1438 Yuan-yuan Xie Xue Xiao Juan-min Luo Chan Fu Qiao-wei Wang Yi-ming Wang Qiong-lin Liang ∗ Guo-an Luo Department of Chemistry, Tsinghua University, Beijing, P. R. China Received February 5, 2014 Revised March 11, 2014 Accepted March 21, 2014
J. Sep. Sci. 2014, 37, 1438–1447
Research Article
Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high-performance liquid chromatography with diode array detection and tandem mass spectrometry The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High-performance liquid chromatography coupled with time-of-flight mass spectrometry and high-performance liquid chromatography with electrospray multistage tandem ion-trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high-performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system. Keywords: Danhong injection / High-performance liquid chromatography / Liquid chromatography with mass spectrometry / Qualitative analysis / Quantitative characterization DOI 10.1002/jssc.201400129
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Traditional Chinese medicine injection (TCMI), which is prepared by extracting an effective substance from a single herb or a group of herbs in a composite formula with the modern scientific technology and method have played an irreplaceable role in the treatment of some emergency or severe illness [1]. However, chemical ingredients in herbal medicines vary greatly with the geographical origin of the species, cultivation practice, harvest time, storage conditions, and processing methods, which contribute to differences in the composition of the final product and bring about instability to the quality of TCMI [2, 3]. Recently, many serious complications of TCMI have been reported, which are due largely to the lack of a practicable and reliable quality standard to monitor the Correspondence: Professor Guo-an Luo, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China E-mail: [email protected] Fax: +86-10-62772264
Abbreviations: DAD, diode array detection; DHI, Danhong injection; TCMI, traditional Chinese medicine injection C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
properties that change in the procedure from preparation, transportation, and storage to clinic usage. So quality control for TCMI containing tens to hundreds of characteristic phytochemicals poses a challenge for developing robust quality control metrics [4, 5]. Since great progress has been made in the field of analysis of TCM formulations, current quality control standards of TCMI are no longer just focused on the absolute quantitation of a single component or a limited number of components, but also try to use complete fingerprint patterns to characterize more completely the multichemical species [6, 7]. Chemical fingerprinting has been introduced and adopted by the World Health Organization, State Food, and Drug Administration and other authorities as a strategy for the quality assessment of TCMI at the turn of the century [8, 9] in order to describe the macroscopic characteristics of all components in TCMI and demonstrate the “similarity” of various samples. Then in order to detect every potential phytochemical class of molecules, on the basis of several hyphenated ∗ Additional corresponding author: Professor Qiong-lin Liang, E-mail: [email protected]
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Figure 1. Roadmap of the integrated strategy on simultaneous qualitative and quantitative characterization of TCMs.
techniques, such as GC–MS, CE–MS, LC–MS, and MS/MS (MSn ), multidimensional fingerprinting have been proposed and applied in the analysis of TCMIs [10, 11]. On-line structure identification of ingredients can be performed by the retention time, UV spectra, m/z values and MS/MS fragmentation pattern, which will provide exact chemical composition information and be convenient to understand the relationships between the fingerprint and activities [12–15]. Meanwhile, multicomponent quantitative analysis with multiwavelength detection has been used to exhibit the “specificity” of different samples, which was summarized as microcosmic features [16–18]. We represent a powerful strategy as an integrated analysis of macroscopic characteristic (chemical fingerprint + on-line structure identification) and microcosmic features (multicomponent quantification) for the quality assessment of TCMIs as well as other complex TCM systems [19]. The application of on-line structure identification technology in the chemical fingerprint may make us understand the chemical composition well, which is a benefit for the selection of the quantified marker. The present study aims to describe and exemplify the integrated strategy as integrating qualitative and quantitative characterization of multicomponent for the quality control of TCMIs using the example of Danhong injection (DHI). The roadmap of this strategy is shown in Fig. 1. DHI, which is made of Radix Salviae Miltiorrhizae and Flos Carthami, is one of the commonly used TCMIs for the treatment of coronary heart disease and angina pectoris in China [20]. Lipophilic diterpenoid quinones and hydrophilic phenolic acids are the most representative components in Radix Salviae Miltiorrhizae; flavonoid glycosides and safflor yellow are the effective constituents in Flos Carthami with similar therapeutic effects to Radix Salviae Miltiorrhizae, such as facilitating blood circulation and dispersing blood stasis [21, 22]. Studies on the quality control of DHI have been reported previously, while most of them only focused on a few specific chemical marker compounds from Radix Salviae Miltiorrhizae [23, 24]. In order to develop a suitable quality control method, the TCM fingerprint and multicomponent quantification were usually used as the supporting technology in some reported studies on DHI. Liu et al. de C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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veloped a comprehensive quality evaluation method combining quantitative analysis with chromatographic fingerprint of Danhong injection using HPLC with diode array detection (DAD). However, the developed method only focused on the salvianolic acids from Radix Salviae Miltiorrhizae, which is short of the evaluation of components from Flos Carthami [25]. Liu et al. developed a dual-standard quality assessment method of Danhong injection by characterizing the antioxidant constituents in vivo. Salvianolic acids from Radix Salviae Miltiorrhizae with potent anti-oxidative activities were selected as key quality markers, while nucleosides from Flos Carthami (Hong-hua) exhibited weak anti-oxidative activities and were used as general quality markers. The bioactivityrelated quality assessment method provided a novel idea, while only a pharmacology indicator as the quality evaluation criteria of the traditional Chinese medicine prescription is not enough [26]. We have established the fingerprint evaluation method for the quality control of DHI previously [27]. In this work, an approach to screen and identify the main constituents in DHI by combining the accurate mass measurement of LC–TOF-MS and the complementary fragmentation data for structure confirmation provided by LC with ion trap MSn is described. Additionally, simultaneous quantitative analysis of seven markers in different batches of DHI, including uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B would be performed. The integrating qualitative and quantitative characterization strategy was subsequently applied to evaluate 20 batches of DHI and testified to be suitable for its quality control, which may provide an effective and reliable pattern for comprehensive and systematic characterization of the complex TCM system.
2 Materials and methods 2.1 Materials and reagents The reference compounds of uridine (1, LOT887– 200202), adenosine (2, LOT110879–200202), guanosine (3, LOTG6752), phenylalanine (4, LOT111615–200301), 5hydroxymethyl-2- furalcdehyde (5, LOT111626–201108), danshensu (6, LOT110855–200809), protocatechuic aldehyde (8, LOT110810–200506), caffeic acid (11, LOT110885– 200102), hydroxysafflor yellow A (10, LOT111637–201207), p-coumaric acid (18, LOT18279), rosmarinic acid (23, LOT111871–201203), salvianolic acid A (24), and salvianolic acid B (25, LOT 111562–201212) were purchased from the National Institute for Food and Drug control (Beijing, China). And the purity of these reference compounds was more than 98% to meet the need of content determination. HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany) and formic acid was obtained from Fluka (Buchs, Switzerland). Ultrapure water (18.2 M⍀) was prepared with a Milli-Q water purification system (Millipore Corporation, MA, USA). Other reagents were of analytical www.jss-journal.com
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grade. Twenty batches of DHI commercial products were supplied by Heze Buchang Pharmaceutical (Heze, China).
2.2 Preparation of standard solutions and sample solutions Each reference was accurately weighed, dissolved in methanol and deionized water, and diluted to the appropriate concentration. The mixed standard solution was prepared by dissolving standards with a fixed weight in methanol, and then stored at 4⬚C. The solutions were brought to room temperature, diluted, and filtered through a 0.22 m membrane filter before analysis. The samples of DHI for HPLC–DAD analysis were diluted to 1:5 v/v with ultrapure water, and then filtered through a 0.22 m membrane filter before injection; an aliquot of 10 L filtrate solution was injected for HPLC–DAD and LC– MS analysis.
The HPLC conditions for the LC–TOF-MS analysis were the same as the HPLC method, except for that one-third of the eluent was introduced into the TOF-MS system with a split valve. TOF-MS analysis was performed in both positive (ESI+ ) and negative (ESI− ) ion mode over m/z 50–1500 under the following operation parameters: capillary voltage 3500 V (ESI− ) or 4000 V (ESI+ ); drying gas 8.0 L/min; nebulizer pressure 40 psig; gas temperature 325⬚C; fragmentor voltage 175 V (ESI+ ) and 190 V (ESI− ); skimmer voltage 60 V; octopole dcl 37.5 V (ESI+ ) and –38.0 (ESI− ); octopole RF 250 V. The reference solution was used as a continuous calibration using the following reference masses: 121.0509 and 922.0098 m/z. Analyst QS software (Applied Biosystems, Framingham, MA, USA) was used to process the accurate mass data. Exact masses corresponding to particular elemental compositions were also calculated by the formula calculator in this software. Daily instrument tuning was carried out using the tuning solution (G1969–85000, Agilent, USA) to ensure no more than 5 ppm mass error prior to run samples. 2.4.2 Ion trap MSn system
2.3 HPLC apparatus and conditions HPLC–DAD analysis was performed using an Agilent 1100 Series HPLC–DAD system (Agilent Series 1100, Palo Alto, CA, USA) compressing a vacuum degasser, binary pump, autosampler, thermostatted column compartment, and a diode array detector, scanning from 200–400 nm, the wavelength was then selected and fixed at 254, 280, and 310 nm for qualitative and quantitative analysis, considering the variety of constituents in DHI. Separation was performed on an AlltimaTM C18 analytical column (250 mm × 4.6 mm i.d., 5 m, Grace Davison Discovery Science, USA) at 30⬚C. The mobile phase consisted of two solvents: 0.4% aqueous formic acid (solvent A) and acetonitrile (solvent B) with gradient elution (0–20 min, 3–12% B; 20–35 min, 12–20% B; 35–55 min, 20–38% B; 55–75 min, 38–80% B). Re-equilibration duration was 10 min between individual runs. The flow rate was kept at 1.0 mL/min and 10 L of sample solution was injected in each run [27].
2.4 LC–MS apparatus and conditions 2.4.1 TOF-MS system The above HPLC system was interfaced with an Agilent 1100 LC/MSD TOF equipped with an electrospray interface (Agilent, Waldbronn, Germany). The electrospray source includes dual nebulizers, one nebulizer for the LC eluent and the other for the internal reference solution. The reference nebulizer, along with the LC/MSD TOF’s automated calibrant delivery system, provides continuous introduction of reference mass standards into the ion source for automated mass calibration. Accurate mass measurements were obtained with this calibrant delivery system and thus enhanced accuracy was achieved. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The HPLC system was also connected to Agilent 1100 LC/MSD Trap (Agilent, Santa Clara, CA, USA) equipped with an electrospray interface to provide sufficient fragmentation data. The ion trap MS analysis was carried out in both positive and negative mode over m/z 50–1500 using the following operation parameters: capillary voltage 3500 V (ESI− ) or 4000 V (ESI+ ); skimmer voltage 40 V; capillary exit voltage: 137 V; nebulizer pressure 30 psig; drying gas: 8 L/min; gas temperature 325⬚C; target mass: 622 m/z; compound stability: 100%; trap drive level: 60%; threshold: 50 000 (ESI+ ) and 10 000 (ESI− ); ion charged control (ICC): on; target: 10 000; accumulation time: 200 ms. An amplitude voltage of 1.0 V was typically used for fragmentation in the ion trap auto MS2 and MS3 experiments. Data were processed by Agilent Chemstation Rev. A. 09.01 software (Agilent, Palo Alto, CA, USA).
3 Results and discussion 3.1 Optimization of HPLC conditions Column types, mobile phase compositions, gradient elution procedure, flow rate of the mobile phase, and column temperature were optimized respectively to achieve good separation of as many peaks as possible within a short-analysis time. The applicability of the established analysis method to different HPLC instrument systems has been verified. The detection wavelength was selected with the use of a DAD detector. In order to detect more common peaks while achieving precise detection of them, the most appropriate wavelength for fingerprinting was set as 254 nm. The detection wavelengths for quantitative analysis were selected according to the maximum adsorption wavelengths of uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B at 260, 260, 280, 280, www.jss-journal.com
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Figure 2. HPLC chromatograms of standard solutions as well as DHI at five detection wavelengths. HPLC chromatogram of mixed standards including uridine (1), adenosine (2), danshensu (3), protocatechuic aldehyde (4), p-coumaric acid (5), rosmarinic acid (6), and salvianolic acid B (7) detected at 260 nm. (B–F) HPLC chromatograms of DHI at five detection wavelengths: 260 nm (B), 280 nm (C), 286 nm (D), 310 nm (E), and 320 nm (F) for different markers.
310, 320, and 286 nm, respectively, shown in UV spectra with three-dimensional chromatograms of DAD, as shown in Fig. 2. The proposed method is therefore acceptable as well as adequate for further MS analysis. 3.2 Identity assignment and confirmation of the detected components in DHI The screening, identification, and further confirmation of components in DHI were performed by LC–TOF-MS to provide the elemental compositions of both the molecular ions and even characteristic fragment ions firstly. Formulae and available fragmentation were then checked by LC with ion trap MS3 to confirm the results of LC–TOF-MS. The total ion chromatograms of DHI were obtained by LC–TOF-MS in both positive and negative ion mode. Most of compositions showed their [M+H]+ and [M+Na]+ ions in positive ion mode, and exhibited quasi-molecular ions [M−H]− , adduct ion [M+HCOO]− in negative ion mode. MS/MS and MSn data were detected by collision-induced dissociation. Twentysix compounds were identified by interpreting their mass spectra obtained by their MS (TOF), MS/MS, and MSn (ion trap), as well as UV spectra, taking into account all the data provided by the literature (Fig. 3). Thirteen compounds were
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accurately identified by comparing the retention time, UV spectra, and m/z values with those of the authentic standards. Information of the 26 components defined is displayed in Table 1 and their chemical structures are shown in Fig. 4 [12, 28–35]. The results exhibited excellent coherence with the two types of MS. The detailed identification of each group of components was outlined as followed. For identification of the nucleotides in DHI, extracted ion chromatograms provided by TOF-MS were applied since relatively lower signals of these compounds in the total ion chromatograms. By extracting ions at m/z 243, 268, 284, and 166, four nucleotides including uridine, adenosine, guanosine, and phenylalanine (peaks 1, 2, 3, 4) could be deduced and ultimately verified with authentic standards. TOF-MS provided us the accurate mass value of m/z 755.2041 and the probable formula of C33 H39 O20 for the peak 13. The proposed formula was searched using “SciFinder Scholar” database (CAS on-line, https://scifinder.cas.org) and several matching results were obtained in November 2011. Then the MS spectra from ion trap delivered a molecular mass of m/z 754.8 for this compound, together with two main diagnostic fragments at m/z 593.0 (loss of a glucosyl group, 162 mass units) and 284.8 (loss of a glucosyl group and arutinosyl moiety, 470 mass units), suggesting that peak 13 is a flavone diglycoside and the aglycone moiety should be
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Figure 3. HPLC–UV profile of DHI at 254 nm. The number of peaks marked in Fig. 3 is corresponding to Table 1.
kaempferol. Thus, peak 13 was identified as kaempferol-3-Orutinoside-7-glucopyranoside [29]. Peak 11 showed the same UV absorptions as peak 13 at 254, 267(sh), and 348 nm, and which was identified as 6-hydroxykaempferol-3,6,7-O-D-glucosidetentatively using this combinative method according to the accurate mass value and trap fragmentation data [30]. The MS spectrum of peak 10 exhibited a negative molecular ion at m/z 611.1, together with two diagnostic fragments at m/z 491 (loss of a p-vinylphenol group, 120 mass units) and 329 (loss of a p-vinylphenol group and a glucose, 282 mass units). This represents the precursor→product transition of [M-H]− → [M-H-120]− → [M–H–120–glu]− , which indicated the neutral loss of a p-vinylphenol group and then a glucose. Peak 10, thus, was identified as a hydroxysafflor yellow A [33], and the identification of this compound was ultimately verified with authentic standards. Fourteen phenolic acids mainly from Radix Salviae Miltiorrhizae were deduced in turn by comparing their retention times or MS spectra with those of authentic compounds or literature data [31,32,34–39]. The ESI-MS/MS spectra from ion trap of salvianolic acids exhibited the fragment ions derived from neutral loss of one or two molecules of danshensu moiety corresponding to 198 mass units. The fragment [M– 44–H]− corresponds to the loss of a CO2 molecule is also present in the spectra of most of the phenolics including caffeic acid monomer and its metabolites due to the existence of a –COOH group in all of these compounds. The other phenolic acids were characterized as in Table 1, referring to previous data reported in the literature. And peaks 6, 8, 11, 18, 23, and 25 were unambiguously characterized by comparison with reference compounds. In conclusion, 26 main components including nucleotides, phenolic acids, and flavonoid glycosides have been detected and identified in DHI. What need to be emphasized was that 5-hydroxymethylfurfural (Peak 5) may be a byproduct from the preparation of DHI, which has been verified when the established chemical profile was used for the production process monitoring the analysis of DHI.
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3.3 Method validation of quantitative analysis The precision and repeatability of the chemical profiling method by HPLC–DAD were validated by the reduplicate analysis of six injections of the same DHI samples and six parallel samples prepared using the same preparation protocol, respectively. The relative standard deviations of the peak retention time and area value were less than 5.0%. The resulting data showed that the precision and repeatability of the proposed method were satisfactory for chemical profiling analysis. The method validation of quantitative analysis is described below. 3.3.1 Calibration curves, LODs, and LOQs The HPLC method was validated defining the linearity, limits of quantification, and detection, identification and quantification of the analytes, repeatability, precision, stability, and recovery. All calibration curves were plotted based on linear regression analysis of the integrated peak areas (y) versus concentrations (x, g/mL) of the seven marker constituents in the standard solution at seven different concentrations. The regression equations, correlation coefficients, and linear ranges for the analysis of the seven marker constituents are shown in Table 2. The LOD was calculated as the amount of the injected sample that gave a single-to-noise ratio of 3 (S/N = 3), and the LOQ was calculated as the amount of the injected sample that gave a single-to-noise ratio of 10 (S/N = 3). The LOD and LOQ values of the method for the seven components are listed in Table 2. 3.3.2 Precision, repeatability, stability, and recovery Intra- and interday variations were chosen to determine the precision of the developed assay. For intraday variability test, the DHI sample solutions were analyzed for six replicates within one day, while for interday variability test, the solutions were examined in duplicate for three consecutive
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611.172 179.0304 431.1824 755.2041
C10 H14 N5 O4 C10 H14 N5 O5 C9 H12 NO2 C6 H7 O3 C9 H9 O5 C7 H5 O4 C7 H5 O3 C33 H41 O22
C27 H31 O16
C27 H31 O16 C18 H15 O8 C19 H17 O8 C19 H17 O8 C9 H7 O3 C27 H21 O12 C27 H21 O12 C20 H17 O10 C36 H29 O16 C18 H15 O8 C26 H21 O10 C36 H29 O16 C26 H21 O10
[M–H] − [M–H] − [M+H]+
[M–H] − C9 H7 O4 [M–H] − [M–H+HCOOH]− C20 H31 O10 C33 H39 O20
[M+H]+ [M–H] − [M+H]+ [M–H] − [M–H] −
[M–H] − [M–H] − [M+H]+ [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] −
7.0 9.3 11.9 12.5 14.6
17.2 22.5 24.7
2* 3* 4* 5* 6*
7 8* 9
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Caffeic acid Roseoside
Kaempferol-3-rutinoside-7glucopyranoside 4’,5,6,7-Tetrahydroxyflavanone6,7-di-O--D-glucoside Prolithospermic acid Methyl rosmarinate/isomer
11* 28.9 12 30.1
31.8
32.3
34.7 35.4
38.3
13
14
15 16
17
Alkannic acid/salvianolic acid J
42.6
45.0
46.9
20
21
22
Salvianolic acid A Salvianolic acid B
Salvianolic acid A isomer
24* 48.4 25* 50.4
26
493.1041
493.1022 717.132
359.0804
717.1309
417.0735
537.0949
163.0454 537.0947
373.0945
359.0804 373.0945
611.1579
153.0149 137.0214 789.2157
268.1007 284.0916 166.0840 127.0395 197.0411
493.1135
493.1135 717.1456
359.0767
717.1456
417.0822
537.1033
163.0395 537.1033
373.0923
359.0767 373.0923
611.1612
755.2035
179.0344 431.1917
611.1612
153.0188 137.0239 789.2089
268.1046 284.0995 166.0868 127.0395 197.0450
243.0617
–19.0066
–22.8598 –18.9092
10.3244
–20.4431
–20.7927
–15.6425
36.0685 –16.0149
5.7819
10.3244 5.7819
–5.4167
0.8350
–22.5316 –21.6211
17.6538
–25.3816 –18.0200 8.5544
–14.4690 –27.7861 –16.8816 7.7203 –19.7855
–17.3263
16.5
16.5 22.5
11.5
22.5
12.5
17.5
6.5 17.5
11.5
11.5 11.5
12.5
14.5
6.5 5.5
12.5
5.5 5.5 13.5
6.5 6.5 4.5 3.5 5.5
5.5
492.9 [M–H] −
492.9 [M–H] − 717.1 [M–H] −
719.0 [2M–H] −
717.1 [M–H] −
416.9 [M–H] −
537.0 [M–H] −
162.8 [M–H] − 537.0 [M–H] −
372.9 [M–H] −
359.2 [M+H]+ 373.0 [M–H] −
611.1 [M–H] −
754.8 [M–H] −
178.7 [M–H] − 431.0 [M–H+HCOOH] −
611.1 [M–H] −
153.0 [M–H] − 136.8 [M–H]− 789.3 [M+H]+
394.8 [2M–H]−
268.1 [M+H]+ 282.1 [M–H] − 166.1 [M+H]+
242.8 [M–H] −
627.2 [M+H–glu]+ 456.1 [M+H–glu–glu]+ 303.1 [M+H–glu–glu–glu]+ 490.9 [M–H–120]− 328.7 [M–H–120–glu]− 134.8 [M–H–COO] − 385.0 [M–H] − 223.0 [M–H–glu] − 593.0 [M–H–glu] − 284.7 [M–H–glu–rutin]− 448.9 [M–H–glu] − 286.8 [M–H–glu–glu] − 311.8 [M+H–COO]+ 178.8 [M–H–194]− 142.8 [M–H–194–2H2 O]− 178.8 [M–H–194]− 142.8 [M–H–194–2H2 O]− 118.9[M–H–COO] − 492.9 [M–H–COO] − 294.8 [M–H–COO–198] − 492.9 [M–H–COO] − 294.8 [M–H–COO–198] − 372.8 [M–H–COO] − 156.8 [M–H–COO–198] − 519.0 [M–H–198] − 320.8 [M–H–198–198] − 358.8 [M–H] − 160.7 [M–H–198] − 294.8 [M–H–198] − 518.9 [M–H–198] − 320.8 [M–H–198–198] − 294.8 [M–H–198] −
196.8 [M–H]− 178.7 [M–H–H2 O]−
150.1 [M–H–rib]− 120.0 [M+H–HCOO]+
199.7 [M–H–HNCO]− 109.8 [M–H–rib]−
MS2 –MS3 (m/z)
Danshen
Danshen Danshen
Danshen
Danshen
Danshen
Danshen
Honghua Danshen
Danshen
Danshen Danshen
Honghua
Honghua
Danshen Honghua
Honghua
Danshen Danshen Honghua
Honghua Honghua Honghua Byproduct Danshen
Honghua
Origin
Liquid Chromatography
a) Compared with authentic compounds; *a&b compounds were not detected in DAD detector, only found in TOF-MS detector; Honghua: Flos Carthami; Danshen: Radix Salviae Miltiorrhizae. “DBE”: “degree of unsaturation.”
52.7
Rosmarinic acid
23* 47.8
Salvianolic acid E
Salvianolic acid D
p-Coumaric acid Alkannic acid/salvianolic acid J
18* 39.0 19 41.5
Methyl rosmarinate/isomer
6-Hydroxysafflor yellow A
10* 27.5
Protocatechuic acid Protocatechuic aldehyde 6-Hydroxykaempferol-3,6,7-O-D-glucoside
Adenosine Guanosine Phenylalanine 5-Hydroxymethylfurfural Danshensu
243.0575
C9 H11 N2 O6
[M–H] −
Uridine
6.2
1*
Measured Calculated Error (ppm) DBE MS (pos/neg) mass (m/z) mass (m/z) MS (m/z)
Formula
Selected ion
No. tR (min) Identification
Table 1. Identification of multicomponent from DHI by LC–MS (MSn ) J. Sep. Sci. 2014, 37, 1438–1447
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Figure 4. The structures of components identified in DHI.
days. Variations were expressed by the RSD for intra- and interday, which were both less than 3.0%. Six different sample solutions prepared from the same sample were analyzed to confirm the repeatability of the developed assay. RSD values of the seven determined compounds’ contents were all less than 3.0%, which satisfied the criteria of quantitative analysis. For the stability test, peak areas of seven compounds in sample solution, which was stored at room temperature in the dark, were analyzed at 0, 4, 8, 12, 16, 24 h. RSD values of the contents of seven compounds were less than 3.0%, respectively. These results suggested that it was feasible to analyze
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the samples within one day. The percentage difference between amounts determined and spiked was considered to be a measure of accuracy. Known amounts (low, medium, and high) of the seven standard references were spiked into samples and then prepared as test solutions. The determination was performed in triplicate, and the average recoveries and RSD were calculated. The developed method had good accuracy with the overall recovery of 95.6 to 102.9%, with the RSD ranging from 0.42 to 3.36%. These results are listed in the Supporting Information, which indicates that the HPLC–UV
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Table 2. Regression equation, linear range, and LOD of the developed method
Compound
Regression equationa)
Correlation factors (r)
Linearity range (g/mL)
LOQ (ng/mL)b)
LOD (ng/mL)b)
Uridine Adenosine Danshensu Protocatechuic aldehyde p-Coumaric acid Rosmarinic acid Salvianolic acid B
Y = 2.42 × 103 X +2.65 Y = 3.08 × 103 X +3.26 Y = 0.66 × 103 X +5.53 Y = 4.12 × 103 X +7.60
1.000 1.000 1.000 1.000
1.078–107.8 1.36–136.2 8.46–1692 2.89–2890
6.67 × 10−3 1.46 × 10−2 9.17 × 10−1 3.38 × 10−2
2.00 × 10−3 4.39 × 10−3 2.75 × 10−1 1.02 × 10−2
Y = 6.81 × 103 X +22.5 Y = 2.71 × 103 X +7.93 Y = 1.12 × 103 X – 0.40
1.000 1.000 1.000
1.92–1922 2.51–2512 11.59–2318
1.06 × 10−2 1.12 × 10−1 1.08 × 10−1
3.19 × 10−3 3.36 × 10−2 3.23 × 10−2
a) Y and X are, respectively, the peak areas and concentrations (g/mL) of the analytes. b) The LOQ was defined as the concentration for which the signal-to-noise ratio was 10 and the LOD was defined as the concentration for which the signal-to-noise ratio was 3.
method is precise, accurate, and sensitive for the quantitative determination of seven components in DHI samples [40].
3.4 Chemical profiling and quantitation Chemical profiling and assay of 20 different commercial batches of DHI were carried out. A reference fingerprint of DHI was generated on the basis of the chromatogram fingerprints of all samples using the software of the similarity evaluation system and the peak from Salvianolic acid B was used as reference. And then the similarity of the chromatogram from a sample with the reference fingerprint was assessed by calculating the correlation coefficient and/or angle cosine value of the original data [41]. As shown in Fig. 5, the chemical fingerprint similarity of these determined samples ranged from 0.91 to 0.99, indication a good consistency among the 20 batches products in qualitative evaluation. As for the quantitative analysis, danshensu, and protocatechuic aldehyde were used as the marker components in
the enacted quality standard of DHI recorded in the National Standard for Chinese Patent Medicine. And some specific chemical marker compounds from Radix Salviae Miltiorrhizae were usually used as the quality evaluation indicators [25]. Characteristic components from Flos Carthami were ignored in most cases. Flavonoid glycosides, nucleosides, and safflor yellow are the effective constituents in Flos Carthami with some activities such as facilitating blood circulation and dispersing blood stasis. In this study, ten components originated from Flos Carthami had been detected and identified by LC–MS, including four nucleosides, four flavonoid glycosides, one safflor yellow, and one coumarin. As we know, for the selection of marker components, the representativeness of the derived medicinal materials, the determination accuracy and the availability of the reference substances were taken into account. Since the absence of reference substances of flavonoid glycosides, and the low content of 6-hydroxysafflor yellow A detected in DHI, two typical nucleosides (uridine and adenosine) as well as p-coumaric acid from Flos Carthami were selected as the marker components. Furthermore, four typical components hydrophilic phenolic acids from Radix Salviae Miltiorrhizae were also determined. The developed multiwavelength multicomponent determination method was subsequently applied to simultaneous determination of the seven marker constituents in 20 different commercial batches of DHI. The results are shown in Table 3. The quality fluctuations between batches were discussed using the parameter P. The closer the P value is to 100%, the better the consistency between batches. The formula is given as follows: P=
Figure 5. Box chart of contents of seven components and similarity from 20 batches of DHI. Uri, uridine (1); Ade, adenosine (2); DSS, danshensu (3); PA, protocatechuic aldehyde (4); p-C, p-coumaric acid (5); RA, rosmarinic acid (6); SaB, salvianolic acid B (7).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Ci Ci
× 100%
(1)
Where Ci denotes the measured concentration of certain component, while C¯ i denotes the average concentration of 20 batches of DHI. A box chart of the seven components from 20 batches of DHI, as well as the similarity of these samples was shown www.jss-journal.com
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Table 3. Contents of the seven active components and the fingerprint similarity in 20 batches of DHI
LOT
Content (g/100ml)
Similarity
uridine (1)
adenosine (2)
danshensu (3)
protocatechuic aldehyde (4)
p-coumaric acid (5)
rosmarinic acid (6)
salvianolic acid B (7)
90606 90934 91004 91106 91219 91221 91253 91255 100202 100121 100128 100141 100213 100231 100244 100306 100311 100347 100401 100426
80.19 84.38 79.62 77.67 69.91 74.37 78.36 76.97 75.12 64.56 74.00 69.65 73.76 64.91 69.97 52.61 61.42 70.89 68.96 79.52
8.27 14.73 14.09 12.97 17.52 14.21 13.73 12.70 17.32 11.78 12.58 13.46 16.20 14.20 15.64 12.65 13.27 15.40 20.27 15.21
1434.87 1225.86 1338.55 1348.11 1097.10 998.72 1271.99 1252.94 976.30 1022.82 1317.59 1216.45 1092.89 1192.43 941.88 862.85 905.92 1061.45 1002.39 1371.29
244.57 180.80 213.33 229.35 154.54 143.80 191.85 208.10 123.02 158.13 206.68 116.14 146.75 199.36 155.01 127.50 143.08 177.02 155.98 247.09
26.92 32.75 32.36 32.36 26.91 24.45 28.96 27.20 20.13 20.49 23.54 21.08 22.94 29.97 29.34 23.09 24.78 30.26 32.19 31.62
213.02 207.98 209.58 194.77 243.25 217.83 217.10 190.21 170.22 197.52 237.22 157.61 201.31 242.20 168.25 150.25 162.60 192.74 150.51 189.17
390.37 662.24 555.21 414.26 675.44 520.83 523.35 432.82 509.75 443.32 529.35 542.73 543.81 581.76 457.70 330.21 364.73 456.57 377.44 429.71
0.981 0.973 0.986 0.975 0.936 0.959 0.959 0.907 0.982 0.982 0.961 0.972 0.983 0.981 0.984 0.978 0.987 0.986 0.965 0.973
max min average S.D. RSD (%)
84.38 52.61 72.34 7.51 10.38
20.27 8.27 14.31 2.49 17.39
1434.87 862.85 1146.62 171.75 14.98
247.09 116.14 176.10 39.82 22.61
32.75 20.13 27.07 4.27 15.79
243.25 150.25 195.67 28.94 14.79
675.44 330.21 487.08 93.76 19.25
0.907 0.987 0.97 0.02 2.02
in Fig. 5. Different from the good consistency of 20 batches products in chemical fingerprint (similarity ranged from 0.91 to 0.99), the contents of these seven determined compounds varied greatly, the RSD (%) of contents ranged from 10.38 to 22.61%, especially protocatechuic aldehyde and salvianolic acid B. As reported previously, salvianolic acid B has been found to have potent anti-oxidative capabilities, as well as reduction of leukocyte-endothelial adherence, inhibition of inflammation and metalloproteinases expression from aortic smooth muscle cells, and indirect regulation of immune function [42]. Due to these activities, salvianolic acid B exhibited its great clinical impact on cardiovascular protection, and as a “key component” in many TCM prescriptions including DHI for cardiovascular diseases. So the fluctuation of its content would inevitably affect the therapeutic efficacy of DHI. Therefore, more attention should be paid to study the content change tendency of these compounds during the whole product process, and find out the key processing point and parameters may affect the content of these marker components. In addition, the integrative strategy also contained the simultaneous qualification and quantification analysis of other components in DHI without UV absorption, such as amino acids and sugars. So a precolumn derivatization method with o-phthaldialdehyde and fluorenylmethyl chloroformate C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(OPA-FMOC) to determine the contents of amino acids in DHI and the UV/Vis method to determine the contents of reducing sugars have also been established previously [43]. It is expected that more useful information about the overall quality of DHI would be provided to characterize the overall quality of DHI.
4 Concluding remarks A comprehensive quality control standard was usually compared to a “bright eyes,” which would be used to find out the variation of detected products and ensure the consistency of TCM product quality. So the integrality and specificity should be taken into account when develop a quality evaluation method. In this study, a reliable and powerful analytical method by using the integrative strategy of simultaneous qualification and quantification of multiple components for the comprehensive quality evaluation of a TCMI (Danhong injection) has been established. TCM fingerprinting together with the identification of unknown components on-line were used to character the holistic profiles of the DHI, the “similarity” of products from different manufactured batches was used to evaluate the “consistency,” and the structure information of 26 components in DHI may provide www.jss-journal.com
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detailed information for us to understand its efficacy as well as the safety. With the guidance of macroscopic characteristic research, seven main representative components from the two raw medicinal materials were used as the markers of multicomponent quantification determination, which was used to present the differences among the products. The macroscopic characteristic (chemical fingerprint) and microcosmic features (multicomponent quantification) of 20 batches DHI was subsequently described with the established method. The developed method has been testified to be an effective and reliable pattern for comprehensive and systematic characterization of DHI, and which is recommendable for the quality assessment of complex TCM systems.
[16] Hu, P., Luo, G. A., Zhao, Z. Z., Jiang, Z. H., Chem. Pharm. Bull. 2005, 53, 481.
This work was sponsored by the International Cooperation Projects of Ministry of Science and Technology (MOST) in China (No. 2010DFA32420) and the National Natural Sciences Foundation of China (No. 90917003, 81130066, 81102766). The authors acknowledge the manufacturer of Danhong Injection for supplying the test samples.
[23] Wang, S. M., LiShiZhen Med. Mat. Med. Res. 2006, 17, 989.
The authors have declared no conflict of interest.
5 References [1] Shi, H. S., Feng, J. C., Chin. J. Med. Hist. 1995, 25, 107. [2] Li, X. L., Song, L. G., Liu, Y., Bo, Y. J., J. Chin. Mater. Med. 2009, 34, 1326–1328. [3] Xie, P. S., Chen, S. B., Liang, T. Z., Wang, X. H., Tian, R. T., Upton R., J. Chromatogr. A 2006, 1112, 171–180. [4] Van Breemaen, R. B., Fong, H. H. S., Farnsworth, N. R., Chem. Res. Toxicol. 2007, 20, 577–582.
[17] Yan, S. K., Luo, G. A., Wang, Y. M., Cheng, Y. Y., J. Pharm. Biomed. Anal. 2006, 40, 889. [18] Ruan, M., Li, Y., Li, X., Luo, J. G., Kong, L. Y., J. Pharm. Biomed. Anal. 2012, 59, 184. [19] Luo, G. A., Wang, Y. M., Liang, Q. L., Xie, Y. Y., Fan, X. M., Tradit. Chin. Med. Mater. Med. 2011, 13, 1. [20] Sun, M., Zhang, J. J., Shan, J., Zhang, Z. H., Jin, C., Xu, Y. S., Wang, Y. L., Phytomedicine 2009, 16, 683. [21] Tai, M. H., Liu, L. M., Ma, R. Q., J. First Mil. Med. Univ. 2005, 25, 335. [22] Xin, Q., Li, X. F., Si, D. Y., Chin. Trad. Pat. Med. 2004, 26, 222.
[24] You, H. L., Xi, C. P., Lin, Y. Q., Xu, L. H., Qilu Pharm. Aff. 2011, 30, 139. [25] Liu, X. S., Wu, Z. Z., Yang, K., Ding, H. Y., Wu, Y. J., J. Pharm. Biomed. Ana. 2013, 73, 70. [26] Liu, H. T., Wang, Y. F., Olaleye, O., Zhu, Y., Gao, X. M., Kang, L. Y., Zhao, T., Biomed. Chromatogr. 2013, 27, 655. [27] Luo, J. M., Xiao, X., Liang, Q. L., Luo, G. A., Wang, Y. M., Chin. Trad. Pat. Med. 2011, 33, 1277. [28] Zhou, J. J., Xie, G. R., Yan, X. J., Dictionary of Phytochemical Compositions of Traditional Chinese Medicine, Science Press, Beijing 2010. [29] Djoukeng, J. D., Arbona, V., Argamasilla, R., Cadenas, A. G., J. Agric. Food Chem. 2008, 56, 11087. [30] Huang, H., Ji, L. X., Song, S. Y., Wang, J., Wei, N., Jiang, M., Bai, G., Luo, G. A., Phytochem. Anal. 2011, 22, 330. [31] Qiao, X., Han, J., Xu, M., Zhang, Z. C., Ye, M., Ye, G., Guo, D. A., J. Pharm. Bio. Anal. 2007, 54, 789–798.
[5] Khan, I. A., Life Science 2006, 78, 2033–2038.
[32] Qiao, X., Han, J., Zhang, Z. C., Sun, S. F., Li, H. F., Wang, B. R., Ye, M., Guo, D. A., Planta Med. 2008, 74, 1720–1729.
[6] Xia, J. F., Liang, Q. L., Luo, G. A., Sci. Sin. Chim. 2010, 40, 641.
[33] Li, F., Pu, R., Zhao, H. Y., Liu, X., Ma, C., Wang, B. R., Guo, D. A., J. Chin. Pharm. Sci. 2011, 20, 47.
[7] Tilton, R., Paiva, A. A., Guan, J. Q., Marathe, R., Jiang, Z. L., Eyndhoven, W. V., Bjoraker, J., Prusoff, Z., Wang, H. L., Liu, S. H., Cheng, Y. C., Chin. Med. 2010, 5, 30–36.
[34] Zhu, Z. Y., Zhang, H., Zhao, L., Dong, X., Li, X., Chai, Y. F., Zhang, G. Q., Rapid Commun. Mass Spectrom. 2007, 21, 1855.
[8] State Food and Drug Administration of China, Technical Requirements for the Development of Fingerprints of TCM Injections, Beijing 2000.
[35] Liu, A. H., Guo, H., Ye, M., Lin, Y. H., Sun, J. H., Xu, M., Guo, D. A., J. Chromatogr. A 2007, 1161, 170.
[9] World Health Organization, WHO Guidelines for the Assessment of Herbal Medicine, Munich 1991. [10] Luo, G. A., Wang, Y. M., Cao, J., Chin. Trad. Pat. Med. 2000, 6, 395. [11] Yan, S. K., Xin, W. F., Luo, G. A., Wang, Y. M., Cheng, Y. Y., J. Chromatogr. A 2005, 1090, 90. [12] Zhang, H. Y., Hu, P., Luo, G. A., Liang, Q. L., Wang, Y. L., Yan, S. K., Wang, Y. M., Chim. Acta. 2006, 577, 190. [13] Qu, J., Wang, Y. M., Luo, G. A., Wu, Z. P., J. Chromatogr. A 2011, 928, 155. [14] Qu, J., Wang, Y. M., Luo, G. A., J. Chromatogr. A 2001, 919, 437. [15] Huang, X., Song, F. R., Liu, Z. Q., Liu, S. Y., Chim. Acta. 2008, 615, 124.
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[36] Hu, P., Liang, Q. L., Luo, G. A., Zhao, Z. Z., Jiang, Z. H., Chem. Pharm. Bull. 2005, 53, 677. [37] Hu, P., Luo, G. A., Zhao, Z. Z., Jiang, Z. H., Chem. Pharm. Bull. 2005, 53, 705. [38] Liu, M., Li, Y. G., Zhang, F. Yang, L., Chou, G. X., Wang, Z. T., Hu, Z. B., J. Sep. Sci. 2007, 30, 2256. [39] Xia, L., Liu, H. L., Li, P., Zhou, J. L., Qi, L. W., Yi, L., Chen, J. J. Sep. Sci. 2008, 31, 3156. [40] Fu, C., Luo, J. M., Wang, Y. M., Liang, Q. L., Luo, G. A., Chin. J. New Drugs 2012, 21, 2817. [41] Wang, X. M., Xie, Y. Y., Hu, X. M., Li, Y. K., Hu, P., Wang, Y. M., Liang, Q. L., Luo, G. A., Acta. Pharm. Sinica. B 2013, 3, 337. [42] Ho, J. H., Hong, C. Y., J. Biomed. Sci. 2011, 18, 30. [43] Pei, Y., Xiao, X., Fu, C., Ying, X. H., Liang, Q. L., Wang, Y. M., Luo, G. A., Chin. Trad. Pat. Med. 2012, 34, 1703. www.jss-journal.com
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Research Article
Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high-performance liquid chromatography with diode array detection and tandem mass spectrometry The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High-performance liquid chromatography coupled with time-of-flight mass spectrometry and high-performance liquid chromatography with electrospray multistage tandem ion-trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high-performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system. Keywords: Danhong injection / High-performance liquid chromatography / Liquid chromatography with mass spectrometry / Qualitative analysis / Quantitative characterization DOI 10.1002/jssc.201400129
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Traditional Chinese medicine injection (TCMI), which is prepared by extracting an effective substance from a single herb or a group of herbs in a composite formula with the modern scientific technology and method have played an irreplaceable role in the treatment of some emergency or severe illness [1]. However, chemical ingredients in herbal medicines vary greatly with the geographical origin of the species, cultivation practice, harvest time, storage conditions, and processing methods, which contribute to differences in the composition of the final product and bring about instability to the quality of TCMI [2, 3]. Recently, many serious complications of TCMI have been reported, which are due largely to the lack of a practicable and reliable quality standard to monitor the Correspondence: Professor Guo-an Luo, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China E-mail: [email protected] Fax: +86-10-62772264
Abbreviations: DAD, diode array detection; DHI, Danhong injection; TCMI, traditional Chinese medicine injection C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
properties that change in the procedure from preparation, transportation, and storage to clinic usage. So quality control for TCMI containing tens to hundreds of characteristic phytochemicals poses a challenge for developing robust quality control metrics [4, 5]. Since great progress has been made in the field of analysis of TCM formulations, current quality control standards of TCMI are no longer just focused on the absolute quantitation of a single component or a limited number of components, but also try to use complete fingerprint patterns to characterize more completely the multichemical species [6, 7]. Chemical fingerprinting has been introduced and adopted by the World Health Organization, State Food, and Drug Administration and other authorities as a strategy for the quality assessment of TCMI at the turn of the century [8, 9] in order to describe the macroscopic characteristics of all components in TCMI and demonstrate the “similarity” of various samples. Then in order to detect every potential phytochemical class of molecules, on the basis of several hyphenated ∗ Additional corresponding author: Professor Qiong-lin Liang, E-mail: [email protected]
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Figure 1. Roadmap of the integrated strategy on simultaneous qualitative and quantitative characterization of TCMs.
techniques, such as GC–MS, CE–MS, LC–MS, and MS/MS (MSn ), multidimensional fingerprinting have been proposed and applied in the analysis of TCMIs [10, 11]. On-line structure identification of ingredients can be performed by the retention time, UV spectra, m/z values and MS/MS fragmentation pattern, which will provide exact chemical composition information and be convenient to understand the relationships between the fingerprint and activities [12–15]. Meanwhile, multicomponent quantitative analysis with multiwavelength detection has been used to exhibit the “specificity” of different samples, which was summarized as microcosmic features [16–18]. We represent a powerful strategy as an integrated analysis of macroscopic characteristic (chemical fingerprint + on-line structure identification) and microcosmic features (multicomponent quantification) for the quality assessment of TCMIs as well as other complex TCM systems [19]. The application of on-line structure identification technology in the chemical fingerprint may make us understand the chemical composition well, which is a benefit for the selection of the quantified marker. The present study aims to describe and exemplify the integrated strategy as integrating qualitative and quantitative characterization of multicomponent for the quality control of TCMIs using the example of Danhong injection (DHI). The roadmap of this strategy is shown in Fig. 1. DHI, which is made of Radix Salviae Miltiorrhizae and Flos Carthami, is one of the commonly used TCMIs for the treatment of coronary heart disease and angina pectoris in China [20]. Lipophilic diterpenoid quinones and hydrophilic phenolic acids are the most representative components in Radix Salviae Miltiorrhizae; flavonoid glycosides and safflor yellow are the effective constituents in Flos Carthami with similar therapeutic effects to Radix Salviae Miltiorrhizae, such as facilitating blood circulation and dispersing blood stasis [21, 22]. Studies on the quality control of DHI have been reported previously, while most of them only focused on a few specific chemical marker compounds from Radix Salviae Miltiorrhizae [23, 24]. In order to develop a suitable quality control method, the TCM fingerprint and multicomponent quantification were usually used as the supporting technology in some reported studies on DHI. Liu et al. de C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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veloped a comprehensive quality evaluation method combining quantitative analysis with chromatographic fingerprint of Danhong injection using HPLC with diode array detection (DAD). However, the developed method only focused on the salvianolic acids from Radix Salviae Miltiorrhizae, which is short of the evaluation of components from Flos Carthami [25]. Liu et al. developed a dual-standard quality assessment method of Danhong injection by characterizing the antioxidant constituents in vivo. Salvianolic acids from Radix Salviae Miltiorrhizae with potent anti-oxidative activities were selected as key quality markers, while nucleosides from Flos Carthami (Hong-hua) exhibited weak anti-oxidative activities and were used as general quality markers. The bioactivityrelated quality assessment method provided a novel idea, while only a pharmacology indicator as the quality evaluation criteria of the traditional Chinese medicine prescription is not enough [26]. We have established the fingerprint evaluation method for the quality control of DHI previously [27]. In this work, an approach to screen and identify the main constituents in DHI by combining the accurate mass measurement of LC–TOF-MS and the complementary fragmentation data for structure confirmation provided by LC with ion trap MSn is described. Additionally, simultaneous quantitative analysis of seven markers in different batches of DHI, including uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B would be performed. The integrating qualitative and quantitative characterization strategy was subsequently applied to evaluate 20 batches of DHI and testified to be suitable for its quality control, which may provide an effective and reliable pattern for comprehensive and systematic characterization of the complex TCM system.
2 Materials and methods 2.1 Materials and reagents The reference compounds of uridine (1, LOT887– 200202), adenosine (2, LOT110879–200202), guanosine (3, LOTG6752), phenylalanine (4, LOT111615–200301), 5hydroxymethyl-2- furalcdehyde (5, LOT111626–201108), danshensu (6, LOT110855–200809), protocatechuic aldehyde (8, LOT110810–200506), caffeic acid (11, LOT110885– 200102), hydroxysafflor yellow A (10, LOT111637–201207), p-coumaric acid (18, LOT18279), rosmarinic acid (23, LOT111871–201203), salvianolic acid A (24), and salvianolic acid B (25, LOT 111562–201212) were purchased from the National Institute for Food and Drug control (Beijing, China). And the purity of these reference compounds was more than 98% to meet the need of content determination. HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany) and formic acid was obtained from Fluka (Buchs, Switzerland). Ultrapure water (18.2 M⍀) was prepared with a Milli-Q water purification system (Millipore Corporation, MA, USA). Other reagents were of analytical www.jss-journal.com
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grade. Twenty batches of DHI commercial products were supplied by Heze Buchang Pharmaceutical (Heze, China).
2.2 Preparation of standard solutions and sample solutions Each reference was accurately weighed, dissolved in methanol and deionized water, and diluted to the appropriate concentration. The mixed standard solution was prepared by dissolving standards with a fixed weight in methanol, and then stored at 4⬚C. The solutions were brought to room temperature, diluted, and filtered through a 0.22 m membrane filter before analysis. The samples of DHI for HPLC–DAD analysis were diluted to 1:5 v/v with ultrapure water, and then filtered through a 0.22 m membrane filter before injection; an aliquot of 10 L filtrate solution was injected for HPLC–DAD and LC– MS analysis.
The HPLC conditions for the LC–TOF-MS analysis were the same as the HPLC method, except for that one-third of the eluent was introduced into the TOF-MS system with a split valve. TOF-MS analysis was performed in both positive (ESI+ ) and negative (ESI− ) ion mode over m/z 50–1500 under the following operation parameters: capillary voltage 3500 V (ESI− ) or 4000 V (ESI+ ); drying gas 8.0 L/min; nebulizer pressure 40 psig; gas temperature 325⬚C; fragmentor voltage 175 V (ESI+ ) and 190 V (ESI− ); skimmer voltage 60 V; octopole dcl 37.5 V (ESI+ ) and –38.0 (ESI− ); octopole RF 250 V. The reference solution was used as a continuous calibration using the following reference masses: 121.0509 and 922.0098 m/z. Analyst QS software (Applied Biosystems, Framingham, MA, USA) was used to process the accurate mass data. Exact masses corresponding to particular elemental compositions were also calculated by the formula calculator in this software. Daily instrument tuning was carried out using the tuning solution (G1969–85000, Agilent, USA) to ensure no more than 5 ppm mass error prior to run samples. 2.4.2 Ion trap MSn system
2.3 HPLC apparatus and conditions HPLC–DAD analysis was performed using an Agilent 1100 Series HPLC–DAD system (Agilent Series 1100, Palo Alto, CA, USA) compressing a vacuum degasser, binary pump, autosampler, thermostatted column compartment, and a diode array detector, scanning from 200–400 nm, the wavelength was then selected and fixed at 254, 280, and 310 nm for qualitative and quantitative analysis, considering the variety of constituents in DHI. Separation was performed on an AlltimaTM C18 analytical column (250 mm × 4.6 mm i.d., 5 m, Grace Davison Discovery Science, USA) at 30⬚C. The mobile phase consisted of two solvents: 0.4% aqueous formic acid (solvent A) and acetonitrile (solvent B) with gradient elution (0–20 min, 3–12% B; 20–35 min, 12–20% B; 35–55 min, 20–38% B; 55–75 min, 38–80% B). Re-equilibration duration was 10 min between individual runs. The flow rate was kept at 1.0 mL/min and 10 L of sample solution was injected in each run [27].
2.4 LC–MS apparatus and conditions 2.4.1 TOF-MS system The above HPLC system was interfaced with an Agilent 1100 LC/MSD TOF equipped with an electrospray interface (Agilent, Waldbronn, Germany). The electrospray source includes dual nebulizers, one nebulizer for the LC eluent and the other for the internal reference solution. The reference nebulizer, along with the LC/MSD TOF’s automated calibrant delivery system, provides continuous introduction of reference mass standards into the ion source for automated mass calibration. Accurate mass measurements were obtained with this calibrant delivery system and thus enhanced accuracy was achieved. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The HPLC system was also connected to Agilent 1100 LC/MSD Trap (Agilent, Santa Clara, CA, USA) equipped with an electrospray interface to provide sufficient fragmentation data. The ion trap MS analysis was carried out in both positive and negative mode over m/z 50–1500 using the following operation parameters: capillary voltage 3500 V (ESI− ) or 4000 V (ESI+ ); skimmer voltage 40 V; capillary exit voltage: 137 V; nebulizer pressure 30 psig; drying gas: 8 L/min; gas temperature 325⬚C; target mass: 622 m/z; compound stability: 100%; trap drive level: 60%; threshold: 50 000 (ESI+ ) and 10 000 (ESI− ); ion charged control (ICC): on; target: 10 000; accumulation time: 200 ms. An amplitude voltage of 1.0 V was typically used for fragmentation in the ion trap auto MS2 and MS3 experiments. Data were processed by Agilent Chemstation Rev. A. 09.01 software (Agilent, Palo Alto, CA, USA).
3 Results and discussion 3.1 Optimization of HPLC conditions Column types, mobile phase compositions, gradient elution procedure, flow rate of the mobile phase, and column temperature were optimized respectively to achieve good separation of as many peaks as possible within a short-analysis time. The applicability of the established analysis method to different HPLC instrument systems has been verified. The detection wavelength was selected with the use of a DAD detector. In order to detect more common peaks while achieving precise detection of them, the most appropriate wavelength for fingerprinting was set as 254 nm. The detection wavelengths for quantitative analysis were selected according to the maximum adsorption wavelengths of uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B at 260, 260, 280, 280, www.jss-journal.com
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Figure 2. HPLC chromatograms of standard solutions as well as DHI at five detection wavelengths. HPLC chromatogram of mixed standards including uridine (1), adenosine (2), danshensu (3), protocatechuic aldehyde (4), p-coumaric acid (5), rosmarinic acid (6), and salvianolic acid B (7) detected at 260 nm. (B–F) HPLC chromatograms of DHI at five detection wavelengths: 260 nm (B), 280 nm (C), 286 nm (D), 310 nm (E), and 320 nm (F) for different markers.
310, 320, and 286 nm, respectively, shown in UV spectra with three-dimensional chromatograms of DAD, as shown in Fig. 2. The proposed method is therefore acceptable as well as adequate for further MS analysis. 3.2 Identity assignment and confirmation of the detected components in DHI The screening, identification, and further confirmation of components in DHI were performed by LC–TOF-MS to provide the elemental compositions of both the molecular ions and even characteristic fragment ions firstly. Formulae and available fragmentation were then checked by LC with ion trap MS3 to confirm the results of LC–TOF-MS. The total ion chromatograms of DHI were obtained by LC–TOF-MS in both positive and negative ion mode. Most of compositions showed their [M+H]+ and [M+Na]+ ions in positive ion mode, and exhibited quasi-molecular ions [M−H]− , adduct ion [M+HCOO]− in negative ion mode. MS/MS and MSn data were detected by collision-induced dissociation. Twentysix compounds were identified by interpreting their mass spectra obtained by their MS (TOF), MS/MS, and MSn (ion trap), as well as UV spectra, taking into account all the data provided by the literature (Fig. 3). Thirteen compounds were
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
accurately identified by comparing the retention time, UV spectra, and m/z values with those of the authentic standards. Information of the 26 components defined is displayed in Table 1 and their chemical structures are shown in Fig. 4 [12, 28–35]. The results exhibited excellent coherence with the two types of MS. The detailed identification of each group of components was outlined as followed. For identification of the nucleotides in DHI, extracted ion chromatograms provided by TOF-MS were applied since relatively lower signals of these compounds in the total ion chromatograms. By extracting ions at m/z 243, 268, 284, and 166, four nucleotides including uridine, adenosine, guanosine, and phenylalanine (peaks 1, 2, 3, 4) could be deduced and ultimately verified with authentic standards. TOF-MS provided us the accurate mass value of m/z 755.2041 and the probable formula of C33 H39 O20 for the peak 13. The proposed formula was searched using “SciFinder Scholar” database (CAS on-line, https://scifinder.cas.org) and several matching results were obtained in November 2011. Then the MS spectra from ion trap delivered a molecular mass of m/z 754.8 for this compound, together with two main diagnostic fragments at m/z 593.0 (loss of a glucosyl group, 162 mass units) and 284.8 (loss of a glucosyl group and arutinosyl moiety, 470 mass units), suggesting that peak 13 is a flavone diglycoside and the aglycone moiety should be
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Figure 3. HPLC–UV profile of DHI at 254 nm. The number of peaks marked in Fig. 3 is corresponding to Table 1.
kaempferol. Thus, peak 13 was identified as kaempferol-3-Orutinoside-7-glucopyranoside [29]. Peak 11 showed the same UV absorptions as peak 13 at 254, 267(sh), and 348 nm, and which was identified as 6-hydroxykaempferol-3,6,7-O-D-glucosidetentatively using this combinative method according to the accurate mass value and trap fragmentation data [30]. The MS spectrum of peak 10 exhibited a negative molecular ion at m/z 611.1, together with two diagnostic fragments at m/z 491 (loss of a p-vinylphenol group, 120 mass units) and 329 (loss of a p-vinylphenol group and a glucose, 282 mass units). This represents the precursor→product transition of [M-H]− → [M-H-120]− → [M–H–120–glu]− , which indicated the neutral loss of a p-vinylphenol group and then a glucose. Peak 10, thus, was identified as a hydroxysafflor yellow A [33], and the identification of this compound was ultimately verified with authentic standards. Fourteen phenolic acids mainly from Radix Salviae Miltiorrhizae were deduced in turn by comparing their retention times or MS spectra with those of authentic compounds or literature data [31,32,34–39]. The ESI-MS/MS spectra from ion trap of salvianolic acids exhibited the fragment ions derived from neutral loss of one or two molecules of danshensu moiety corresponding to 198 mass units. The fragment [M– 44–H]− corresponds to the loss of a CO2 molecule is also present in the spectra of most of the phenolics including caffeic acid monomer and its metabolites due to the existence of a –COOH group in all of these compounds. The other phenolic acids were characterized as in Table 1, referring to previous data reported in the literature. And peaks 6, 8, 11, 18, 23, and 25 were unambiguously characterized by comparison with reference compounds. In conclusion, 26 main components including nucleotides, phenolic acids, and flavonoid glycosides have been detected and identified in DHI. What need to be emphasized was that 5-hydroxymethylfurfural (Peak 5) may be a byproduct from the preparation of DHI, which has been verified when the established chemical profile was used for the production process monitoring the analysis of DHI.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.3 Method validation of quantitative analysis The precision and repeatability of the chemical profiling method by HPLC–DAD were validated by the reduplicate analysis of six injections of the same DHI samples and six parallel samples prepared using the same preparation protocol, respectively. The relative standard deviations of the peak retention time and area value were less than 5.0%. The resulting data showed that the precision and repeatability of the proposed method were satisfactory for chemical profiling analysis. The method validation of quantitative analysis is described below. 3.3.1 Calibration curves, LODs, and LOQs The HPLC method was validated defining the linearity, limits of quantification, and detection, identification and quantification of the analytes, repeatability, precision, stability, and recovery. All calibration curves were plotted based on linear regression analysis of the integrated peak areas (y) versus concentrations (x, g/mL) of the seven marker constituents in the standard solution at seven different concentrations. The regression equations, correlation coefficients, and linear ranges for the analysis of the seven marker constituents are shown in Table 2. The LOD was calculated as the amount of the injected sample that gave a single-to-noise ratio of 3 (S/N = 3), and the LOQ was calculated as the amount of the injected sample that gave a single-to-noise ratio of 10 (S/N = 3). The LOD and LOQ values of the method for the seven components are listed in Table 2. 3.3.2 Precision, repeatability, stability, and recovery Intra- and interday variations were chosen to determine the precision of the developed assay. For intraday variability test, the DHI sample solutions were analyzed for six replicates within one day, while for interday variability test, the solutions were examined in duplicate for three consecutive
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611.172 179.0304 431.1824 755.2041
C10 H14 N5 O4 C10 H14 N5 O5 C9 H12 NO2 C6 H7 O3 C9 H9 O5 C7 H5 O4 C7 H5 O3 C33 H41 O22
C27 H31 O16
C27 H31 O16 C18 H15 O8 C19 H17 O8 C19 H17 O8 C9 H7 O3 C27 H21 O12 C27 H21 O12 C20 H17 O10 C36 H29 O16 C18 H15 O8 C26 H21 O10 C36 H29 O16 C26 H21 O10
[M–H] − [M–H] − [M+H]+
[M–H] − C9 H7 O4 [M–H] − [M–H+HCOOH]− C20 H31 O10 C33 H39 O20
[M+H]+ [M–H] − [M+H]+ [M–H] − [M–H] −
[M–H] − [M–H] − [M+H]+ [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] − [M–H] −
7.0 9.3 11.9 12.5 14.6
17.2 22.5 24.7
2* 3* 4* 5* 6*
7 8* 9
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Caffeic acid Roseoside
Kaempferol-3-rutinoside-7glucopyranoside 4’,5,6,7-Tetrahydroxyflavanone6,7-di-O--D-glucoside Prolithospermic acid Methyl rosmarinate/isomer
11* 28.9 12 30.1
31.8
32.3
34.7 35.4
38.3
13
14
15 16
17
Alkannic acid/salvianolic acid J
42.6
45.0
46.9
20
21
22
Salvianolic acid A Salvianolic acid B
Salvianolic acid A isomer
24* 48.4 25* 50.4
26
493.1041
493.1022 717.132
359.0804
717.1309
417.0735
537.0949
163.0454 537.0947
373.0945
359.0804 373.0945
611.1579
153.0149 137.0214 789.2157
268.1007 284.0916 166.0840 127.0395 197.0411
493.1135
493.1135 717.1456
359.0767
717.1456
417.0822
537.1033
163.0395 537.1033
373.0923
359.0767 373.0923
611.1612
755.2035
179.0344 431.1917
611.1612
153.0188 137.0239 789.2089
268.1046 284.0995 166.0868 127.0395 197.0450
243.0617
–19.0066
–22.8598 –18.9092
10.3244
–20.4431
–20.7927
–15.6425
36.0685 –16.0149
5.7819
10.3244 5.7819
–5.4167
0.8350
–22.5316 –21.6211
17.6538
–25.3816 –18.0200 8.5544
–14.4690 –27.7861 –16.8816 7.7203 –19.7855
–17.3263
16.5
16.5 22.5
11.5
22.5
12.5
17.5
6.5 17.5
11.5
11.5 11.5
12.5
14.5
6.5 5.5
12.5
5.5 5.5 13.5
6.5 6.5 4.5 3.5 5.5
5.5
492.9 [M–H] −
492.9 [M–H] − 717.1 [M–H] −
719.0 [2M–H] −
717.1 [M–H] −
416.9 [M–H] −
537.0 [M–H] −
162.8 [M–H] − 537.0 [M–H] −
372.9 [M–H] −
359.2 [M+H]+ 373.0 [M–H] −
611.1 [M–H] −
754.8 [M–H] −
178.7 [M–H] − 431.0 [M–H+HCOOH] −
611.1 [M–H] −
153.0 [M–H] − 136.8 [M–H]− 789.3 [M+H]+
394.8 [2M–H]−
268.1 [M+H]+ 282.1 [M–H] − 166.1 [M+H]+
242.8 [M–H] −
627.2 [M+H–glu]+ 456.1 [M+H–glu–glu]+ 303.1 [M+H–glu–glu–glu]+ 490.9 [M–H–120]− 328.7 [M–H–120–glu]− 134.8 [M–H–COO] − 385.0 [M–H] − 223.0 [M–H–glu] − 593.0 [M–H–glu] − 284.7 [M–H–glu–rutin]− 448.9 [M–H–glu] − 286.8 [M–H–glu–glu] − 311.8 [M+H–COO]+ 178.8 [M–H–194]− 142.8 [M–H–194–2H2 O]− 178.8 [M–H–194]− 142.8 [M–H–194–2H2 O]− 118.9[M–H–COO] − 492.9 [M–H–COO] − 294.8 [M–H–COO–198] − 492.9 [M–H–COO] − 294.8 [M–H–COO–198] − 372.8 [M–H–COO] − 156.8 [M–H–COO–198] − 519.0 [M–H–198] − 320.8 [M–H–198–198] − 358.8 [M–H] − 160.7 [M–H–198] − 294.8 [M–H–198] − 518.9 [M–H–198] − 320.8 [M–H–198–198] − 294.8 [M–H–198] −
196.8 [M–H]− 178.7 [M–H–H2 O]−
150.1 [M–H–rib]− 120.0 [M+H–HCOO]+
199.7 [M–H–HNCO]− 109.8 [M–H–rib]−
MS2 –MS3 (m/z)
Danshen
Danshen Danshen
Danshen
Danshen
Danshen
Danshen
Honghua Danshen
Danshen
Danshen Danshen
Honghua
Honghua
Danshen Honghua
Honghua
Danshen Danshen Honghua
Honghua Honghua Honghua Byproduct Danshen
Honghua
Origin
Liquid Chromatography
a) Compared with authentic compounds; *a&b compounds were not detected in DAD detector, only found in TOF-MS detector; Honghua: Flos Carthami; Danshen: Radix Salviae Miltiorrhizae. “DBE”: “degree of unsaturation.”
52.7
Rosmarinic acid
23* 47.8
Salvianolic acid E
Salvianolic acid D
p-Coumaric acid Alkannic acid/salvianolic acid J
18* 39.0 19 41.5
Methyl rosmarinate/isomer
6-Hydroxysafflor yellow A
10* 27.5
Protocatechuic acid Protocatechuic aldehyde 6-Hydroxykaempferol-3,6,7-O-D-glucoside
Adenosine Guanosine Phenylalanine 5-Hydroxymethylfurfural Danshensu
243.0575
C9 H11 N2 O6
[M–H] −
Uridine
6.2
1*
Measured Calculated Error (ppm) DBE MS (pos/neg) mass (m/z) mass (m/z) MS (m/z)
Formula
Selected ion
No. tR (min) Identification
Table 1. Identification of multicomponent from DHI by LC–MS (MSn ) J. Sep. Sci. 2014, 37, 1438–1447
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Figure 4. The structures of components identified in DHI.
days. Variations were expressed by the RSD for intra- and interday, which were both less than 3.0%. Six different sample solutions prepared from the same sample were analyzed to confirm the repeatability of the developed assay. RSD values of the seven determined compounds’ contents were all less than 3.0%, which satisfied the criteria of quantitative analysis. For the stability test, peak areas of seven compounds in sample solution, which was stored at room temperature in the dark, were analyzed at 0, 4, 8, 12, 16, 24 h. RSD values of the contents of seven compounds were less than 3.0%, respectively. These results suggested that it was feasible to analyze
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the samples within one day. The percentage difference between amounts determined and spiked was considered to be a measure of accuracy. Known amounts (low, medium, and high) of the seven standard references were spiked into samples and then prepared as test solutions. The determination was performed in triplicate, and the average recoveries and RSD were calculated. The developed method had good accuracy with the overall recovery of 95.6 to 102.9%, with the RSD ranging from 0.42 to 3.36%. These results are listed in the Supporting Information, which indicates that the HPLC–UV
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Table 2. Regression equation, linear range, and LOD of the developed method
Compound
Regression equationa)
Correlation factors (r)
Linearity range (g/mL)
LOQ (ng/mL)b)
LOD (ng/mL)b)
Uridine Adenosine Danshensu Protocatechuic aldehyde p-Coumaric acid Rosmarinic acid Salvianolic acid B
Y = 2.42 × 103 X +2.65 Y = 3.08 × 103 X +3.26 Y = 0.66 × 103 X +5.53 Y = 4.12 × 103 X +7.60
1.000 1.000 1.000 1.000
1.078–107.8 1.36–136.2 8.46–1692 2.89–2890
6.67 × 10−3 1.46 × 10−2 9.17 × 10−1 3.38 × 10−2
2.00 × 10−3 4.39 × 10−3 2.75 × 10−1 1.02 × 10−2
Y = 6.81 × 103 X +22.5 Y = 2.71 × 103 X +7.93 Y = 1.12 × 103 X – 0.40
1.000 1.000 1.000
1.92–1922 2.51–2512 11.59–2318
1.06 × 10−2 1.12 × 10−1 1.08 × 10−1
3.19 × 10−3 3.36 × 10−2 3.23 × 10−2
a) Y and X are, respectively, the peak areas and concentrations (g/mL) of the analytes. b) The LOQ was defined as the concentration for which the signal-to-noise ratio was 10 and the LOD was defined as the concentration for which the signal-to-noise ratio was 3.
method is precise, accurate, and sensitive for the quantitative determination of seven components in DHI samples [40].
3.4 Chemical profiling and quantitation Chemical profiling and assay of 20 different commercial batches of DHI were carried out. A reference fingerprint of DHI was generated on the basis of the chromatogram fingerprints of all samples using the software of the similarity evaluation system and the peak from Salvianolic acid B was used as reference. And then the similarity of the chromatogram from a sample with the reference fingerprint was assessed by calculating the correlation coefficient and/or angle cosine value of the original data [41]. As shown in Fig. 5, the chemical fingerprint similarity of these determined samples ranged from 0.91 to 0.99, indication a good consistency among the 20 batches products in qualitative evaluation. As for the quantitative analysis, danshensu, and protocatechuic aldehyde were used as the marker components in
the enacted quality standard of DHI recorded in the National Standard for Chinese Patent Medicine. And some specific chemical marker compounds from Radix Salviae Miltiorrhizae were usually used as the quality evaluation indicators [25]. Characteristic components from Flos Carthami were ignored in most cases. Flavonoid glycosides, nucleosides, and safflor yellow are the effective constituents in Flos Carthami with some activities such as facilitating blood circulation and dispersing blood stasis. In this study, ten components originated from Flos Carthami had been detected and identified by LC–MS, including four nucleosides, four flavonoid glycosides, one safflor yellow, and one coumarin. As we know, for the selection of marker components, the representativeness of the derived medicinal materials, the determination accuracy and the availability of the reference substances were taken into account. Since the absence of reference substances of flavonoid glycosides, and the low content of 6-hydroxysafflor yellow A detected in DHI, two typical nucleosides (uridine and adenosine) as well as p-coumaric acid from Flos Carthami were selected as the marker components. Furthermore, four typical components hydrophilic phenolic acids from Radix Salviae Miltiorrhizae were also determined. The developed multiwavelength multicomponent determination method was subsequently applied to simultaneous determination of the seven marker constituents in 20 different commercial batches of DHI. The results are shown in Table 3. The quality fluctuations between batches were discussed using the parameter P. The closer the P value is to 100%, the better the consistency between batches. The formula is given as follows: P=
Figure 5. Box chart of contents of seven components and similarity from 20 batches of DHI. Uri, uridine (1); Ade, adenosine (2); DSS, danshensu (3); PA, protocatechuic aldehyde (4); p-C, p-coumaric acid (5); RA, rosmarinic acid (6); SaB, salvianolic acid B (7).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Ci Ci
× 100%
(1)
Where Ci denotes the measured concentration of certain component, while C¯ i denotes the average concentration of 20 batches of DHI. A box chart of the seven components from 20 batches of DHI, as well as the similarity of these samples was shown www.jss-journal.com
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Table 3. Contents of the seven active components and the fingerprint similarity in 20 batches of DHI
LOT
Content (g/100ml)
Similarity
uridine (1)
adenosine (2)
danshensu (3)
protocatechuic aldehyde (4)
p-coumaric acid (5)
rosmarinic acid (6)
salvianolic acid B (7)
90606 90934 91004 91106 91219 91221 91253 91255 100202 100121 100128 100141 100213 100231 100244 100306 100311 100347 100401 100426
80.19 84.38 79.62 77.67 69.91 74.37 78.36 76.97 75.12 64.56 74.00 69.65 73.76 64.91 69.97 52.61 61.42 70.89 68.96 79.52
8.27 14.73 14.09 12.97 17.52 14.21 13.73 12.70 17.32 11.78 12.58 13.46 16.20 14.20 15.64 12.65 13.27 15.40 20.27 15.21
1434.87 1225.86 1338.55 1348.11 1097.10 998.72 1271.99 1252.94 976.30 1022.82 1317.59 1216.45 1092.89 1192.43 941.88 862.85 905.92 1061.45 1002.39 1371.29
244.57 180.80 213.33 229.35 154.54 143.80 191.85 208.10 123.02 158.13 206.68 116.14 146.75 199.36 155.01 127.50 143.08 177.02 155.98 247.09
26.92 32.75 32.36 32.36 26.91 24.45 28.96 27.20 20.13 20.49 23.54 21.08 22.94 29.97 29.34 23.09 24.78 30.26 32.19 31.62
213.02 207.98 209.58 194.77 243.25 217.83 217.10 190.21 170.22 197.52 237.22 157.61 201.31 242.20 168.25 150.25 162.60 192.74 150.51 189.17
390.37 662.24 555.21 414.26 675.44 520.83 523.35 432.82 509.75 443.32 529.35 542.73 543.81 581.76 457.70 330.21 364.73 456.57 377.44 429.71
0.981 0.973 0.986 0.975 0.936 0.959 0.959 0.907 0.982 0.982 0.961 0.972 0.983 0.981 0.984 0.978 0.987 0.986 0.965 0.973
max min average S.D. RSD (%)
84.38 52.61 72.34 7.51 10.38
20.27 8.27 14.31 2.49 17.39
1434.87 862.85 1146.62 171.75 14.98
247.09 116.14 176.10 39.82 22.61
32.75 20.13 27.07 4.27 15.79
243.25 150.25 195.67 28.94 14.79
675.44 330.21 487.08 93.76 19.25
0.907 0.987 0.97 0.02 2.02
in Fig. 5. Different from the good consistency of 20 batches products in chemical fingerprint (similarity ranged from 0.91 to 0.99), the contents of these seven determined compounds varied greatly, the RSD (%) of contents ranged from 10.38 to 22.61%, especially protocatechuic aldehyde and salvianolic acid B. As reported previously, salvianolic acid B has been found to have potent anti-oxidative capabilities, as well as reduction of leukocyte-endothelial adherence, inhibition of inflammation and metalloproteinases expression from aortic smooth muscle cells, and indirect regulation of immune function [42]. Due to these activities, salvianolic acid B exhibited its great clinical impact on cardiovascular protection, and as a “key component” in many TCM prescriptions including DHI for cardiovascular diseases. So the fluctuation of its content would inevitably affect the therapeutic efficacy of DHI. Therefore, more attention should be paid to study the content change tendency of these compounds during the whole product process, and find out the key processing point and parameters may affect the content of these marker components. In addition, the integrative strategy also contained the simultaneous qualification and quantification analysis of other components in DHI without UV absorption, such as amino acids and sugars. So a precolumn derivatization method with o-phthaldialdehyde and fluorenylmethyl chloroformate C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(OPA-FMOC) to determine the contents of amino acids in DHI and the UV/Vis method to determine the contents of reducing sugars have also been established previously [43]. It is expected that more useful information about the overall quality of DHI would be provided to characterize the overall quality of DHI.
4 Concluding remarks A comprehensive quality control standard was usually compared to a “bright eyes,” which would be used to find out the variation of detected products and ensure the consistency of TCM product quality. So the integrality and specificity should be taken into account when develop a quality evaluation method. In this study, a reliable and powerful analytical method by using the integrative strategy of simultaneous qualification and quantification of multiple components for the comprehensive quality evaluation of a TCMI (Danhong injection) has been established. TCM fingerprinting together with the identification of unknown components on-line were used to character the holistic profiles of the DHI, the “similarity” of products from different manufactured batches was used to evaluate the “consistency,” and the structure information of 26 components in DHI may provide www.jss-journal.com
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detailed information for us to understand its efficacy as well as the safety. With the guidance of macroscopic characteristic research, seven main representative components from the two raw medicinal materials were used as the markers of multicomponent quantification determination, which was used to present the differences among the products. The macroscopic characteristic (chemical fingerprint) and microcosmic features (multicomponent quantification) of 20 batches DHI was subsequently described with the established method. The developed method has been testified to be an effective and reliable pattern for comprehensive and systematic characterization of DHI, and which is recommendable for the quality assessment of complex TCM systems.
[16] Hu, P., Luo, G. A., Zhao, Z. Z., Jiang, Z. H., Chem. Pharm. Bull. 2005, 53, 481.
This work was sponsored by the International Cooperation Projects of Ministry of Science and Technology (MOST) in China (No. 2010DFA32420) and the National Natural Sciences Foundation of China (No. 90917003, 81130066, 81102766). The authors acknowledge the manufacturer of Danhong Injection for supplying the test samples.
[23] Wang, S. M., LiShiZhen Med. Mat. Med. Res. 2006, 17, 989.
The authors have declared no conflict of interest.
5 References [1] Shi, H. S., Feng, J. C., Chin. J. Med. Hist. 1995, 25, 107. [2] Li, X. L., Song, L. G., Liu, Y., Bo, Y. J., J. Chin. Mater. Med. 2009, 34, 1326–1328. [3] Xie, P. S., Chen, S. B., Liang, T. Z., Wang, X. H., Tian, R. T., Upton R., J. Chromatogr. A 2006, 1112, 171–180. [4] Van Breemaen, R. B., Fong, H. H. S., Farnsworth, N. R., Chem. Res. Toxicol. 2007, 20, 577–582.
[17] Yan, S. K., Luo, G. A., Wang, Y. M., Cheng, Y. Y., J. Pharm. Biomed. Anal. 2006, 40, 889. [18] Ruan, M., Li, Y., Li, X., Luo, J. G., Kong, L. Y., J. Pharm. Biomed. Anal. 2012, 59, 184. [19] Luo, G. A., Wang, Y. M., Liang, Q. L., Xie, Y. Y., Fan, X. M., Tradit. Chin. Med. Mater. Med. 2011, 13, 1. [20] Sun, M., Zhang, J. J., Shan, J., Zhang, Z. H., Jin, C., Xu, Y. S., Wang, Y. L., Phytomedicine 2009, 16, 683. [21] Tai, M. H., Liu, L. M., Ma, R. Q., J. First Mil. Med. Univ. 2005, 25, 335. [22] Xin, Q., Li, X. F., Si, D. Y., Chin. Trad. Pat. Med. 2004, 26, 222.
[24] You, H. L., Xi, C. P., Lin, Y. Q., Xu, L. H., Qilu Pharm. Aff. 2011, 30, 139. [25] Liu, X. S., Wu, Z. Z., Yang, K., Ding, H. Y., Wu, Y. J., J. Pharm. Biomed. Ana. 2013, 73, 70. [26] Liu, H. T., Wang, Y. F., Olaleye, O., Zhu, Y., Gao, X. M., Kang, L. Y., Zhao, T., Biomed. Chromatogr. 2013, 27, 655. [27] Luo, J. M., Xiao, X., Liang, Q. L., Luo, G. A., Wang, Y. M., Chin. Trad. Pat. Med. 2011, 33, 1277. [28] Zhou, J. J., Xie, G. R., Yan, X. J., Dictionary of Phytochemical Compositions of Traditional Chinese Medicine, Science Press, Beijing 2010. [29] Djoukeng, J. D., Arbona, V., Argamasilla, R., Cadenas, A. G., J. Agric. Food Chem. 2008, 56, 11087. [30] Huang, H., Ji, L. X., Song, S. Y., Wang, J., Wei, N., Jiang, M., Bai, G., Luo, G. A., Phytochem. Anal. 2011, 22, 330. [31] Qiao, X., Han, J., Xu, M., Zhang, Z. C., Ye, M., Ye, G., Guo, D. A., J. Pharm. Bio. Anal. 2007, 54, 789–798.
[5] Khan, I. A., Life Science 2006, 78, 2033–2038.
[32] Qiao, X., Han, J., Zhang, Z. C., Sun, S. F., Li, H. F., Wang, B. R., Ye, M., Guo, D. A., Planta Med. 2008, 74, 1720–1729.
[6] Xia, J. F., Liang, Q. L., Luo, G. A., Sci. Sin. Chim. 2010, 40, 641.
[33] Li, F., Pu, R., Zhao, H. Y., Liu, X., Ma, C., Wang, B. R., Guo, D. A., J. Chin. Pharm. Sci. 2011, 20, 47.
[7] Tilton, R., Paiva, A. A., Guan, J. Q., Marathe, R., Jiang, Z. L., Eyndhoven, W. V., Bjoraker, J., Prusoff, Z., Wang, H. L., Liu, S. H., Cheng, Y. C., Chin. Med. 2010, 5, 30–36.
[34] Zhu, Z. Y., Zhang, H., Zhao, L., Dong, X., Li, X., Chai, Y. F., Zhang, G. Q., Rapid Commun. Mass Spectrom. 2007, 21, 1855.
[8] State Food and Drug Administration of China, Technical Requirements for the Development of Fingerprints of TCM Injections, Beijing 2000.
[35] Liu, A. H., Guo, H., Ye, M., Lin, Y. H., Sun, J. H., Xu, M., Guo, D. A., J. Chromatogr. A 2007, 1161, 170.
[9] World Health Organization, WHO Guidelines for the Assessment of Herbal Medicine, Munich 1991. [10] Luo, G. A., Wang, Y. M., Cao, J., Chin. Trad. Pat. Med. 2000, 6, 395. [11] Yan, S. K., Xin, W. F., Luo, G. A., Wang, Y. M., Cheng, Y. Y., J. Chromatogr. A 2005, 1090, 90. [12] Zhang, H. Y., Hu, P., Luo, G. A., Liang, Q. L., Wang, Y. L., Yan, S. K., Wang, Y. M., Chim. Acta. 2006, 577, 190. [13] Qu, J., Wang, Y. M., Luo, G. A., Wu, Z. P., J. Chromatogr. A 2011, 928, 155. [14] Qu, J., Wang, Y. M., Luo, G. A., J. Chromatogr. A 2001, 919, 437. [15] Huang, X., Song, F. R., Liu, Z. Q., Liu, S. Y., Chim. Acta. 2008, 615, 124.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[36] Hu, P., Liang, Q. L., Luo, G. A., Zhao, Z. Z., Jiang, Z. H., Chem. Pharm. Bull. 2005, 53, 677. [37] Hu, P., Luo, G. A., Zhao, Z. Z., Jiang, Z. H., Chem. Pharm. Bull. 2005, 53, 705. [38] Liu, M., Li, Y. G., Zhang, F. Yang, L., Chou, G. X., Wang, Z. T., Hu, Z. B., J. Sep. Sci. 2007, 30, 2256. [39] Xia, L., Liu, H. L., Li, P., Zhou, J. L., Qi, L. W., Yi, L., Chen, J. J. Sep. Sci. 2008, 31, 3156. [40] Fu, C., Luo, J. M., Wang, Y. M., Liang, Q. L., Luo, G. A., Chin. J. New Drugs 2012, 21, 2817. [41] Wang, X. M., Xie, Y. Y., Hu, X. M., Li, Y. K., Hu, P., Wang, Y. M., Liang, Q. L., Luo, G. A., Acta. Pharm. Sinica. B 2013, 3, 337. [42] Ho, J. H., Hong, C. Y., J. Biomed. Sci. 2011, 18, 30. [43] Pei, Y., Xiao, X., Fu, C., Ying, X. H., Liang, Q. L., Wang, Y. M., Luo, G. A., Chin. Trad. Pat. Med. 2012, 34, 1703. www.jss-journal.com
