Method for determining content of ethanol in arginine

Abstract

The invention discloses a method for determining the content of ethanol in arginine, which is a headspace sampling gas chromatography method and comprises the following steps: preparing a test solution; preparing a reference solution; performing gas chromatographic analysis; and calculating the ethanol residual quantity by adopting an external standard method. The method for determining the content of ethanol in arginine has the advantages of short sample pretreatment time, high detection efficiency, no need of using an organic solvent, environmental protection and economy; the accuracy and the reliability of a detection result are ensured by precisely optimizing the headspace equilibrium temperature, the headspace equilibrium time and the gas chromatography separation condition design; the method disclosed by the invention is high in detection sensitivity, high in accuracy, good in precision, stable in determination result, suitable for a wide-range concentration range from a quantitation limit to a high concentration, very flexible in application range, and integrated with simplicity, convenience, exclusiveness, sensitivity and accuracy, and has a significant industrial application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis and detection technology, and in particular to a method for determining the ethanol content in arginine. Background Technology

[0002] Arginine is an important amino acid widely used in pharmaceutical preparations, especially for injection. In the production of arginine, ethanol is commonly used as a process solvent, detergent, or crystallizing agent; therefore, its residual amount must be strictly controlled to ensure medication safety. Pharmacopoes of various countries have strict regulations regarding the detection of residual solvents in pharmaceuticals.

[0003] Currently, gas chromatography is the commonly used method for detecting residual ethanol. However, for amino acid matrices such as arginine, which have certain polarity and thermal instability, direct injection may lead to problems such as matrix interference, injection port contamination, or target analyte decomposition. Traditional direct solution injection methods involve complex pretreatment and are prone to inaccurate quantification due to matrix effects.

[0004] Therefore, there is an urgent need in this field for a method for detecting ethanol residues that is specific to the arginine matrix, has simple pretreatment, can effectively avoid matrix interference, and has high sensitivity and good reproducibility. Summary of the Invention

[0005] This invention provides a method for determining the ethanol content in arginine, which solves the problem of interference from the arginine matrix in ethanol determination, achieving rapid, accurate, and sensitive quantitative detection, and has good durability to meet stringent pharmaceutical quality control requirements.

[0006] To address the aforementioned technical problems, this invention provides a method for determining the ethanol content in arginine. The method is a headspace gas chromatography method, comprising the following steps: (1) Preparation of test solution: Accurately weigh the arginine sample, place it in a headspace vial, add water to dissolve it and seal it; (2) Preparation of reference solution: Accurately weigh the ethanol reference standard, prepare a reference solution of known concentration with water, measure a certain volume and place it in a headspace bottle and seal it; (3) Gas chromatography analysis: Place the headspace vials containing the test solution and the reference solution from steps (1) and (2) and the headspace vials containing only water into the headspace vial injector, equilibrate at the set equilibrium temperature for a period of time, and take headspace samples for gas chromatography analysis. (4) Calculation of ethanol residue: Using the external standard method, based on the calculation results of the peak area of ​​the reference solution in step (2), the ethanol residue in the arginine sample in step (1) is calculated. The calculation formula is as follows: ； Where w is the ethanol content in the test sample, % Ru represents the peak area of ​​ethanol in the test solution; Rs is the peak area of ​​ethanol in the reference solution; m is the sample weight, in grams; Vu is the volume of the test solution, in ml; Cs represents the concentration of ethanol in the reference solution, in mg / ml.

[0007] In a preferred embodiment of the present invention, in step (1), the amount of arginine sample is 40 mg and the amount of water added is 1.0 ml.

[0008] In a preferred embodiment of the present invention, in step (2), the concentration of the reference solution is 0.2 mg / ml.

[0009] In a preferred embodiment of the present invention, in step (3), the headspace injection conditions are: headspace furnace temperature 65-75℃, equilibration time 15-25min, and injection volume 1.0ml.

[0010] In a preferred embodiment of the present invention, the headspace furnace temperature is 70°C and the balancing time is 20 min.

[0011] In a preferred embodiment of the present invention, in step (3), the conditions for the gas chromatography analysis are as follows: Capillary chromatography column; The injection port temperature is 190-210℃; Injection method: Split injection mode, split ratio of 50:1; Detector temperature: 230-250℃; Carrier gas: nitrogen, flow rate 1.8-2.2 ml / min; Temperature program: Initial temperature 35-45℃, hold for 2-4 min; then increase to 210-230℃ at a rate of 10-20℃ / min, hold for 4-6 min.

[0012] In a preferred embodiment of the present invention, the chromatographic column has dimensions of 30m × 0.53mm × 3.0μm.

[0013] In a preferred embodiment of the present invention, the injection port temperature is 200°C and the detector temperature is 240°C.

[0014] In a preferred embodiment of the present invention, the flow rate of the carrier gas is 2.0 ml / min.

[0015] In a preferred embodiment of the present invention, the heating process is as follows: the initial temperature is 40°C, held for 3 minutes; then the temperature is increased to 220°C at a rate of 15°C / min, and held for 5 minutes.

[0016] The beneficial effects of this invention are as follows: The method for determining the ethanol content in arginine has a short sample pretreatment time, high detection efficiency, and eliminates the need for organic solvents, making it environmentally friendly and economical. Through precise optimization of headspace equilibrium temperature, time, and gas chromatography separation conditions, the accuracy and reliability of the detection results are ensured. The method exhibits high detection sensitivity, high accuracy, and good precision, providing stable results suitable for a wide concentration range from the limit of quantitation to high concentrations. Its application is highly flexible, combining simplicity, specificity, sensitivity, and accuracy, making it of significant industrial application value. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the process flow for a method of determining the ethanol content in arginine according to the present invention. Detailed Implementation

[0018] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, 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.

[0019] Example 1 A method for determining the ethanol content in arginine 1. Instruments and reagents used Gas chromatograph: Model 6890N, Agilent.

[0020] Chromatographic column: Capillary column (30 m × 0.53 mm × 3.0 μm, serial number: US2698735H), model DB-624, Agilent.

[0021] Electronic balance: Model AB135-S, METTLER TOLEDO.

[0022] Ethanol reference standard: batch number 0311220301, purity 99.99%, purchased from Shanghai Xingke High Purity Solvent Co., Ltd.

[0023] Experimental water: Batch number 20220901, Wahaha purified water.

[0024] Test sample: Arginine for injection, batch number 20230201.

[0025] 2. Solution preparation: Blank solution: Accurately measure 1.0 ml of water and place it in a 20 ml headspace vial, then seal it quickly.

[0026] Reference stock solution (2 mg / ml): Accurately weigh 50 mg of ethanol reference standard, place it in a 25 ml volumetric flask, dissolve and dilute with water to the mark, and shake well.

[0027] Reference solution (0.2 mg / ml): Accurately measure 2.5 ml of the above reference stock solution into a 25 ml volumetric flask, dilute to the mark with water, and mix well. Accurately measure 1.0 ml of this solution into a headspace vial and seal quickly.

[0028] Test solution: Accurately weigh 40 mg of arginine and place it in a 25 ml headspace vial. Accurately add 1.0 ml of water to dissolve it, seal it quickly, and prepare two parallel solutions.

[0029] 3. Chromatographic conditions (1) Headspace conditions: Furnace temperature: 70 ℃; Metering loop temperature: 80 ℃; Transmission line temperature: 105 ℃; Equilibrium time: 20 min; Injection volume: 1.0 mL; (2) Gas chromatography conditions: Inlet temperature: 200 ℃ Flow split ratio: 50:1 Detector (FID) temperature: 240 ℃ Carrier gas: High-purity nitrogen (≥99.999%), constant flow rate: 2.0 mL / min Column temperature program: Initial temperature 40 ℃, hold for 3 min; increase to 220 ℃ at a rate of 15 ℃ / min, hold for 5 min.

[0030] 4. Determination method Inject the blank solution, the reference working solution (5 consecutive injections), and the test solution sequentially. Record the chromatogram and integrate the ethanol peak area.

[0031] 5. Results and Calculations System suitability: The RSD of the ethanol peak area in the reference solution was 3.07% (n=5), which meets the requirement of less than 10.0%, indicating good system precision. The blank solution chromatogram showed no interference at the ethanol peak position.

[0032] Test results: No ethanol peaks were detected in the chromatograms of the two test solutions (below the detection limit), indicating that the residual ethanol content in this batch of arginine raw materials is extremely low and meets the quality requirements.

[0033] Content calculation: Calculated using the external standard method formula: Where: w is the content of the impurity to be detected in the test 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 represents the concentration of the impurity to be tested in the reference solution, in mg / ml.

[0034] In Example 1, the method described above was successfully applied to determine the residual ethanol content in arginine. This method features simple pretreatment, good system applicability, and strong specificity, making it suitable for quality control of this product.

[0035] Example 2 Analytical Methodology Validation 1. Specificity and System Applicability Preparation of ethanol stock solution: Accurately weigh 5g of ethanol, place it in a 100ml volumetric flask, dilute with water to the 100ml mark, shake well, and the ethanol standard stock solution with a concentration of 50mg / ml is obtained.

[0036] To prepare the local solution: Accurately transfer 1 ml of the above ethanol stock solution into a 100 ml volumetric flask, add water to the mark, and shake well.

[0037] Take 2 ml each of the blank solvent (water), ethanol positioning solution, and reference solution (0.2 mg / mL), and place them in separate 20 ml headspace vials. Seal the vials. Inject and analyze the samples according to the method described in Example 1 above, and record the chromatograms.

[0038] The injection sequence and acceptable criteria are shown in Table 1 below, and the test results are shown in Table 2 below.

[0039] Table 1 Table 2 The test results showed that the blank solvent chromatogram had no interference at the ethanol peak position, indicating good method specificity. The retention time of ethanol in the ethanol positioning solution and the reference solution was consistent. After five consecutive injections of the reference solution, the relative standard deviation (RSD) of the ethanol peak area was 3.07%, meeting the system suitability requirements (RSD ≤ 10%).

[0040] 2. Limit of Detection and Limit of Quantification Take an appropriate amount of ethanol and dissolve it in water to prepare a solution of suitable concentration. Dilute stepwise and inject into the gas chromatograph to confirm the limit of detection and limit of quantitation.

[0041] The limit of quantitation (LOQ) of this method was determined by serial dilution at a signal-to-noise ratio of 10:1. A confirmatory solution of 10% of the solvent limit concentration was then prepared and injected into the gas chromatograph. The limit of quantitation was determined at a signal-to-noise ratio ≥10, and the limit of detection (LOD) was determined at a signal-to-noise ratio ≥3.

[0042] The injection sequence and acceptable criteria are shown in Table 3 below, the results of the limit of quantitation test are shown in Table 4 below, and the results of the limit of diagnostic test are shown in Table 5 below.

[0043] Table 3 Table 4 Table 5 The baseline noise was measured to be 0.11 pA, the signal-to-noise ratio of ethanol in the limit of quantitation solution was 10.46, and the RSD% of the 6-needle limit of quantitation precision test was 5.45%, which met the acceptable standard; the signal-to-noise ratio of ethanol in the limit of detection solution was 3.98, which also met the acceptable standard.

[0044] The detection limit for ethanol is 0.048% (2.4 × 10⁻⁻⁴). 4 mg / mL), the limit of quantitation was 0.16% (8.0 × 10⁻). 4 (mg / mL). Six consecutive injections of the solution at the limit of quantitation concentration showed an RSD of 5.45% for the peak area, demonstrating that the method still has good precision and sensitivity at low concentration levels.

[0045] 3. Linearity and Range Preparation: Linear stock solution: Accurately weigh 5g of ethanol into a 100ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0046] Linear 1# solution: Prepare a standard solution with a specified concentration limit.

[0047] 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.

[0048] Linear 3# solution: Accurately measure 0.8 ml of linear stock solution into a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0049] Linear 4# solution: Accurately measure 1.0 ml of linear stock solution into a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0050] Linear 5# solution: Accurately measure 1.2 ml of linear stock solution into a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0051] Linear 6# solution: Accurately measure 1.5 ml of linear stock solution into a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0052] Accurately measure 2 ml of each of the linear solutions 1# to 5# (prepare two copies of each), place them in 20 ml headspace vials, seal them, and inject them sequentially from low concentration to high concentration according to chromatographic conditions, recording 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.

[0053] The injection sequence and acceptable criteria are shown in Table 5 below, and the test results are shown in Table 6 below. Linear regression was performed with concentration (X) as the abscissa and peak area (Y) as the ordinate. The obtained linear regression equation and correlation coefficient are shown in Table 7 below.

[0054] Table 6 Table 7 The results showed that the linear range of ethanol was 0.80024 ~ 750.315 μg / mL, and the peak area showed a good linear relationship within the linear concentration range. The linear regression equation was y = 3.1403x + 12.3868, the correlation coefficient r was 0.999, and the ratio of the absolute value of the ordinate intercept to the ethanol limit concentration was 0.78%, which was much less than 25% and met the acceptable standard.

[0055] 4. Accuracy (Recovery Rate) The accuracy was examined using the spiking recovery method.

[0056] Unspecified test solution: Weigh 1g of the test sample accurately, place it in a 10ml volumetric flask, dissolve and dilute with water to the mark, and shake well to obtain the solution.

[0057] Test solution (80% limit concentration): Accurately weigh 1g of the test sample, place it in a 10ml volumetric flask, dissolve and dilute to the mark with linear solution #3, and shake well. Prepare 3 portions using the same method.

[0058] Test solution (100% limit concentration): Accurately weigh 1g of the test sample, place it in a 10ml volumetric flask, dissolve and dilute to the mark with linear 4# solution, and shake well. Prepare 3 portions using the same method.

[0059] Test solution (120% limit concentration): Accurately weigh 1g of the test sample, place it in a 10ml volumetric flask, dissolve and dilute to the mark with linear 5# solution, and shake well. Prepare 3 portions using the same method.

[0060] Take 2 ml each of the blank solution, reference solution, unspecified test solution, and 9 spiked test solutions, and place them in 20 ml headspace vials. Seal the vials and inject them under the chromatographic conditions described above. 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.

[0061] The formula for calculating the recovery rate is: In the formula: ŋ is the recovery rate, %; 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.

[0062] The injection sequence and acceptable criteria are shown in Table 8 below, and the test results are shown in Table 9 below.

[0063] Table 8 Table 9 The results showed that the average recovery rate of the nine samples was 100.51%, with an RSD of 2.50% (n=9). All recovery rates were between 80% and 120%, demonstrating the excellent accuracy of the method.

[0064] 5. Precision 5.1 Repeatability Take the same batch of samples and test them in parallel for three different sample concentrations according to the above method. Calculate the content of residual solvent by peak area using the external standard method, and calculate the RSD of the measured values.

[0065] Specifically, prepare the test solution (80% test concentration): accurately weigh 0.8 g of the 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 this as the test solution. Prepare 3 parallel solutions.

[0066] Test solution (100% test concentration): Accurately weigh 1.0 g of the 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.

[0067] Test solution (120% test concentration): Accurately weigh 1.2 g of the 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.

[0068] The injection sequence and acceptable criteria are shown in Table 10 below.

[0069] Table 10 The ethanol content in all nine samples was "not detected" (ND), indicating that the method yielded consistent results at different sample concentrations and had good repeatability.

[0070] 5.2 Intermediate Precision The same batch of samples were taken and measured by different analysts on different dates using the above method. Each analyst conducted 6 parallel tests, for a total of 18 tests. The residual solvent content of each sample was calculated, and the RSD of the measured values ​​was calculated.

[0071] The injection sequence, testing time, and acceptable criteria are shown in Table 11 below, and the injection sequence, tester, and acceptable criteria are shown in Table 12 below.

[0072] Table 11 Table 12 All results were "Not Detected" (ND), meaning that the same batch of samples, measured by different analysts on different dates using the above method, showed no detectable ethanol content in arginine in any of the six samples. This indicates that the method is not affected by changes in time or personnel and has good intermediate precision.

[0073] 6. Durability The robustness of the method was examined by varying the chromatographic conditions.

[0074] Initial column temperatures were set at 38℃, 40℃, and 42℃. 2 ml of the control solution was placed in a 20 ml headspace vial, sealed, and injected under the chromatographic conditions described above. Chromatograms were recorded, and the robustness of the method was evaluated based on the resolution and measurement results.

[0075] Using column flow rates of 1.6 ml / min, 1.8 ml / min, and 2.0 ml / min, 2 ml of the control solution was placed in a 20 ml headspace vial, sealed, and injected under the chromatographic conditions described above. The chromatograms were recorded, and the robustness of the method was evaluated based on the resolution and measurement results.

[0076] Acceptable conditions: Under each chromatographic condition, the RSD between the test results of each solvent is ≤10%.

[0077] The test results are shown in Table 13 below.

[0078] Table 13 The results showed that the measurement results were unaffected under all varying conditions (all were ND), and the system suitability met the requirements, proving that the method is insensitive to changes in operating conditions and has strong robustness.

[0079] This invention provides a method for determining the ethanol content in arginine, which uses headspace gas chromatography to determine the residual ethanol in arginine, and has the following significant advantages: This invention allows for headspace sampling analysis of arginine samples by directly dissolving them in water. This process eliminates the need for organic solvents, shortens sample pretreatment time, increases detection efficiency, reduces operator workload and potential errors, and is also more environmentally friendly and economical.

[0080] By precisely optimizing the headspace equilibrium temperature, time, and gas chromatographic separation conditions, the interference of the complex matrix of arginine on the determination of ethanol was successfully eliminated. The blank solution showed no interfering peaks, and the target analyte exhibited sharp, symmetrical peaks with excellent separation, ensuring the accuracy and reliability of the detection results. This method is particularly suitable for the detection of amino acid-based drugs with complex compositions.

[0081] The method of this invention has extremely low limits of detection (LOD) and quantitation (LOQ), reaching 0.048% and 0.16%, respectively. This high sensitivity can accurately capture and quantify trace amounts of ethanol residue in arginine, fully meeting the increasingly stringent limits for residual solvents in domestic and international pharmacopoeias, and providing a solid technical guarantee for drug safety.

[0082] The method exhibits high accuracy and precision: Spiking recovery tests demonstrate that the average recovery rate of this method reaches 100.51%, with all recovery results falling within the ideal range of 80%–120%, and a relative standard deviation (RSD) of 2.50% (n=9), indicating extremely high accuracy. Furthermore, repeatability and intermediate precision test results meet the requirements (RSD ≤ 10%), indicating good reproducibility and consistent, reliable results obtainable by different personnel at different times.

[0083] The results obtained by this method remain stable. Even when the chromatographic conditions change slightly (such as column temperature ±2°C or flow rate ±0.2 mL / min), the system still meets the requirements. This ensures that the method has good transferability and reproducibility in different laboratories and different models of instruments, and is easy to standardize and promote.

[0084] The method of this invention exhibits good linearity (r=0.999) in nearly three orders of magnitude concentration range from 0.80024 μg / mL to 750.315 μg / mL, covering a wide range from the limit of quantitation to high concentrations. It can handle the detection of trace residues as well as the analysis of potentially high-concentration samples without dilution or method changes, making its application range very flexible.

[0085] 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's specification and drawings, 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 method for determining the ethanol content in arginine, characterized in that, The method is headspace gas chromatography, which includes the following steps: (1) Preparation of test solution: Accurately weigh the arginine sample, place it in a headspace vial, add water to dissolve it and seal it; (2) Preparation of reference solution: Accurately weigh the ethanol reference standard, prepare a reference solution of known concentration with water, measure a certain volume and place it in a headspace bottle and seal it; (3) Gas chromatography analysis: Place the headspace vials containing the test solution and the reference solution from steps (1) and (2) and the headspace vials containing only water into the headspace vial injector, equilibrate at the set equilibrium temperature for a period of time, and take headspace samples for gas chromatography analysis. (4) Calculation of ethanol residue: Using the external standard method, based on the calculation results of the peak area of ​​the reference solution in step (2), the ethanol residue in the arginine sample in step (1) is calculated. The calculation formula is as follows: ； Where w is the ethanol content in the test sample, % Ru represents the peak area of ​​ethanol in the test solution; Rs is the peak area of ​​ethanol in the reference solution; m is the sample weight, in grams; Vu is the volume of the test solution, in ml; Cs represents the concentration of ethanol in the reference solution, in mg / ml.

2. The method according to claim 1, characterized in that, In step (1), the amount of arginine sample is 40 mg, and the amount of water added is 1.0 ml.

3. The method according to claim 1, characterized in that, In step (2), the concentration of the reference solution is 0.2 mg / ml.

4. The method according to claim 1, characterized in that, In step (3), the headspace injection conditions are: headspace furnace temperature 65-75℃, equilibration time 15-25min, and injection volume 1.0ml.

5. The method according to claim 4, characterized in that, The headspace furnace temperature is 70℃ and the balancing time is 20 minutes.

6. The method according to claim 1, characterized in that, In step (3), the conditions for gas chromatography analysis are as follows: Capillary chromatography column; The injection port temperature is 190-210℃; Injection method: Split injection mode, split ratio of 50:1; Detector temperature: 230-250℃; Carrier gas: nitrogen, flow rate 1.8-2.2 ml / min; Temperature program: Initial temperature 35-45℃, hold for 2-4 min; then increase to 210-230℃ at a rate of 10-20℃ / min, hold for 4-6 min.

7. The method according to claim 6, characterized in that, The chromatographic column has dimensions of 30m × 0.53mm × 3.0μm.

8. The method according to claim 6, characterized in that, The temperature of the injection port is 200°C, and the temperature of the detector is 240°C.

9. The method according to claim 6, characterized in that, The flow rate of the carrier gas is 2.0 ml / min.

10. The method according to claim 6, characterized in that, The heating program is as follows: the initial temperature is 40℃, held for 3 minutes; then the temperature is increased to 220℃ at a rate of 15℃ / min, and held for 5 minutes.

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