isomer of loganic acid
—
213.0765

169.0867

151.0759
Prototype component

4
4.20
—
375.1287

Loganic acid
—
213.0778

169.0853

151.0773
Prototype component

5
4.24
—
353.0873
C₁₆H₁₈O₉
3-O-Caffeoylquinic acid
—
191.0569

179.0365
Prototype component

6
4.29
—
353.0873
C₁₆H₁₈O₉
4-O-Caffeoylquinic acid
—
173.0450

135.0453
Prototype component

7
4.37
478.1365
—
C₂₂H₂₃NO₁₁
Isorhamnetin 7-glucosamine
316.0847

298.0745

280.0654
—
Metabolite of flavonoids

8
4.42
—
475.1816
C₂₁H₃₂O₁₂
Kanokoside A
—
313.0276

193.0493

123.0452
Metabolite of iridoids

9
4.43
375.1288
373.1129
C₁₆H₂₂O₁₀
Secologanic acid
213.0749

195.0638
193.0494

149.0605
Prototype component

10
4.45
—
389.1074
C₁₆H₂₂O₁₁
Monotropein
—
209.0455
Prototype component

11
4.57
—
369.0815
C₁₆H₁₈O₁₀
Ferulic acid 4-O-glucuronide
—
193.0490

178.0263
Metabolite of quinic acids

12
4.72
—
403.1239
C₁₇H₂₄O₁₁
Isomer of secoxyloganin
—
241.1177
Prototype component

13
4.73
359.1348
—
C₁₆H₂₂O₉
Sweroside
197.0812

151.0400
—
Prototype component

14
5.03
—
731.1866
C₃₁H₄₀O₁₈S
Methylated and sulfated forsythiaside
—
651.2212

457.1421
Metabolite of phenylethanoid glycosides

15
5.06
405.1387
403.1236
C₁₇H₂₄O₁₁
Secoxyloganin
243.0880

211.0612
371.0979

223.0611
Prototype component

16
5.09
—
515.1174
C₂₅H₂₄O₁₂
3,4-Dicaffeoylquinic

acid
—
353.0906

191.0561

135.0446
Prototype component

17
5.16
623.1266
621.1092
C₂₇H₂₆O₁₇
Genistein 4′,7-O-diglucuronide
447.0916

271.0607
445.0765

357.1336

269.0444
Metabolite of flavonoids

18
5.20
—
827.2600
C₃₇H₄₈O₂₁
2-(3,4-Dihydroxyphenyl)ethyl6-deoxy-mannopyranosyl-glucopyranosyl-2-O-acetyl-4-O-[3-(3,4-dihydroxyphenyl)-2-propenoyl]-glucopyranoside
—
520.1801

429.1375

437.0904
Metabolite of phenylethanoid glycosides

19
5.21
—
519.1863
C₂₆H₃₂O₁₁
Pinoresinol 4-O-glucoside
—
357.1336

151.0398

136.0164
Prototype component

20
5.47
623.1250
621.1088
C₂₇H₂₆O₁₇
Baicalein 6,7-diglucuronide
447.0922

271.0605
445.0774

269.0452
Metabolite of flavonoids

21
5.54
609.1461
607.1299
C₂₇H₂₈O₁₆
Luteolin 7-glucuronide-4′-rhamnoside
447.0919

271.0610
431.0965
Metabolite of flavonoids

22
5.54
—
757.2550
C₃₄H₄₆O₁₉
Centauroside
—
525.1569

493.1695

179.0511
Prototype components

23
5.69
—
287.0234
C₁₁H₁₂O₇S
5′-(3′,4′-Dihydroxyphenyl)-gamma-

valerolactone sulfate
—
207.0651

179.0334

135.0437
Metabolite of flavonoids

24
5.78
—
533.2020
C₂₇H₃₄O₁₁
Phillyrin
—
371.1484

356.1257

121.0295
Prototype component

25
5.88
447.0925
445.0771
C₂₁H₁₈O₁₁
Baicalin

271.0603
269.0455

241.0503
Prototype component

26
6.17
431.0969
429.0815
C₂₁H₁₈O₁₀
Chrysin 7-glucuronide
255.0645
253.0505
Prototype component

27
6.23
461.1079
459.0927
C₂₂H₂₀O₁₁
Wogonoside
285.0760
283.0611

268.0375

239.0345
Prototype component

28
6.38
—
445.0779
C₂₁H₁₈O₁₁
Norwogonin-7-O-glucuronide
—
269.0449

131.0625
Prototype component

29
6.41
—
349.0014
C₁₅H₁₀O₈S
Baicalein 7-sulfate
—
269.0449

Metabolite of flavonoids

30
6.43
—
363.0174
C₁₆H₁₂O₈S
Wogonin 7-sulfate
—
283.0606
Metabolite of flavonoids

31
6.46
—
283.0607
C₁₆H₁₂O₅
7,5-Dihydroxy-6-methoxyflavone
—
268.0371

Metabolite of flavonoids

32
6.81
271.0608
269.0446
C₁₅H₁₀O₅
Baicalein
271.0623

253.0498

123.0083
251.0362

223.0379

195.0447
Prototype component

33
7.26
285.0761
283.0602
C₁₆H₁₂O₅
Wogonin
270.0489

268.0377

162.9845
Prototype component

34
7.35
255.0654
—
C₁₅H₁₀O₄
Chrysin
153.0173
—
Prototype component

35
7.41
285.0761
283.0601
C₁₆H₁₂O₅
Wogonin
270.0502
268.0409
Prototype component

3.4. Characterization Analysis of Human Serum Prototype Components and Metabolites of SHLI

In our study, the prototype components and metabolites of SHLI were identified by comparing the accurate mass and fragment information obtained from the technique. Figure 6 showed typical MS/MS spectra of the prototype component 6.23-461.1079 and the flavonoid metabolite 6.46-363.0174. In positive ion mode, the ion at m/z 483.0906 was [M + Na]+ ion. The dominant fragment ion of m/z 285.0763 was produced by loss of m/z 176 (glucuronide-H₂O) fragment from [M + H]⁺ in positive ion mode. The characteristic and abundant fragment ion [M + H-CH₃]+• was generated by loss of CH₃• for the flavones with a methoxyl group on the side chains of an aromatic ring. Its molecular formula was speculated to be C₂₂H₂₁O₁₁ based on the analysis of its elemental composition. Then the ion at m/z 483.0906 was inferred as wogonoside. The ion at m/z 363.0168 was [M − H] ⁻ ion. The major fragment ion of m/z 283.0606 was generated by loss of m/z 80 (sulfate-H₂O) fragment from [M − H]⁻ in negative ion mode. The molecular formula was speculated to be C₁₆H₁₂O₈S, and the fragmentation information and the molecular formula were consistent with wogonin 7-sulfate. Other metabolites were determined by the same method described above and some of them were also supported by the databases such as HMDB (http://www.hmdb.ca/) and METLIN (http://masspec.scripps.edu/). As a result, 23 prototype components and 12 metabolites of SHLI were identified.

(a)

(b)

Figure 6: MS/MS spectra and structures of (a) prototype compound wogonoside in positive ion mode and (b) flavonoids metabolite wogonin 7-sulfate in negative ion mode identified in human serum after SHLI administration. In the tag, glu was the abbreviation of glucuronide-H₂O and sul was the abbreviation of sulfate-H₂O.

3.5. Correlative Analysis of the Prototype Components and Metabolites of SHLI

The prototype herb components could be further metabolized by various drug metabolizing enzymes. Drug metabolism is classified into phase I and phase II reactions. Phase I reactions are mediated primarily by the cytochrome P450 family of microsomal enzymes [25]. Compounds are factionalized by oxidation, hydrolysis, or reduction, leading to the introduction of, for example, hydroxyl, amino, carboxyl, or thiol groups into the molecule. Most compounds undergo phase I oxidation prior to phase II conjugation, but molecules with sites amenable to conjugation may undergo phase II reactions directly. The most relevant phase II drug conjugation reactions are methylation, sulfation, glucuronidation, and glutathione conjugation. There were three types of components found in human serum after SHLI administration: (i) compounds found in their native form; (ii) phase I metabolites formed by chemical modifications, such as hydroxylation (M + OH) and hydration (M + H₂O), and (iii) phase II metabolites formed by conjugation, such as methylation (M + CH₃), glucuronidation (M + C₆H₈O₆), sulfation (M + HSO₃), and other conjugation reactions. In human serum, a large number of phase II metabolites were found. Among them, 8 flavonoids metabolites, 2 phenylephrine glycosides metabolites, 1 iridoid metabolite, and 1 quinic acid metabolite were found.

Some researchers have reported the metabolites of SHL formula in rat plasma [8]. We compared the metabolites differences in human and rats after SHLI administration and found great differences on the types and quantities of the metabolites after SHLI or SHL formula administrated between human and rats. The metabolites of SHLI found in rats and human were listed in Supplementary Material (see Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/241505). Large number of phase I metabolites were detected in rats such as dihydrosecologanic acid and 3,4-dihydroxyphenylethanol, while little was found in the human serum. Besides, sulfated metabolites which were common in human serum were less detected in the rat plasma. Such a discrepancy might be attributed to different species (human or rats), prescription (SHLI or SHL formula), or blood collection time (1 h or shorter time). Further studies of the biological properties of these metabolites would be helpful to understand the pharmacological mechanism of SHLI.

4. Conclusion

In this paper, we developed an unbiased approach for screening the prototype components and metabolites of SHLI in human serum based on metabolomic technique. Employing UPLC-Q-TOF-MS combined with multivariate statistical analysis, 23 prototype components and 12 metabolites of SHLI were rapidly and sensitively identified, which suggested that the metabolomic approach was an effective tool to discover, screen, and analyze the multiple prototype components and metabolites from complicated traditional Chinese preparations in vivo. SHLI in human body mainly caused phase II metabolite reactions such as sulfation, methylation, glucuronidation, and other complex conjugation reactions. This identification and structural elucidation of the chemical compounds provided essential data for further pharmacological and pharmacokinetics study of SHLI. The human serum metabolomic approach avoids the laborious process of predicting possible metabolites and provides information on unexpected reactive metabolites and a type of validated rapid and higher throughput methodology for the identification of constituents of traditional Chinese medicine.

Conflict of Interests

The authors declare that they have no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported in part by National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (Grant no. 2011ZX09201-201-15), National Science Foundation of China (Grant no. 81173562), and in part by the Foundation of Independent Topics at Beijing University of Chinese Medicine (Grant no. 2013-JYBZZ-XS-064).

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