Headspace gas chromatography method for detecting residual quantity of ethanol in gastrodin

Abstract

The invention discloses a headspace gas chromatography method for detecting ethanol residual quantity in gastrodin. The headspace gas chromatography method comprises the following steps: (1) preparing a test solution; preparing a reference solution; (3) performing chromatographic determination; and (4) calculating a result. According to the headspace gas chromatography method for detecting the residual amount of ethanol in gastrodin, the headspace equilibrium temperature and time are optimized, a chromatographic column with specific polarity and programmed heating conditions are adopted, the ethanol residue detection method special for a gastrodin matrix is established, matrix interference is effectively overcome, and the detection accuracy is high. The method has high sensitivity and high accuracy, realizes exclusive, accurate and reliable quantitative control of ethanol residues in gastrodin, and has strong practicability.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a headspace gas chromatography method for detecting ethanol residues in gastrodin. Background Technology

[0002] Gastrodin is an active ingredient extracted from the orchid Gastrodia elata and is widely used in the treatment of nervous system diseases. Ethanol is often used as a solvent in its synthesis or refining process, therefore the residual ethanol content in the finished product must be strictly controlled.

[0003] Headspace gas chromatography is a commonly used technique for detecting residual solvents. Although existing techniques exist for the detection of ethanol in certain drug matrices, they typically employ higher headspace equilibrium temperatures (e.g., 70°C) and shorter equilibrium times (e.g., 20 minutes).

[0004] However, gastrodin, as a glycoside compound, is quite sensitive to heat, and its physicochemical properties differ from those of conventionally synthesized drugs. Directly applying existing methods would present two major technical challenges: Specificity issues: At higher headspace equilibrium temperatures (e.g., ≥70℃), gastrodin molecules are prone to pyrolysis or interaction, generating unknown volatile impurity peaks. These impurity peaks can severely interfere with the accurate quantification and qualitative analysis of ethanol.

[0005] Sensitivity issue: Under conventional headspace equilibrium times (e.g., ≤30 minutes), ethanol is not completely released from the gastrodin lattice or intermolecular forces, resulting in a significant decrease in the method's sensitivity, which fails to meet the pharmacopoeia's requirements for the detection of trace residues.

[0006] Therefore, there is an urgent need in the field for a method for determining ethanol residues that is specifically designed for gastrodin matrix and can simultaneously overcome high matrix interference and achieve high-sensitivity detection. Summary of the Invention

[0007] This invention provides a headspace gas chromatography method for detecting ethanol residues in gastrodin, which can solve the above-mentioned problems existing in the prior art.

[0008] To address the aforementioned technical problems, this invention provides a headspace gas chromatography method for detecting ethanol residues in gastrodin, comprising the following steps: (1) Preparation of test solution: Take gastrodin test sample, dissolve it in water and place it in headspace bottle, seal it to prepare test solution; (2) Preparation of reference solution: Take ethanol reference standard, dilute with water to a concentration of 0.1-0.5 mg / mL, place in headspace vial, seal, and prepare reference solution; (3) Chromatographic determination: The headspace vials of the test solution and the reference solution were placed in a headspace sampler and analyzed by a gas chromatograph equipped with a flame ionization detector under the following chromatographic conditions: Chromatographic column: Capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as stationary phase; Programmed temperature rise: Start at 35-45℃, maintain for 8-12 minutes, then increase to 155-165℃ at a rate of 35-45℃ per minute, and maintain for 3-7 minutes; The injection port temperature is 210-230℃; The detector temperature is 190-210℃; Headspace conditions: Equilibrium temperature 55-65℃, equilibrium time 35-45 minutes; (4) Result calculation: Record the chromatogram and calculate the residual amount of ethanol in gastrodin by peak area using the external standard method; The limit of quantitation for the method is no higher than 0.2%.

[0009] In a preferred embodiment of the present invention, the programmed temperature rise is initiated at a temperature of 40°C, maintained for 10 minutes, and then increased to 160°C at a rate of 40°C per minute, and maintained for 5 minutes.

[0010] In a preferred embodiment of the present invention, the chromatographic conditions further include an injection port temperature of 220°C and a detector temperature of 200°C.

[0011] In a preferred embodiment of the present invention, the equilibrium temperature is 60°C and the equilibrium time is 40 minutes.

[0012] In a preferred embodiment of the present invention, the concentration of the reference solution is 0.2 mg / mL.

[0013] In a preferred embodiment of the present invention, in step (1), the preparation of the test solution is as follows: accurately weigh 0.04 g of gastrodin test sample, add 1 mL of water, and seal.

[0014] This invention employs a lower headspace equilibrium temperature (55-65℃), a longer equilibrium time (35-45 minutes), a chromatographic column of specific polarity, and an optimized temperature program. These three elements are interconnected and work synergistically to form a cohesive whole that solves the problem of detecting ethanol residues in the gastrodin matrix. Each element is indispensable, thus successfully solving the problem of ethanol detection in the specific matrix of gastrodin.

[0015] The beneficial effects of this invention are as follows: This invention provides a headspace gas chromatography method for detecting ethanol residues in gastrodin. By optimizing the headspace equilibrium temperature and time, using a chromatographic column of specific polarity, and employing programmed temperature conditions, a method specifically designed for detecting ethanol residues in a gastrodin matrix has been established. This method effectively overcomes matrix interference, possesses both high sensitivity and high accuracy, and achieves specific, precise, and reliable quantitative control of ethanol residues in gastrodin, making it highly practical. Detailed Implementation

[0016] The preferred embodiments of the present invention will now be described in detail so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0017] Example 1 A headspace gas chromatography method for detecting ethanol residue in gastrodin, the specific steps of which are as follows.

[0018] 1. Solution preparation Test solution: Accurately weigh 0.04 g of gastrodin test sample and place it in a 20 mL headspace vial. Accurately add 1 mL of water, shake or vortex to dissolve, and quickly cap and seal. Prepare two parallel solutions for testing.

[0019] Reference solution: Accurately weigh 0.05 g of anhydrous ethanol and place it in a 25 mL volumetric flask. Dissolve and dilute to the mark with water, and shake well to obtain the reference stock solution. Accurately measure 2.5 mL of the above reference stock solution and place it in a 25 mL volumetric flask. Dilute to the mark with water, and shake well to obtain a reference solution with a concentration of 0.2 mg / mL. Accurately measure 1 mL of this solution and place it in a 20 mL headspace vial. Seal the vial.

[0020] Blank solution: Measure 1 mL of ultrapure water and place it in a 20 mL headspace vial. Quickly cap and seal the vial to obtain the blank solution.

[0021] 2. Chromatographic determination Place the prepared headspace vials containing the blank solution, reference solution, and test solution into the headspace sampler tray in sequence. First, inject the blank solution to confirm there is no interference at the ethanol peak position. Then, inject five consecutive reference solutions, calculating the relative standard deviation (RSD) of their peak areas. The RSD should not exceed 10.0% to confirm system suitability. Finally, inject the test solution sequentially.

[0022] The gas chromatograph used was a gas chromatograph equipped with a flame ionization detector and coupled with an autosampler.

[0023] The chromatographic conditions are as follows: Chromatographic column: Capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as stationary phase.

[0024] Carrier gas: high-purity nitrogen, constant flow mode, flow rate 3.0 mL / min.

[0025] Programmed temperature rise: Start at 40℃ and hold for 10 minutes; then rise to 160℃ at a rate of 40℃ / minute and hold for 5 minutes.

[0026] Inlet temperature: 220℃.

[0027] Split injection with a split ratio of 10:1.

[0028] Detector temperature: 200℃.

[0029] Headspace conditions: Equilibrium temperature 60℃, equilibrium time 40 minutes.

[0030] 3. Result calculation: Record the chromatogram and calculate the residual amount of ethanol in gastrodin using the external standard method according to the following formula. The limit of quantitation should not be higher than 0.2%.

[0031] Where w represents the content of the impurity to be tested in the sample, % . Ru represents the peak area of ​​the impurity to be tested in the test solution; Rs is the peak area of ​​the impurity to be tested in the reference solution; m is the sample weight, in grams; Vu is the volume of the test solution, in mL; Cs — the concentration of the impurity to be tested in the reference solution, in mg / mL.

[0032] Specifically, three batches of gastrodin raw material were taken, and test solutions were prepared and measured according to the above method. Reference solutions were also prepared and measured simultaneously. The calculated ethanol residue levels in all three batches of samples were below the limit of quantitation (0.16%), meeting the product quality standard requirements.

[0033] Example 2 Methodological Validation 1. Specificity and System Applicability Preparation of ethanol stock solution: Accurately weigh 5g of ethanol, dilute with water to 100mL, shake well to obtain ethanol standard stock solution with a concentration of 50mg / mL.

[0034] To prepare the ethanol reference standard positioning solution: Accurately transfer 1 mL of the ethanol stock solution into a 100 mL volumetric flask, add water to the mark, and shake well.

[0035] Take 2 mL each of the blank solution, the ethanol reference standard positioning solution, and the reference solution from Example 1, and place them in 20 mL headspace vials. Seal the vials and inject them according to the chromatographic conditions in Example 1. The injection order is as follows: inject the blank solution once, inject the ethanol reference solution once, and repeat the injection of the reference solution 5 times. Record the chromatograms.

[0036] The test results showed that the blank aqueous solution did not interfere with the ethanol peak. The retention times of ethanol in the ethanol positioning solution and the reference solution were 1.133 minutes and 1.134 minutes, respectively, and the RSD of the peak area of ​​the reference solution was 1.25%, which met the requirements.

[0037] 2. Limit of Detection and Limit of Quantification Ethanol was diluted with water and injected into a gas chromatograph. Under a signal-to-noise ratio (S / N) of 10, the limit of quantification of ethanol was 0.00080012 mg / mL (based on solution), equivalent to 0.16% of the sample. Under a signal-to-noise ratio (S / N) of 3, the limit of detection of ethanol was 0.000240036 mg / mL, equivalent to 0.048% of the sample.

[0038] 3. Linear Linear stock solution: Accurately weigh 5g of ethanol, place it in a 100mL volumetric flask, dilute with water to the mark, and shake well.

[0039] Linear 1# solution: Prepare a standard solution with a concentration at the limit of quantitation.

[0040] Linear No. 2 solution: Accurately measure 0.5 mL of the linear stock solution into a 100 mL volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0041] Linear 3# solution: Accurately measure 0.8 mL of the linear stock solution into a 100 mL volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0042] Linear 4# solution: Accurately measure 1.0 mL of the linear stock solution into a 100 mL volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0043] Linear 5# solution: Accurately measure 1.2 mL of the linear stock solution into a 100 mL volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0044] Accurately measure 2 mL of solutions #1 through #5 and place them in separate 20 mL headspace vials. Seal the vials and inject the solutions sequentially from low to high concentrations under chromatographic conditions. Record the chromatograms. Plot a linear graph with concentration on the ordinate and peak area on the abscissa, and calculate the regression equation and correlation coefficient r.

[0045] The injection sequence and acceptable criteria are shown in Table 1 below, and the test results are shown in Table 2 below.

[0046] Table 1 Table 2 The test results in Table 2 show that the linear range of ethanol in gastrodin is 0.80024 μg / mL to 750.315 μg / mL. Within this linear range, there is a good linear relationship between the ethanol concentration and the peak area.

[0047] 4. Recovery rate Unspecified test solution: Accurately weigh 1g of gastrodin test sample, place it in a 10mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and the solution is ready.

[0048] Test solution (80% limit concentration): Accurately weigh 1g of gastrodin test sample, place it in a 10mL volumetric flask, dissolve and dilute to the mark with the linear solution No. 3 prepared under '3. Linearity' in Example 2, shake well, and the solution is ready. Prepare 3 portions in the same manner.

[0049] Test solution (100% limit concentration): Accurately weigh 1g of gastrodin test sample, place it in a 10mL volumetric flask, dissolve and dilute to the mark with the linear solution No. 4 prepared under '3. Linearity' in Example 2, shake well, and the solution is ready. Prepare 3 portions in the same manner.

[0050] Test solution (120% limit concentration): Accurately weigh 1g of gastrodin test sample, place it in a 10mL volumetric flask, dissolve and dilute to the mark with the linear 5# solution prepared under '3. Linearity' in Example 2, shake well, and the solution is ready. Prepare 3 portions in the same manner.

[0051] Take 2 mL each of the blank solution, the reference solution from Example 1, the unspecified test solution, 3 test solutions (80% limit concentration), 3 test solutions (100% limit concentration), and 3 test solutions (120% limit concentration), and place them in 20 mL headspace vials respectively. Seal the vials and inject them according to the chromatographic conditions in Example 1. Record the chromatograms. Calculate the content of each residual solvent in the unspecified test solution and the 9 spiked test solutions using the external standard method based on the peak area, and calculate the recovery rate ŋ.

[0052] The formula for calculating the recovery rate is: In the formula, C 总 To determine the total amount of residual solvent, in μg / mL; C 底样 The residual solvent content in the background of the sample is expressed in μg / mL. C 加 The amount of residual solvent added is expressed in μg / mL.

[0053] The injection sequence and acceptable criteria are shown in Table 3 below, and the test results are shown in Table 4 below.

[0054] Table 3 Table 4 The test results above show that the blank solution does not interfere with the detection. The recovery rate of ethanol in gastrodin is in the range of 80-120%, with an average recovery rate of 100.51% and a relative standard deviation (RSD) of 2.50%, which meets the acceptable standard.

[0055] 5. Method reproducibility Test solution (80% test concentration): Accurately weigh 0.8 g of gastrodin test sample into a 10 mL volumetric flask, dissolve and dilute to the mark, accurately measure 2 mL into a 20 mL headspace vial, cap and seal, and use as the test solution. Prepare 3 parallel solutions.

[0056] Test solution (100% test concentration): Accurately weigh 1.0 g of gastrodin test sample into a 10 mL volumetric flask, dissolve and dilute to the mark, accurately measure 2 mL into a 20 mL headspace vial, cap and seal, and use as the test solution. Prepare 3 parallel solutions.

[0057] Test solution (120% test concentration): Accurately weigh 1.2 g of gastrodin test sample into a 10 mL volumetric flask, dissolve and dilute to the mark, accurately measure 2 mL into a 20 mL headspace vial, cap and seal, and use as the test solution. Prepare 3 parallel solutions.

[0058] The injection sequence and acceptable criteria are shown in Table 5 below.

[0059] Table 5 The test results showed that the same person prepared nine test solutions of different concentrations on the same day, and all of them were not detected (ND), indicating that the method itself is stable.

[0060] Furthermore, measurements were performed by different personnel on different dates, and all results showed no change (ND), indicating that the method has good reproducibility under different conditions. The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A headspace gas chromatography method for detecting residual ethanol in gastrodin, characterized in that, Includes the following steps: (1) Preparation of test solution: Take gastrodin test sample, dissolve it in water and place it in headspace bottle, seal it to prepare test solution; (2) Preparation of reference solution: Take ethanol reference standard, dilute with water to a concentration of 0.1-0.5 mg / mL, place in headspace vial, seal, and prepare reference solution; (3) Chromatographic determination: The headspace vials of the test solution and the reference solution were placed in a headspace sampler and analyzed by a gas chromatograph equipped with a flame ionization detector under the following chromatographic conditions: Chromatographic column: Capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as stationary phase; Programmed temperature rise: Start at 35-45℃, maintain for 8-12 minutes, then increase to 155-165℃ at a rate of 35-45℃ per minute, and maintain for 3-7 minutes; The injection port temperature is 210-230℃; The detector temperature is 190-210℃; Headspace conditions: Equilibrium temperature 55-65℃, equilibrium time 35-45 minutes; (4) Calculation of results: Record the chromatogram and use the external standard method to calculate the residual amount of ethanol in gastrodin based on the peak area.

2. The method according to claim 1, characterized in that, The program starts at a temperature of 40°C, maintains it for 10 minutes, and then increases the temperature to 160°C at a rate of 40°C per minute, maintaining it for 5 minutes.

3. The method according to claim 1, characterized in that, The chromatographic conditions also include an injection port temperature of 220°C and a detector temperature of 200°C.

4. The method according to claim 1, characterized in that, The equilibrium temperature is 60℃ and the equilibrium time is 40 minutes.

5. The method according to claim 1, characterized in that, The concentration of the reference solution was 0.2 mg / mL.

6. The method according to claim 1, characterized in that, In step (1), the test solution is prepared by accurately weighing 0.04 g of gastrodin test sample, adding 1 mL of water, and sealing.

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