Determination of Curcumol in Baofu Kangshuan by Capillary Gas Chromatography - Master's thesis - Dissertation

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This study presents a method for determining the curcumol content in Baofukang suppositories using capillary gas chromatography. Baofukang is a traditional Chinese medicine formulation included in the Chinese Pharmacopoeia, containing zedoary turmeric oil and borneol. Among the active components of zedoary turmeric oil, elemene, curcumol, and zedoary diketone are known for their anti-inflammatory, antibacterial, and anticancer properties. However, the Chinese Pharmacopoeia does not provide quantitative analysis for these compounds. This paper introduces a reliable and accurate method using gas chromatography to measure curcumol content, thereby enhancing the quality control standards of the preparation.

1. Instruments and Reagents

1.1 The GC7800 gas chromatograph (Puri) was used, equipped with a SE30 capillary column (30 m × 0.25 mm × 0.25 μm).

1.2 The reference material for the determination of alcohol content in the test drug was obtained from the China National Institute for the Control of Pharmaceutical and Biological Products. Zedoary turmeric oil was sourced from Zhejiang Rui'an Medicinal Oil Plant, and Baofukang suppositories were commercially available. All other reagents were of analytical grade.

2. Methods and Results

2.1 Chromatographic conditions: The column temperature was programmed as follows: initial temperature 130°C for 40 minutes, then increased at 32°C/min to 250°C, held for 16 minutes. The detector was a flame ionization detector (FID), with both the injection port and detector maintained at 280°C. Nitrogen was used as the carrier gas, with a column pressure of 0.5 kg/cm² and a split ratio of 1:100. The theoretical plate count was no less than 25,000 based on the peak of zedoary alcohol.

2.2 Internal standard stock solution: n-Tridecane and ethyl acetate were accurately weighed and dissolved in a 1 mg/mL solution, then thoroughly mixed.

2.3 Calibration factor determination: 3 mg of curcumol was accurately weighed and placed in a 10 mL volumetric flask. Then, 2 mL of internal standard solution was added, and the volume was adjusted with ethyl acetate. A 1 μL sample was injected into the gas chromatograph, repeated 3–5 times, and the average peak area was calculated to determine the correction factor.

2.3.1 Test solution preparation: Ten samples of the product were placed in a round-bottom flask connected to a volatile oil extractor. 5 mL of ethyl acetate was added to the side tube, and the oil was collected following the volatile oil extraction method. Every 2 hours, the ethyl acetate layer was separated, and another 5 mL of ethyl acetate was added. This process was repeated three times. The collected ethyl acetate layer was transferred to a 25 mL volumetric flask. After extraction, the volume was adjusted with ethyl acetate. Then, 2.5 mL of the solution was transferred to a 10 mL volumetric flask, 2 mL of internal standard was added, and the volume was made up with ethyl acetate. The resulting solution was used for analysis.

2.3.2 Separation and specificity: A blank preparation without zedoary turmeric oil was prepared according to the pharmacopoeia method. It was analyzed using the same procedure as 2.3.1, and no interference was observed. The resolution between curcumol, the internal standard, and other potential interferents was greater than 1.5, with symmetric peaks.

2.3.3 Linear relationship: A stock solution of 1 mg/mL was prepared, and 5.0, 4.0, 3.0, 2.0, and 1.0 mL were taken into 10 mL volumetric flasks. Each was diluted with 2 mL of internal standard solution and topped up with ethyl acetate. A series of standard solutions with concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL were prepared. Each solution was injected into the gas chromatograph, and the peak areas were measured. The calibration curve was plotted with the peak area ratio of curcumol to internal standard on the y-axis and the concentration of curcumol on the x-axis. The regression equation was found to be Y = 3.54X - 2.48×10⁻³, with a correlation coefficient r = 0.9995.

2.3.4 Recovery test: Known samples were prepared, and half the amount was quantitatively spiked with 5 mL of 3.0 mg/mL curcumol reference solution. The test solution was prepared according to 2.3.1, and the recovery rate was calculated. The average recovery was 102.3%, with an RSD of 2.92% (n=5).

2.3.5 Accuracy and stability: A 0.3 mg/mL reference solution was injected 5 times at 1-hour intervals. The peak area ratio of curcumol to internal standard was 0.845, with an RSD of 2.6%.

2.3.6 Reproducibility: Five samples from the same batch of Baofukang suppository were prepared and analyzed. The RSD was 2.9%.

2.3.7 Sample analysis: Three batches of samples were prepared and analyzed according to the method described in 2.3.1.

2.4 Discussion

2.4.1 In this experiment, it's crucial that the curcumol peak elutes before the temperature program is initiated. Otherwise, the resolution may be compromised. If the temperature increases too quickly, other components may elute later, leading to overlapping peaks and reduced resolution.

2.4.2 Borneol, another component in the formulation, is also amenable to gas chromatography. Under the current experimental conditions, it elutes early and cannot be separated from curcumol. However, if a multi-stage temperature-programmed gas chromatograph is used, simultaneous quantification could be possible. The chromatogram shows several well-separated, symmetric peaks, indicating potential for further research and optimization.