β-elemene (β-elemene) is an effective monomer extracted from the rhizome of Chinese medicine Wenzhuzhu (C.wenyujin Chen.etC. Ling) [1], and has anti-tumor effect [2]. There is no report on the determination method of β-elemene in biological fluids at home and abroad. For the first time, we established a method for separation and determination of β-elemene in biological samples by gas chromatography, and used this method to study the physiological disposal of this product in animals (to be published).
1 Materials and methods
1.1 Drugs and reagents β-elemene standard product, purity 98.8%, 1% β-elemene emulsion, batch number 940828, all donated by the Chinese Academy of Science and Technology Development Institute of Pharmaceutical Technology Development; Gift from the Institute of Chemical Physics; Ether: Shanghai Malu Pharmaceutical Factory, analytically pure; Carbon Disulfide, Shenyang Chemical Reagent Factory, analytically pure.
1.2 Animal Wistar rats, ♂, body weight: (180 ± 5) g, provided by the Laboratory Animal Center of Dalian Medical University.
1.3 Instrument Shimadzu GC-7AG gas chromatograph, hydrogen flame ionization detector (FID), ChromatopacC-R2A data processor; GCD-300B hydrogen generator (Beijing China HP Analytical Technology Institute).
1.4 Chromatographic conditions Capillary chromatographic column: stainless steel column (2.1m × 3 mm), stationary phase: 5% SE—30 / Gas Chrom Q 80 ～ 100 mesh; column temperature: 115 ℃, gasification chamber temperature 210 ℃, detector: FID; gas flow rate: high purity N2 30 mL / min, H2 40 mL / min, Air 400mL / min; range: 10; attenuation: 23; paper speed: 2.5 mm / min, injection volume 2 μL.
1.5 Preparation of standard solution Accurately weigh 50 mg of β-elemene standard product, place it in a 100 mL volumetric flask, dilute to the mark with carbon disulfide, and shake to obtain 0.05% β-elemene mother liquor. Accurately measure 10 mL of β-elemene mother liquor, place it in a 50 mL volumetric flask, dilute to the mark with carbon disulfide, and shake to obtain 0.01% β-elemene working solution. The mother liquor and working fluid are sealed and stored at -4 ℃.
1.6 Preparation of internal standard solution Accurately weigh 350 mg of n-tridecane, place in a 100 mL volumetric flask, dilute to the mark with carbon disulfide, and shake to obtain 0.35% internal standard mother solution. Accurately measure 10 mL of the internal standard mother liquor, place it in a 50 mL volumetric flask, dilute to the mark with carbon disulfide, and shake to obtain 0.07% of the internal standard working solution. After the mother liquor and working fluid are sealed, store at -4 ℃.
1.7 Biological sample preparation Take 1 mL each of rat plasma, fecal homogenate (1:20) and bile, add 10 μL of internal standard working solution, 3 drops of saturated sodium chloride solution, and 200 μL of 0.1mol / L sodium hydroxide solution After mixing, let stand at room temperature for 5 min, extract with ether (2 mL × 2), vortex to mix for 30 s, centrifuge (3000 r / min, 10 min), combine ether extracts, evaporate under constant temperature water bath at 35 ℃, use 100 μL carbon disulfide was dissolved, and 2 μL was injected for determination.
2 Biological sample standard curve and recovery rate: Take 1 mL each of rat blank plasma, fecal homogenate and bile, put them in glass centrifuge test tubes, add 10 μL of internal standard working solution each, and β-elemene working solution 2, 4. 6, 10, 20, 40, 80 μL, operate according to the above biological sample preparation method, dilute 100 μL under ice bath, and inject 2 μL. The linear regression of the peak area ratio (As / Ai, Y) of β-elemene to the internal standard to the concentration (X) of β-elemene shows that the standard curve of each biological sample passes through the origin and is within The linear relationship is good in the mL range. The recovery rate was calculated from the ratio of the peak area of ​​each point of the standard curve of each biological sample to the peak area of ​​the corresponding point of the absolute standard curve. The recovery rate of each sample was between 97.50% and 99.16% (Table 1).
Table 1 Biological sample regression equation and recovery rate (n = 3)
Biological sample regression equation
r
Recovery rate ± S /%
plasma
Y ï¼ 0.5240X + 0.0123
0.9999
99.16 ± 1.6
Fecal homogenate
Y ï¼ 0.5372X + 0.0042
0.9998
98.64 ± 4.6
bile
Y ï¼ 0.5260X + 0.0056
0.9998
97.50 ± 3.4
3 Precise preparation of different concentrations of β-elemene biological samples for intra-day and intra-day precision experiments. The RSD of As / Ai of each biological sample was less than 10% (Tables 2 and 3). The detection limit of the method is 2ng / 2 μL (S / N = 2), which is equivalent to 0.1 μg / mL plasma.
Table 2 Intra-day precision of biological samples (n = 5)
concentration/
(μg / mL)
RSD /%
Plasma fecal homogenate bile
0.1
8.39
8.24
8.73
1.0
3.99
5.72
4.24
4.0
2.60
3.14
2.07
4 Methods Specific rat iv β-elemene milk 75mg / kg, plasma, fecal homogenate and bile samples were prepared and measured. The results show that the tR of the internal standard and β-elemene are 8.4 and 13.3 min, respectively, which is consistent with the standard. The two are well separated and are not interfered by impurities in the biological sample, indicating that the analyte in each biological sample It is a β-elemene prototype (Figures 1 to 3).
Table 3 Daytime precision of biological samples (n = 5)
concentration/
(μg / mL)
RSD /%
Plasma fecal homogenate bile
0.1
5.95
9.99
8.03
1.0
7.98
7.96
6.16
4.0
2.20
6.41
5.75
Figure 1 Chromatograms of rat blank plasma (A), blank plasma + internal standard + β-elemene standard (B), rat iv β-elemene plasma (C)
1. Internal standard 2. β-elemene
Figure 2 Chromatograms of rat blank bile (A), blank bile + internal standard + β-elemene standard (B), and rat iv β-elemene bile (C)
1. Internal standard 2. β-elemene 3. Unknown â…
Figure 3 Chromatograms of rat blank fecal homogenate (A), blank fecal homogenate + internal standard + β-elemene standard (B), rat iv β-elemene post-fecal homogenate (C)
1. Internal standard 2. β-elemene 3. Unknown Ⅱ 4. Unknown Ⅲ
5 Discussion In this paper, a method for separating and determining β-elemene in biological samples by gas chromatography is established for the first time at home and abroad. In the experiment, ethanol and cyclohexane were used as volume stabilizers, but they all showed long retention time, wide peak shape, and serious tailing, which affected the measurement. After that, carbon disulfide was used as the volume stabilizer. Narrow, to avoid affecting the measurement due to the solvent peak is too wide, and shorten the measurement time, improve sensitivity and work efficiency. The addition of saturated sodium chloride to the plasma sample can increase the polarity, reduce emulsification, and improve the recovery rate. The addition of sodium hydroxide eliminates the organic acid impurities in the plasma that are prone to peak in gas chromatography and reduces its interference with the determination. This method is simple, sensitive, reproducible and specific, and can meet the needs of kinetic research (this method has been used to study the pharmacokinetics and physiological treatment of β-elemene in animals, to be published).
references
1 Guo Yong
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