J. Cent. South Univ. Technol. (2007)01-0057-05

DOI: 10.1007/s11771-007-0012-5               

Analysis of volatile chemical components of Radix Paeoniae Rubra by gas chromatography-mass spectrometry and chemometric resolution

LI Xiao-ru(李晓如), LAN Zheng-gang(兰正刚), LIANG Yi-zeng(梁逸曾)

(School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China)

Abstract: The volatile chemical components of Radix Paeoniae Rubra (RPR) were analyzed by gas chromatography-mass spectrometry with the method of heuristic evolving latent projections and overall volume integration. The results show that 38 volatile chemical components of RPR are determined, accounting for 95.21% of total contents of volatile chemical components of RPR. The main volatile chemical components of RPR are (Z, Z)-9,12-octadecadienoic acid, n-hexadecanoic acid, 2-hydroxy- benzaldehyde, 1-(2-hydroxy-4-methoxyphenyl)-ethanone, 6,6-dimethyl-bicyclo[3.1.1] heptane-2-methanol, 4,7-dimethyl-benzofuran, 4-(1-methylethenyl)-1-cyclohexene-1-carboxaldehyde, and cyclohexadecane.

Key words: Radix Paeoniae Rubra; volatile chemical components; gas chromatography-mass spectrometry; heuristic evolving latent projections

1 Introduction

Radix Paeoniae Rubra (RPR), one of the common traditional Chinese medicines (TCMs) of buttercup plant, is the dried root of Paeonia lactiflora Pall and Paeonia veitchii Lynch, and has the function of removing pathogenic heat from the blood and dispating blood stasis and relieving pain^[1]. LANG et al^[2] studied its chemical components, but its volatile chemical components has not been found so far. Usually, the gas chromatography-mass spectrometry (GC-MS) method can be used to analyze the volatile chemical components of TCMs. But TCMs usually contain too many natural compounds, and the components in TCMs are very complicated, the retention time of some similar com- ponents is very approximately similar. The hyphenated chromatography instruments combined with the related chemometric methods provide powerful tools for the resolution of such complex systems. Heuristic evolving latent projections (HELP) is the effective method to analyze the multidimensional data^[3-5], and has been successfully employed to analyze the volatile components in single herbs such as Rhizoma Aractylodis, Cortex Magnoliae officinalis and recipe peptic powder^[6-18]. In this study, the volatile components in RPR were extracted and then detected by GC-MS, and the obtained two-dimensional data were analyzed by HELP method. The pure gas chromatography spectra and mass spectra were obtained according to the retention time and MS, and the similar searching and qualitative analysis were carried out and then the method of overall volume integration was used to quantitatively analysis.

2 Theory

Here concise theoretical explanation is shown for the sake of brevity^[3-5].

According to the Lambert-Beer law, a two dimensional data X_m×n produced by the GC-MS can be expressed as follows:

X_m×n=CS ^T+E

X_m×n= (i=1, 2, 3, …)

where  X_m×n denotes an absorbance matrix representing N components of m chromatographic scan points at n atom mass units(amu) or wavelength points, C is the pure chromatographic matrix,          S is the pure mass spectral matrix, and E denotes the noise.

The unique resolution of a two-dimensional data into chromatograms and spectra of the pure chemical components is obtained with HELP method. The whole procedure goes in the following steps:

1) Pretreat the original matrix. The measured matrix is divided into different submatrices by zero component regions. Then, subtract the background out using method based on principal component analysis;

2) Establish a rank map and determine number of pure components in the target cluster;

3) Estimate the pure spectra of each component indicated by the rank map with the help of HELP method;

4) Verify the reliability of the resolved results;

5) Qualitative analysis is performed by similarity searches in the National Institute of Standards and Technology (NIST) mass database and quantitative results are obtained by the overall volume integration method.

3 Experimental

3.1 Instrumentals and reagents

The 6890/5973N GC-MS spectrometer was employed in this experiment. The single herb was purchased from Chongqing municipal medicine company, and identified to be the dry root and rootstock of Radix Paeoniae Rubra (RPR) by a researcher from Institute of Materia Medica, Hunan Academy of Traditional Chinese Medicine and Materia Medica.

3.2 Extraction of essential oil

50 g RPR powder was put into extraction apparatus and 350 mL water was added. The essential oil was prepared according to the Chinese pharmacopoeia (2000 version)^[19]. 0.9 mL canary clear oil-like essential oil was gained, and the obtained essential oil was dried over anhydrous sodium sulfate and stored in fridge for the following analysis.

3.3 Analytical conditions

Chromatogram conditions were as follows: a capillary column with 30 m in length and 0.25 mm in inner diameter was used. Temperature-increasing procedure: started at 60 ℃, then heated at 4 ℃/min to 250 ℃, maintained for 5 min. Carrier gas was He kept at 0.2 mL/min flow-rate; The inlet temperature was    250 ℃，and the interfacial temperature was 280 ℃. The mass spectrometer conditions: the spectrometer was operated in electron-impact (EI) mode, the scan range was 20-400 amu, the ionization energy was 70 eV and the scanning rate was 3.8 scan/s, the temperature of ionization source was kept at 230 ℃.

3.4 Data analyses

Data analyses were performed on a Pentium III 850 (Intel) personal computer, and all programs were coded in Matlab 6.1 for windows. Resolved spectra were identified by matching with the standard mass spectral database of NIST.

4 Results and Discussion

4.1 Qualitative analysis of essential oil

Fig.1 shows the total ionic chromatogram (TIC) curve of volatile chemical components of RPR. It is partly overlapped by some peaks that look like the pure peaks, indicating the RPR essential oil system is very complex. If we analyze the TIC curve according to the NIST MS database, the qualitative result will not very accurate, or some hided peak will be failed to check. Or the low-similarity results will be obtained, so the analyzing can’t be performed.

Fig.1 TIC curve of volatile chemical components of RPR

Now take peak cluster A as an example to show the analytical proceeding with HELP method^[3-15]. The retention time of peak cluster A is between 16.21 min and 16.39 min, as shown in Fig.2. By searches directly in the database of the mass spectra, the left part of the peak cluster A  is 4-(1-methylethyl)-1- cyclohexene- 1-carboxaldehyde (C₁₀H₁₆O) with similarity 60%; the middle part of  the peak cluster A is  p-tert- butyl-phenol (C₁₀H₁₄O) with similarity 70%; the right part of the peak cluster A is 2-methyladamantane (C₁₁H₁₈) with similarity 62%。The peak cluster A, however, can be identified into 3 components using HELP method (shown in Fig.3): 1) Tridecanol (see Fig.4) with similarity 97.14%; 2) 3-hexyl-cyclopetene (see Fig.5) with similarity 96.22%; 3) 6-methyl-spiro[4.5]decan-6-ol (see Fig.6) with similarity 93.73%.

As the same as the analytical procedure of peak cluster A, the other clusters in the TIC curve can be analyzed, one by one, by HELP method. The pure mass spectral curve of the each component can be obtained with HELP method, and then the quantitative search of mass spectra in the MS database is adopted to identify each component. The results are listed in Table 1. Because the pure mass spectrum of each constituent can be obtained by HELP method, so the accuracy and reliability of qualitative analysis can be improved greatly.

Fig.2 TIC of peak cluster A during 16.21-16.39 min

Fig.3 Resolved chromatograms of chemical components in peak cluster A

1—Tridecanol; 2—3-hexyl-cyclopetene;   3—6-methyl-spiro[4,5] decan-6-ol

Fig.4 Resolved mass spectrum(a) and standard mass spectrum(b)of tridecanol

Fig.5 Resolved mass spectrum(a) and standard mass spectrum (b)of 3-hexyl-cyclopetene

Fig.6 Resolved mass spectrum(a) and the standard mass  spectrum (b)of 6-methyl-spiro[4.5]decan-6-ol

4.2 Quantitative analysis of volatile components

The overall volume integration method was used for all the chromatogram peaks in order to obtain the quantitative results of each component. There are 38 components in RPR, and the qualitative chemical constituents are 95.21% of the total content. The main volatile chemical components in RPR are (Z, Z)-9,12- octadecadienoic acid (30.11%), n-hexadecanoic acid (20.18%), 2-hydroxy-benzaldehyde(17.10%), 1-(2- hydroxy-4-methoxyphenyl)-ethanone (5.99%), 6,6- dimethyl-bicyclo[3.1.1]heptane-2-methanol (3.39%), 4, 7-dimethyl-benzofuran (2.41%), 4-(1-methylethenyl)-1- cyclohexene-1-carboxaldehyde (1.55%),  and cyclohe- xadecane (1.46%).

Table 1 Main volatile chemical components of RPR

5 Conclusions

1) 38 volatile chemical components of RPR are analyzed qualitatively and quantitatively by GC-MS together with HELP, accounting for 95.21% of the total contents.

2) The main volatile chemical components in RPR are (Z, Z)-9,12-octadecadienoic acid (30.11%), n-hexade-

canoic acid (20.18%), 2-hydroxy- benzaldehyde (17.10%),

1-(2-hydroxy-4-methoxyphenyl)-ethanone (5.99%), 6,6- dimethyl-bicyclo[3.1.1] heptane-2-methanol (3.39%), 4,7-dimethyl-benzofuran (2.41%), 4-(1-methylethenyl)- 1-cyclohexene-1-carboxaldehyde (1.55%) and cyclohexa-decane (1.46%).

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(Edited by YANG You-ping)

Foundation item: Project(20235020) supported by the National Natural Science Foundation of China

Received date: 2006-05-15; Accepted date: 2006-07-29

Corresponding author: LI Xiao-ru, Professor; Tel: +86-731-8836376; E-mail: xrli@mail.csu.edu.cn