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.

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). MSE technique, a new technique used in deducing the splitting disciplinary of MS, was applied to data collection. MSE 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 MSE 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].

Figure 1

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

Table 1

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

NO.	t R (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	127.040085.0288	Quinic acid
2	3.77	355.1033	353.0873	C16H18O9	Chlorogenic acid	163.0395145.0279135.0454	191.0549179.0341135.0449	Quinic acid
3	3.87	—	375.1287	C16H24O10	Isomer of loganic acid	—	213.0765169.0867151.0759	Iridoid
4	3.90	623.2080	—	C29H34O15	Isomer of suspensaside A	191.0568149.0232	461.1674443.1554205.0319	Phenylethanoid glycoside
5	3.97	—	461.1659	C20H30O12	Forsythoside E	—	315.1076205.0718135.0448	Phenylethanoid glycoside
6	4.20	—	375.1287	C16H24O10	Loganic acid	—	213.0778169.0853151.0773	Iridoid
7	4.24	355.1023	353.0866	C16H18O9	3-O-Caffeoylquinic acid	163.0393145.0286	191.0569179.0365	Quinic acid
8	4.29	—	353.0873	C16H18O9	4-O-Caffeoylquinic acid	—	173.0450135.0453	Quinic acid
9	4.35	—	639.1925	C29H36O16	Suspensaside	—	621.1841469.1273	Phenylethanoid glycoside
10	4.43	375.1288	373.1129	C16H22O10	Secologanic acid	213.0749195.0638	193.0494149.0605	Iridoid
11	4.45	391.1255	389.1074	C16H22O11	Monotropein	211.0586177.0546151.0395	209.0455165.0554149.0605	Iridoid
12	4.58	—	639.1918	C29H36O16	Isomer of suspensaside	—	445.1318205.0318179.0346	Phenylethanoid glycoside
13	4.72	—	403.1239	C17H24O11	Isomer of secoxyloganin	—	241.1177	Iridoid
14	4.73	359.1348	—	C16H22O9	Sweroside	197.0812151.0400	—	Iridoid
15	4.80	625.2124	623.1982	C29H36O15	Acteoside	471.1504325.0927163.0398	461.1671443.1567203.0428	Phenylethanoid glycoside
16	4.89	—	755.2399	C34H44O19	Forsythoside B	—	593.2103447.1500315.1137	Phenylethanoid glycoside
17	4.93	623.1986	621.1816	C29H34O15	Suspensaside A	191.0571149.0234	487.1371469.1180	Phenylethanoid glycoside
18	5.03	625.2133	623.1970	C29H36O15	Forsythoside A	471.1512325.0919163.0398	461.1671443.1567205.0321	Phenylethanoid glycoside
19	5.06	405.1387	403.1236	C17H24O11	Secoxyloganin	243.0880211.0612	371.0979223.0611	Iridoid
20	5.09	—	515.1174	C25H24O12	3,4-Dicaffeoylquinicacid	—	353.0906191.0561135.0446	Quinic acid
21	5.13	—	515.1174	C25H24O12	3,5-Dicaffeoylquinicacid	—	353.0906173.0355135.0446	Quinic acid
22	5.21	—	519.1863	C26H32O11	Pinoresinol 4-O-glucoside	—	357.1336151.0398136.0164	Lignan
23	5.30	463.0876	461.0730	C21H18O12	Luteolin 7-galacturonide	287.0552269.0462241.0493	285.0399211.0400113.0238	Flavonoid
24	5.32	—	447.0927	C21H20O11	5,6-Dihydroxy flavanone-7-O-glucuronide	—	285.0399267.0309239.0356	Flavonoid
25	5.35	—	621.1788	C29H34O15	Suspensaside A	—	487.1510179.0351	Phenylethanoid glycoside
26	5.37	611.1599	609.1453	C27H30O16	Rutin	465.1016303.1489	300.0253271.0236255.0290	Flavonoid
27	5.38	517.1344	515.1186	C25H24O12	4,5-Dicaffeoylquinicacid	499.1206355.1702337.0856	353.0866191.0553173.0451	Quinic acid
28	5.54	—	757.2550	C34H46O19	Centauroside	—	525.1569493.1695179.0511	Iridoid
29	5.78	—	533.2020	C27H34O11	Phillyrin	—	371.1484356.1257121.0295	Lignan
30	5.88	447.0925	445.0771	C21H18O11	Baicalin	271.0603	269.0455241.0503	Flavonoid
31	6.01	477.1027	475.0876	C22H19O12	5,2′-Dihydroxy-6′-methoxyflavone-7-O-glucuronide	301.0713	443.0556299.0546	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	285.0767270.0534	283.0611268.0375239.0345	Flavonoid
34	6.38	—	445.0779	C21H18O11	Norwogonin-7-O-glucuronide	—	269.0422	Flavonoid
35	6.81	271.0608	269.0446	C15H10O5	Baicalein	271.0623253.0498123.0083	251.0362223.0379195.0447	Flavonoid
36	7.26	285.0761	283.0602	C16H12O5	Wogonin	270.0489	268.0377162.9845	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

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 t -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.

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.

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.

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.	t R (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	127.040085.0288	Prototype component
2	3.77	355.1033	353.0873	C16H18O9	Chlorogenic acid	163.0395145.0279135.0454	191.0549179.0341135.0449	Prototype component
3	3.87	—	375.1287	C16H24O10	Isomer of loganic acid	—	213.0765169.0867151.0759	Prototype component
4	4.20	—	375.1287		Loganic acid	—	213.0778169.0853151.0773	Prototype component
5	4.24	—	353.0873	C16H18O9	3-O-Caffeoylquinic acid	—	191.0569179.0365	Prototype component
6	4.29	—	353.0873	C16H18O9	4-O-Caffeoylquinic acid	—	173.0450135.0453	Prototype component
7	4.37	478.1365	—	C22H23NO11	Isorhamnetin 7-glucosamine	316.0847298.0745280.0654	—	Metabolite of flavonoids
8	4.42	—	475.1816	C21H32O12	Kanokoside A	—	313.0276193.0493123.0452	Metabolite of iridoids
9	4.43	375.1288	373.1129	C16H22O10	Secologanic acid	213.0749195.0638	193.0494149.0605	Prototype component
10	4.45	—	389.1074	C16H22O11	Monotropein	—	209.0455	Prototype component
11	4.57	—	369.0815	C16H18O10	Ferulic acid 4-O-glucuronide	—	193.0490178.0263	Metabolite of quinic acids
12	4.72	—	403.1239	C17H24O11	Isomer of secoxyloganin	—	241.1177	Prototype component
13	4.73	359.1348	—	C16H22O9	Sweroside	197.0812151.0400	—	Prototype component
14	5.03	—	731.1866	C31H40O18S	Methylated and sulfated forsythiaside	—	651.2212457.1421	Metabolite of phenylethanoid glycosides
15	5.06	405.1387	403.1236	C17H24O11	Secoxyloganin	243.0880211.0612	371.0979223.0611	Prototype component
16	5.09	—	515.1174	C25H24O12	3,4-Dicaffeoylquinicacid	—	353.0906191.0561135.0446	Prototype component
17	5.16	623.1266	621.1092	C27H26O17	Genistein 4′,7-O-diglucuronide	447.0916271.0607	445.0765357.1336269.0444	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	—	520.1801429.1375437.0904	Metabolite of phenylethanoid glycosides
19	5.21	—	519.1863	C26H32O11	Pinoresinol 4-O-glucoside	—	357.1336151.0398136.0164	Prototype component
20	5.47	623.1250	621.1088	C27H26O17	Baicalein 6,7-diglucuronide	447.0922271.0605	445.0774269.0452	Metabolite of flavonoids
21	5.54	609.1461	607.1299	C27H28O16	Luteolin 7-glucuronide-4′-rhamnoside	447.0919271.0610	431.0965	Metabolite of flavonoids
22	5.54	—	757.2550	C34H46O19	Centauroside	—	525.1569493.1695179.0511	Prototype components
23	5.69	—	287.0234	C11H12O7S	5′-(3′,4′-Dihydroxyphenyl)-gamma-valerolactone sulfate	—	207.0651179.0334135.0437	Metabolite of flavonoids
24	5.78	—	533.2020	C27H34O11	Phillyrin	—	371.1484356.1257121.0295	Prototype component
25	5.88	447.0925	445.0771	C21H18O11	Baicalin	271.0603	269.0455241.0503	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	283.0611268.0375239.0345	Prototype component
28	6.38	—	445.0779	C21H18O11	Norwogonin-7-O-glucuronide	—	269.0449131.0625	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	271.0623253.0498123.0083	251.0362223.0379195.0447	Prototype component
33	7.26	285.0761	283.0602	C16H12O5	Wogonin	270.0489	268.0377162.9845	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 MSE fragment information obtained from the MSE 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.

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.

amComparison of metabolites of SHLI and SHL formula found in human serum and rat plasma based on our research and previous reports.

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