Analysis of the fatty acids from Periploca sepium by GC-MS and GC-FID

Analysis of the fatty acids from Periploca sepium by GC-MS and GC-FID Ling Tong, Lei Zhang, Shuanghui Yu, Xiaohui Chen, Kaishun Bi * Department of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China A simple and rapid gas chromatographic (GC) method with flame ionization detection was developed for the analysis of fatty oil in Periploca sepium. After methyl-esterification, nine components were identified by GC-MS. The derivatization conditions were investigated in order to validate this method. Palmitic acid, oleic acid and linoleic acid were determined simultaneously. The calibration curves were linear in the range of 0.050 0.250 mg ml -1 for palmitic acid, 0.019 0.190 mg ml -1 for oleic acid and 0.087 1.044 mg ml -1 for linoleic acid, respectively. The average recovery of palmitic acid was 99.2 % (RSD = 3.3 %); oleic acid, 99.4 % (RSD = 2.6 %); linoleic acid, 100.2 % (RSD = 2.9 %). The method can be used for the analysis of the fatty acids from Periploca sepium. Key Words: gas chromatography-flame ionization detector; methyl-esterification; fatty acid; Periploca sepium Introduction Periploca sepium is officially listed in the Chinese Pharmacopoeia and has been widely used as a tonic for at least two thousand years. Chemical studies have shown that it mainly contains cardenolides, pregnane glycosides and volatile components [1-3]. Most of the volatile components are long chain unsaturated fatty acids which play very important roles in biological systems because they are structural elements of many valuable compounds as well as being important sources of energy [4]. In recent years, increasing research has been carried out on fatty acids and the results obtained show that they possess significant sedative and hypnotic effects [5]. Although many techniques have been developed for the analysis of fatty acids analysis, GC is still the most widely used. This well-established procedure coupled with flame ionization detection (GC-FID) is * Author to whom correspondence should be addressed. Tel: +86-24-23986016; E-mail: bikaishun@yahoo.com.cn Received: 2007-04-10 Accepted: 2007-07-08 very efficient and rapid when complex mixtures with a broad range of molecular weights have to be analyzed. The separation and analysis of long-chain fatty acid mixtures has been widely used to obtain information about many biological systems. However, the inherent low volatility of fatty acids has been a problem. Consequently, they are converted into more volatile derivatives (such as fatty acid methyl esters) when they are analyzed by GC. No accurate determination of the components in Periploca sepium has been reported so far. Fatty acids are among the main constituents of Periploca sepium which exhibits a variety of pharmacological activities and, consequently, there is a need for a new method for the analysis of these fatty acids. Experimental Materials Methanol, boron trifluoride in ether (47.0 % 47.7 % v/v), sodium hydroxide, hexane, and eicosane were of analytical grade and were purchased from Yu-Wang Chemical Company (Shandong). Palmitic acid methyl ester, oleic acid methyl ester and linoleic 110

acid methyl ester with a purity greater than 99 % were supplied by J & K Chemica. The herb sample was collected from Liaoning province in 2006, and authenticated as Periploca sepium by Prof. Qishi Sun (Shenyang Pharmaceutical University). GC-MS conditions The GC-MS system consisted of a Shimadzu GC-17A chromatograph, a Shimadzu QP5050A mass spectrometer and a Class-5000 data system. Compounds were separated on a 30 m 0.25 mm i.d. DB-1 capillary column coated with a 0.25 μm film of dimethyl polysiloxane. The temperature of the non-polar column was 150 ºC after injection then programmed at 5 ºC min -1 to 250 ºC, and maintained at that temperature for 5 min. Split injection was conducted with a split ratio of 10:1, the flow-rate was, 1.0 ml min -1, helium was used as the carrier gas and the injector temperature was 250 ºC. The MS detection conditions were as follows: interface temperature, 230 ºC; ionization mode, EI + ; electron energy, 70 ev; full scan acquisition mode; mass range, 33-450 amu. Compounds were identified by using online NIST-library spectra and published MS data. GC conditions GC analysis was performed on an Agilent 6890 Gas Chromatograph equipped with FID. Compounds were separated on 30 m 0.25 mm i.d. DB-17 capillary column. The initial temperature of the polar column was 120 ºC then it was programmed to 190 ºC at 2 ºC min -1, and maintained at 190 ºC for 8 min. Split injection was conducted with a split ratio of 100:1, nitrogen was used as a carrier gas at a flow-rate of 1.00 ml min -1 and the injector temperature was 250 ºC and the detector temperature was 250 ºC. Palmitic acid methyl ester, oleic acid methyl ester and linoleic acid methyl ester were identified by co-injection of reference standards and comparison of their retention times. Extraction of fatty acids The pulverized samples (100.0 g) were extracted under reflux with 500 ml petroleum ether for 8 h and then filtered. The petroleum ether was evaporated to dryness and the yield of the fatty acid was 2.2 %. Then the fatty oil was prepared for derivatization. Methylation of fatty acids Fatty acid samples (100 mg) were dissolved in 5 ml 0.5 % (w/v) sodium hydroxide in methanol in a 25 ml flask with a ground-glass stopper and boiled under reflux for 45 min. Then, 2.0 ml boron trifluoride-ether solution was added and the solution boiled for 5 min. After this, 5 ml hexane was added and the solution boiled for 5 min. The solution was cooled, then a quantity of saturated sodium chloride was added and shaken for about 10 s. After this, 1.0 ml of the upper phase was collected and 0.5 ml eicosane solution was diluted to 5 ml with hexane to provide a sample solution. Results and discussion Optimization of methylation conditions In this study, the concentration of sodium hydroxide, hydrolysis time and esterification time had an effect on the yield of the derivatives. In order to obtain the optimal conditions, the above factors were investigated. The contents of target compounds were selected in order to investigate optimal condition as the assessment indexes of derivatization conditions. Nine experiments were performed. According to the results obtained, the optimal derivatization conditions were as follows: A fatty acid sample was dissolved in 5 ml 0.5 % (w/v) solution of sodium hydroxide in methanol in a 25 ml flask with a ground-glass stopper and boiled under reflux for 45 min. Then, 2.0 ml boron trifluoride-ether solution was added and the solution boiled for 5 min. Identification of nine major fatty acids A 1 μl sample was injected manually into the GC- MS system and analyzed under full-scan acquisition mode. Palmitic acid, oleic acid and linoleic acid esters 111

were identified by comparing their retention times and mass spectra with those obtained from the injected standards. The identification of the other seven fatty acids was accomplished by the aid of an MS library. The nine characterized components with their respective retention times and percentage contents are listed in Table 1. Calibration curves The standard solution and 1 μl sample solution were injected into the GC. The chromatograms are shown in Fig. 1. The representative GC-FID chromatogram of mixed standards (A) and samples (B). Stock solutions of palmitic acid (2.640 mg ml -1 ), oleic acid (1.896 mg ml -1 ) and linoleic acid (1.741 mg ml -1 ) were prepared. Calibration curves were calculated by plotting the peak area ratios of the analyte to internal standard versus the analyte concentration. Linear relationships between the concentration and the corresponding peak area ratio were found over the range of 0.050 0.250 mg ml -1 for palmitic acid, 0.019 0.190 mg ml -1 for oleic acid and 0.087 1.044 mg ml -1 for linoleic acid, respectively. The regression equations were as follows: Y = 13.28 X 0.004 r = 0.999 8 (palmitic acid) Y = 9.716 X 0.039 Y = 7.779 X 0.008 r = 0.999 3 (oleic acid) r = 0.999 2 (linoleic acid) Table 1 Compounds Identified in the Test Sample Peak No. Compound Percentage contents (%) 1 Tetradecanoic acid 0.8 2 Pentadecanoic acid 0.1 3 Palmitic acid 40.2 4 11-Hexadecenoic acid 3.5 5 Stearic acid 1.4 6 Oleic acid 7.0 7 8-Octadecenoic acid 2.5 8 Linoleic acid 30.7 9 Linolenic acid 3.8 The LOQ of palmitic acid, oleic acid and linoleic acid was 0.050 mg ml -1, 0.019 mg ml -1, 0.087 mg ml -1, respectively. Precision, repeatability and recovery The intra-day precision was tested using the results of six replicate injections of the standard solution containing the three components. The relative standard deviation (RSD) was found to be 0.5 % for palmitic acid, 0.6 % for oleic acid and 0.7 % for linoleic acid. The repeatability of the total procedure was examined by extracting and methylating six individual samples as described above. The contents were Time (min) (A) Time (min) (B) Fig. 1 The representative GC-FID chromatogram of mixed standards (A) and sample (B). The peaks are palmitic acid (a), oleic acid (b), linoleic acid (c) and internal standard (s), respectively. 112

Table 2 Contents of palmitic acid, oleic acid and linoleic acid in Periploca sepium Origin Contents / % Palmitic acid Oleic acid Linoleic acid Yuci, Shanxi 0.076 0.052 0.274 Lanzhou, Gansu 0.067 0.044 0.212 Xi'an, Shǎnxi 0.056 0.033 0.184 Baoding, Hebei 0.059 0.039 0.247 Hangzhou, Zhejiang 0.055 0.049 0.204 Anhui, Shandong 0.049 0.034 0.186 Changchun, Jilin 0.041 0.028 0.164 Jiaozuo, Henan 0.044 0.029 0.172 Daqing, Heilongjiang 0.081 0.064 0.299 Shenyang, Liaoning 0.083 0.070 0.343 Fushun, Liaoning 0.082 0.069 0.340 calculated and the RSD was 2.1 %, 4.4 % and 2.9 % for palmitic acid, oleic acid and linoleic acid, respectively. The recovery experiment was carried out to evaluate the accuracy of the method, which was defined as the percentage of the spiked amount retrieved. Nine sample solutions were prepared by abstraction of the plant materials, and a selected percentage of the three standards was added. The average recovery of palmitic acid, oleic acid and linoleic acid was 99.2 % (RSD = 3.3 %), 99.4% (RSD = 2.6 %) and 100.2 % (RSD = 2.9 %), respectively. Application The newly developed analytical method was successfully applied to the simultaneous determination of palmitic acid, oleic acid and linoleic acid in 11 samples of Periploca sepium obtained from different provinces and cities in China. The percentage contents of these compounds in Periploca sepium of different origins are listed in Table 2. From the results presented in Table 2, it was found that linoleic acid was the main component with a content which ranged from 0.164 % to 0.340 %. A similar variation was also found for the other two components. The reason for this was mainly due to differences in plant origin and geographical sources. Furthermore, some other factors, such as the drying process and storage conditions, could also affect the contents of these compounds. So, it was necessary to develop an accurate and effective method to assess the quality of Periploca sepium by simultaneous quantification of the major fatty acids. Discussion To assay the fatty acids from Periploca sepium with good resolution and a reasonable elution time, different capillary columns, including DB-17, DB-5 and DB-1, and temperature programs were investigated. It was found that a good resolution was obtained on a DB-17 column with a sample temperature program. Hence, the DB-17 column was used to analyze the fatty acids from Periploca sepium. This was the first time that a GC method had been developed for analysis of the fatty acids in Periploca sepium. The method allowed excellent resolution, high recoveries and good reproducibilities. It can be used as a quality control method for medicinal herbs and preparations containing fatty acid from Periploca sepium. References [1] Deng SX, Wang DC, Wang MD, et al. The cardiac effect of Periploca sepium. Acta Pharmaceutica Sinica, 1964, 11: 75 [2] Hedeji I, Xu J P. Pregnane Glycoside from an Antitumour Fraction of Periploca sepium. Phytochemistry, 1988, 27: 113

1173 [3] Shi QH, Ma YM, et al. Preliminary Study on Chemical Composition and Insecticidal Activity of Root Bark from Periploca sepium. Acta Agriculturae Boreali-occidentalis Sinica, 2005, 14: 141-144 [4] Mu YM, Yanass T, et al. Saturated FFAs, palmitic acid and stearic acid, induced apoptosis in human granulose cells. Endocrinologyl. 2001, 142: 3590-3597 [5] Zhang QX, Wang QL, and Han JH. Effect of fatty oil in Periploca sepium on neural system in mice. Xi an Med Univ, 1995, 16: 43-44 114