Journal of Analytical Methods in Chemistry

Journal of Analytical Methods in Chemistry / 2014 / Article
Special Issue

Quality Control of Natural Product Medicine and Nutrient Supplements 2014

View this Special Issue

Research Article | Open Access

Volume 2014 |Article ID 241505 | 12 pages | https://doi.org/10.1155/2014/241505

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

Academic Editor: Ying-Yong Zhao
Received22 Dec 2013
Accepted19 Jan 2014
Published26 Feb 2014

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 [46], 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 [1013]. 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 [1416]. 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 [1719]. 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].


NO. (min)Positive ion MSNegative ion MSFormulaIdentificationPositive ion
MS/MS
Negative ion
MS/MS
Class

10.88193.0722191.0557C7H12O6Quinic acid112.0521
127.0400
85.0288
Quinic acid
23.77355.1033353.0873C16H18O9Chlorogenic acid163.0395
145.0279
135.0454
191.0549
179.0341
135.0449
Quinic acid
33.87375.1287C16H24O10Isomer of loganic acid213.0765
169.0867
151.0759
Iridoid
43.90623.2080C29H34O15Isomer of suspensaside A191.0568
149.0232
461.1674
443.1554
205.0319
Phenylethanoid glycoside
53.97461.1659C20H30O12Forsythoside E315.1076
205.0718
135.0448
Phenylethanoid glycoside
64.20375.1287C16H24O10Loganic acid
213.0778
169.0853
151.0773
Iridoid
74.24355.1023353.0866C16H18O93-O-Caffeoylquinic acid163.0393
145.0286
191.0569
179.0365
Quinic acid
84.29353.0873C16H18O94-O-Caffeoylquinic acid173.0450
135.0453
Quinic acid
94.35639.1925C29H36O16Suspensaside 621.1841
469.1273
Phenylethanoid glycoside
104.43375.1288373.1129C16H22O10Secologanic acid213.0749
195.0638
193.0494
149.0605
Iridoid
114.45391.1255389.1074C16H22O11Monotropein211.0586
177.0546
151.0395
209.0455
165.0554
149.0605
Iridoid
124.58639.1918C29H36O16Isomer of suspensaside445.1318
205.0318
179.0346
Phenylethanoid glycoside
134.72403.1239C17H24O11Isomer of secoxyloganin241.1177Iridoid
144.73359.1348C16H22O9Sweroside197.0812
151.0400
Iridoid
154.80625.2124623.1982C29H36O15Acteoside471.1504
325.0927
163.0398
461.1671
443.1567
203.0428
Phenylethanoid glycoside
164.89755.2399C34H44O19Forsythoside B593.2103
447.1500
315.1137
Phenylethanoid glycoside
174.93623.1986621.1816C29H34O15Suspensaside A191.0571
149.0234
487.1371
469.1180
Phenylethanoid glycoside
185.03625.2133623.1970C29H36O15Forsythoside A471.1512
325.0919
163.0398
461.1671
443.1567
205.0321
Phenylethanoid glycoside
195.06405.1387403.1236C17H24O11Secoxyloganin243.0880
211.0612
371.0979
223.0611
Iridoid
205.09515.1174C25H24O123,4-Dicaffeoylquinic
acid
353.0906
191.0561
135.0446
Quinic acid
215.13515.1174C25H24O12 3,5-Dicaffeoylquinic
acid
353.0906
173.0355
135.0446
Quinic acid
225.21519.1863C26H32O11Pinoresinol 4-O-glucoside357.1336
151.0398
136.0164
Lignan
235.30463.0876461.0730C21H18O12Luteolin 7-galacturonide287.0552
269.0462
241.0493
285.0399
211.0400
113.0238
Flavonoid
245.32447.0927C21H20O11 5,6-Dihydroxy flavanone-7-O-glucuronide285.0399
267.0309
239.0356
Flavonoid
255.35621.1788C29H34O15Suspensaside A487.1510
179.0351
Phenylethanoid glycoside
265.37611.1599609.1453C27H30O16Rutin465.1016
303.1489
300.0253
271.0236
255.0290
Flavonoid
275.38517.1344515.1186C25H24O124,5-Dicaffeoylquinic
acid
499.1206
355.1702
337.0856
353.0866
191.0553
173.0451
Quinic acid
285.54757.2550C34H46O19Centauroside 525.1569
493.1695
179.0511
Iridoid
295.78533.2020C27H34O11Phillyrin371.1484
356.1257
121.0295
Lignan
305.88447.0925445.0771C21H18O11Baicalin271.0603269.0455
241.0503
Flavonoid
316.01477.1027475.0876C22H19O125,2′-Dihydroxy-6′-methoxyflavone-7-O-glucuronide301.0713
443.0556
299.0546
Flavonoid
326.17431.0978429.0815C21H18O10Chrysin 7-glucuronide255.0658253.0505Flavonoid
336.23461.1079459.0927C22H20O11Wogonoside285.0767
270.0534
283.0611
268.0375
239.0345
Flavonoid
346.38445.0779C21H18O11Norwogonin-7-O-glucuronide269.0422Flavonoid
356.81271.0608269.0446C15H10O5Baicalein271.0623
253.0498
123.0083
251.0362
223.0379
195.0447
Flavonoid
367.26285.0761283.0602C16H12O5Wogonin270.0489
268.0377
162.9845
Flavonoid
377.35255.0654C15H10O4Chrysin153.0173Flavonoid
387.40285.0762283.0601C16H12O5Isomer of wogonin270.0489268.0409Flavonoid

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.

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.

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.

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.


NO. (min)Positive ion MSNegative ion MSFormulaIdentificationPositive ion
MS/MS
Negative ion
MS/MS
Relegation

10.88193.0722191.0557C7H12O6Quinic acid112.0521
127.0400
85.0288
Prototype component
23.77355.1033353.0873C16H18O9Chlorogenic acid163.0395
145.0279
135.0454
191.0549
179.0341
135.0449
Prototype component
33.87375.1287C16H24O10Isomer of loganic acid213.0765
169.0867
151.0759
Prototype component
44.20375.1287Loganic acid213.0778
169.0853
151.0773
Prototype component
54.24353.0873C16H18O93-O-Caffeoylquinic acid191.0569
179.0365
Prototype component
64.29353.0873C16H18O94-O-Caffeoylquinic acid173.0450
135.0453
Prototype component
74.37478.1365C22H23NO11Isorhamnetin 7-glucosamine316.0847
298.0745
280.0654
Metabolite of flavonoids
84.42475.1816C21H32O12Kanokoside A313.0276
193.0493
123.0452
Metabolite of iridoids
94.43375.1288373.1129C16H22O10Secologanic acid213.0749
195.0638
193.0494
149.0605
Prototype component
104.45389.1074C16H22O11Monotropein209.0455Prototype component
114.57369.0815C16H18O10Ferulic acid 4-O-glucuronide193.0490
178.0263
Metabolite of quinic acids
124.72403.1239C17H24O11Isomer of secoxyloganin241.1177Prototype component
134.73359.1348C16H22O9Sweroside197.0812
151.0400
Prototype component
145.03731.1866C31H40O18SMethylated and sulfated forsythiaside651.2212
457.1421
Metabolite of phenylethanoid glycosides
155.06405.1387403.1236C17H24O11Secoxyloganin243.0880
211.0612
371.0979
223.0611
Prototype component
165.09515.1174C25H24O123,4-Dicaffeoylquinic
acid
353.0906
191.0561
135.0446
Prototype component
175.16623.1266621.1092C27H26O17Genistein 4′,7-O-diglucuronide447.0916
271.0607
445.0765
357.1336
269.0444
Metabolite of flavonoids
185.20827.2600C37H48O212-(3,4-Dihydroxyphenyl)ethyl6-deoxy-mannopyranosyl-glucopyranosyl-2-O-acetyl-4-O-[3-(3,4-dihydroxyphenyl)-2-propenoyl]-glucopyranoside520.1801
429.1375
437.0904
Metabolite of phenylethanoid glycosides
195.21519.1863C26H32O11Pinoresinol 4-O-glucoside357.1336
151.0398
136.0164
Prototype component
205.47623.1250621.1088C27H26O17Baicalein 6,7-diglucuronide447.0922
271.0605
445.0774
269.0452
Metabolite of flavonoids
215.54609.1461607.1299C27H28O16Luteolin 7-glucuronide-4′-rhamnoside447.0919
271.0610
431.0965Metabolite of flavonoids
225.54757.2550C34H46O19Centauroside 525.1569
493.1695
179.0511
Prototype components
235.69287.0234C11H12O7S5′-(3′,4′-Dihydroxyphenyl)-gamma-
valerolactone sulfate
207.0651
179.0334
135.0437
Metabolite of flavonoids
245.78533.2020C27H34O11Phillyrin371.1484
356.1257
121.0295
Prototype component
255.88447.0925445.0771C21H18O11Baicalin
271.0603269.0455
241.0503
Prototype component
266.17431.0969429.0815C21H18O10Chrysin 7-glucuronide255.0645253.0505Prototype component
276.23461.1079459.0927C22H20O11Wogonoside285.0760283.0611
268.0375
239.0345
Prototype component
286.38445.0779C21H18O11Norwogonin-7-O-glucuronide269.0449
131.0625
Prototype component
296.41349.0014C15H10O8SBaicalein 7-sulfate269.0449
Metabolite of flavonoids
306.43363.0174C16H12O8SWogonin 7-sulfate283.0606Metabolite of flavonoids
316.46283.0607C16H12O57,5-Dihydroxy-6-methoxyflavone268.0371
Metabolite of flavonoids
326.81271.0608269.0446C15H10O5Baicalein271.0623
253.0498
123.0083
251.0362
223.0379
195.0447
Prototype component
337.26285.0761283.0602C16H12O5Wogonin270.0489
268.0377
162.9845
Prototype component
347.35255.0654C15H10O4Chrysin153.0173Prototype component
357.41285.0761283.0601C16H12O5Wogonin270.0502268.0409Prototype 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.

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).

Supplementary Materials

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

  1. Supplementary Material

References

  1. H. Zhang, Q. Chen, W. Zhou et al., “Chinese medicine injection Shuanghuanglian for treatment of acute upper respiratory tract infection: a systematic review of randomized controlled trials,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 987326, 7 pages, 2013. View at: Publisher Site | Google Scholar
  2. Y. Cao, L. Wang, X. Yu, and J. Ye, “Development of the chromatographic fingerprint of herbal preparations Shuang-Huang-Lian oral liquid,” Journal of Pharmaceutical and Biomedical Analysis, vol. 41, no. 3, pp. 845–856, 2006. View at: Publisher Site | Google Scholar
  3. The State Commission of Chinese Pharmacopoeia, Pharmacopoeia of People's Republic of China, part 1, Chemical Industry Press, Beijing, China, 2005.
  4. Y. M. Zhang, D. Yan, P. Zhang et al., “Quality control of Shuanghuanglian freeze-dried powder for injection based on its HPLC-ELSD fingerprints and biological profiles,” Acta Pharmaceutica Sinica, vol. 45, no. 1, pp. 93–97, 2010. View at: Google Scholar
  5. L. Liu, Z. Suo, and J. Zheng, “Simultaneous determination of four compounds in Sanjing Shuanghuanglian oral liquid by high performance liquid chromatography-diode array detection-electrochemical detection,” Chinese Journal of Chromatography, vol. 24, no. 3, pp. 247–250, 2006. View at: Google Scholar
  6. H. Cao and Y. Zhu, “Content determination of chlorogenic acid and baicalin in Shuanghuanglian oral solution by HPLC,” Drug Stands in China, vol. 8, no. 2, pp. 44–46, 2007. View at: Google Scholar
  7. G. L. Yan, A. H. Zhang, H. Sun et al., “An effective method for determining the ingredients of Shuanghuanglian formula in blood samples using high-resolution LC-MS coupled with background subtraction and a multiple data processing approach,” Journal of Separation Science, vol. 36, no. 19, pp. 3191–3199, 2013. View at: Google Scholar
  8. M. J. Zamek-Gliszczynski, K. A. Hoffmaster, K. I. Nezasa, M. N. Tallman, and K. L. R. Brouwer, “Integration of hepatic drug transporters and phase II metabolizing enzymes: mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites,” European Journal of Pharmaceutical Sciences, vol. 27, no. 5, pp. 447–486, 2006. View at: Publisher Site | Google Scholar
  9. H. Ishizuka, K. Konno, T. Shiina et al., “Species differences in the transport activity for organic anions across the bile canalicular membrane,” Journal of Pharmacology and Experimental Therapeutics, vol. 290, no. 3, pp. 1324–1330, 1999. View at: Google Scholar
  10. G. B. Scarfe, J. C. Lindon, J. K. Nicholson et al., “Investigation of the metabolism of 14C/13C-practolol in rat using directly coupled radio-HPLC-NMR-MS,” Xenobiotica, vol. 30, no. 7, pp. 717–729, 2000. View at: Google Scholar
  11. J. Iwabu, J. Watanabe, K. Hirakura, Y. Ozaki, and K. Hanazaki, “Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine, daikenchuto,” Drug Metabolism and Disposition, vol. 38, no. 11, pp. 2040–2048, 2010. View at: Publisher Site | Google Scholar
  12. I. Iswaldi, D. Arráez-Román, A. M. Gómez-Caravaca et al., “Identification of polyphenols and their metabolites in human urine after cranberry-syrup consumption,” Food and Chemical Toxicology, vol. 55, pp. 484–492, 2013. View at: Publisher Site | Google Scholar
  13. G. Tan, W. Liao, X. Dong et al., “Metabonomic profiles delineate the effect of traditional Chinese medicine Sini decoction on myocardial infarction in rats,” PLoS ONE, vol. 7, no. 4, Article ID e34157, 2012. View at: Publisher Site | Google Scholar
  14. O. Corcoran and M. Spraul, “LC-NMR-MS in drug discovery,” Drug Discovery Today, vol. 8, no. 14, pp. 624–631, 2003. View at: Publisher Site | Google Scholar
  15. M. S. Lee and E. H. Kerns, “LC/MS applications in drug development,” Mass Spectrometry Reviews, vol. 18, no. 3-4, pp. 187–279, 1999. View at: Google Scholar
  16. Y. Wu, “The use of liquid chromatography-mass spectrometry for the identification of drug degradation products in pharmaceutical formulations,” Biomedical Chromatography, vol. 14, no. 6, pp. 384–396, 2000. View at: Google Scholar
  17. X. Wang, W. Sun, H. Sun et al., “Analysis of the constituents in the rat plasma after oral administration of Yin Chen Hao Tang by UPLC/Q-TOF-MS/MS,” Journal of Pharmaceutical and Biomedical Analysis, vol. 46, no. 3, pp. 477–490, 2008. View at: Publisher Site | Google Scholar
  18. S. L. Li, S. F. Lai, J. Z. Song et al., “Decocting-induced chemical transformations and global quality of Du-Shen-Tang, the decoction of ginseng evaluated by UPLC-Q-TOF-MS/MS based chemical profiling approach,” Journal of Pharmaceutical and Biomedical Analysis, vol. 53, no. 4, pp. 946–957, 2010. View at: Publisher Site | Google Scholar
  19. L. Li, G. A. Luo, Q. L. Liang, P. Hu, and Y. M. Wang, “Rapid qualitative and quantitative analyses of Asian ginseng in adulterated American ginseng preparations by UPLC/Q-TOF-MS,” Journal of Pharmaceutical and Biomedical Analysis, vol. 52, no. 1, pp. 66–72, 2010. View at: Publisher Site | Google Scholar
  20. C. Chen, F. J. Gonzalez, and J. R. Idle, “LC-MS-based metabolomics in drug metabolism,” Drug Metabolism Reviews, vol. 39, no. 2-3, pp. 581–597, 2007. View at: Publisher Site | Google Scholar
  21. Y. Y. Zhao, X. L. Cheng, F. Wei, X. Bai, and R. C. Lin, “Application of faecal metabonomics on an experimental model of tubulointerstitial fibrosis by ultra performance liquid chromatography/high-sensitivity mass spectrometry with MSE data collection technique,” Biomarkers, vol. 17, no. 8, pp. 7221–7729, 2012. View at: Google Scholar
  22. P. D. Rainville, C. L. Stumpf, J. P. Shockcor, R. S. Plumb, and J. K. Nicholson, “Novel application of reversed-phase UPLC-oaTOF-MS for lipid analysis in complex biological mixtures: a new tool for lipidomics,” Journal of Proteome Research, vol. 6, no. 2, pp. 552–558, 2007. View at: Publisher Site | Google Scholar
  23. J. Han, M. Ye, H. Guo, M. Yang, B. R. Wang, and D. A. Guo, “Analysis of multiple constituents in a Chinese herbal preparation Shuang-Huang-Lian oral liquid by HPLC-DAD-ESI-MSn,” Journal of Pharmaceutical and Biomedical Analysis, vol. 44, no. 2, pp. 430–438, 2007. View at: Publisher Site | Google Scholar
  24. Q. Z. Luo, J. B. Luo, and Y. Z. Wang, “Qualitative analysis of the main chemical constituents of Shuanghuanglian injection powder and their origin by HPLC-ESI/MS/MS spectrometry,” Acta Pharmaceutica Sinica, vol. 44, no. 12, pp. 1391–1396, 2009. View at: Google Scholar
  25. M. J. Zamek-Gliszczynski, K. A. Hoffmaster, K. I. Nezasa, M. N. Tallman, and K. L. R. Brouwer, “Integration of hepatic drug transporters and phase II metabolizing enzymes: mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites,” European Journal of Pharmaceutical Sciences, vol. 27, no. 5, pp. 447–486, 2006. View at: Publisher Site | Google Scholar

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.


More related articles

1364 Views | 660 Downloads | 9 Citations
 PDF  Download Citation  Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly and safely as possible. Any author submitting a COVID-19 paper should notify us at help@hindawi.com to ensure their research is fast-tracked and made available on a preprint server as soon as possible. We will be providing unlimited waivers of publication charges for accepted articles related to COVID-19. Sign up here as a reviewer to help fast-track new submissions.