Method for determining ethylene glycol and diethylene glycol in dosage forms

Abstract

FIELD: analytical chemistry.SUBSTANCE: used to control the quality of medicinal products. A method for determining ethylene glycol and diethylene glycol in medicinal products, including the preparation of standard solutions of ethylene glycol and diethylene glycol, test solutions, test solutions with the addition of a standard solution, solutions for checking the sensitivity of the chromatographic system, chromatography, calculations for the reliable determination of ethylene glycol and diethylene glycol qualitatively and quantitatively in solutions of medicinal products.EFFECT: increase in the reliability of the determination of ethylene glycol and diethylene glycol, including in trace quantities.1 cl, 13 dwg, 6 tbl

Description

[0001] The invention relates to methods for studying materials by determining their chemical properties, specifically to studying or analyzing materials by separating them into their constituent parts (components). It can be used in the quality control of medicines and pharmaceuticals.

[0002] Prior art methods for determining ethylene glycol and diethylene glycol reflect general recommendations for determining the specified impurities in glycerin, which is part of cough syrups and solutions (USP & NF - MZ5420-04-01 "Glycerin", published 08 / 17 / 2023; European pharmacopoeia 11.0 07 / 2022: 0496 "Glycerol", published 07 / 2022; FS.2.2.0006.15 "Glycerin", published 01 / 01 / 2016).

[0003] The disadvantage of the above-described methods is that currently available methods for determining ethylene glycol and diethylene glycol are not designed to detect their presence directly in medicinal products. They merely provide general recommendations for determining these impurities in glycerin. Glycerin is an ingredient in cough syrups and solutions. However, existing methods do not take into account the complex composition of real samples, which, in addition to glycerin, includes active ingredients and excipients.

[0004] The following terms are used in this invention:

[0005] A standard sample is a metrological standard in the form of a substance, material or other object, the composition and / or properties of which are known and certified in accordance with the established procedure.

[0006] Test sample is a real sample of a medicinal product that is subject to analysis (testing).

[0007] Mass-to-charge ratio (m / z) is the ratio of the mass of an ion to its charge, used in mass spectrometry to identify particles by determining their mass at a given charge.

[0008] Matrix is ​​the sum of all sample components, excluding the target substance or substances that enter the chromatographic system along with the sample. The matrix defines the physicochemical "environment" of the analyte: solvent or solvents, excipients, impurities, degradation products, etc.

[0009] Analyte - the target chemical compound (or group of compounds) to be detected and / or quantified by the method.

[0010] The problem to be solved by the claimed invention is to develop a method for determining impurities of ethylene glycol and diethylene glycol in cough syrups and solutions for internal use, which makes it possible to avoid obtaining false positive and false negative results.

[0011] The technical result is the reliable determination of ethylene glycol and diethylene glycol even in trace amounts in cough syrups and solutions for internal use with high sensitivity and selectivity.

[0012] The technical result is achieved due to the fact that the method for determining ethylene glycol and diethylene glycol in dosage forms includes the following stages: preparation of standard solutions of ethylene glycol and diethylene glycol, including the preparation of initial standard solutions, intermediate standard solutions, working standard solutions, wherein the solutions are obtained by dissolving ethylene glycol and diethylene glycol in ethanol, followed by mixing the intermediate standard solutions to obtain a working standard solution; preparation of test solutions of the drug, including dilution with ethanol and water, followed by cooling, heating to room temperature, filtration;preparation of test solutions with the addition of a standard solution, including dilution of the test sample of the medicinal product with ethanol and water, followed by the addition of standard solutions of ethylene glycol and diethylene glycol, cooling, heating to room temperature, filtration; preparation of solutions for testing the sensitivity of the chromatographic system, including the preparation of initial standard solutions of ethylene glycol and diethylene glycol for testing the sensitivity of the chromatographic system, a solution for testing the sensitivity of the chromatographic system, wherein the solutions are obtained by diluting the initial standard solutions of ethylene glycol and diethylene glycol with ethanol and then mixing them to obtain a solution for testing the sensitivity of the chromatographic system;Chromatography, including sequential chromatography of a solvent, a solution for checking the sensitivity of the chromatographic system, a working standard solution, test solutions, test solutions with the addition of a standard solution, followed by obtaining chromatograms and mass spectra; checking the suitability of the chromatographic system, including an assessment of the relative standard deviation of the peak area of ​​ethylene glycol and diethylene glycol, the relative deviation of the retention time of the peak of ethylene glycol and diethylene glycol, the signal-to-noise ratio for the peak of ethylene glycol and diethylene glycol in the chromatogram of the solution for checking the sensitivity of the chromatographic system; calculations, including calculations of the concentrations of ethylene glycol and diethylene glycol, calculation of the degree of extraction for ethylene glycol and diethylene glycol.

[0013] Ethylene glycol and diethylene glycol are toxic compounds found in medicinal products (especially cough syrups and solutions) due to manufacturing violations or counterfeiting. Their organoleptic similarity to glycerin and propylene glycol makes detection impossible without physicochemical analysis. This method involves the determination of ethylene glycol and diethylene glycol in solutions and syrups using gas chromatography with mass spectrometric detection (GC-MS). The use of a flame ionization detector (GC-FID) in complex syrup matrices carries the risk of interference between target analyte signals and matrix component signals, reducing the reliability of the results. The proposed method, using GC-MS, exhibits high selectivity and sensitivity, making it a universal screening procedure regardless of matrix complexity.The method has been validated for the following parameters: specificity, linearity, accuracy, precision, sensitivity, and stability. The developed method is universal and allows for the simultaneous determination of EG and DEG, both qualitatively and quantitatively.

[0014] Equipment. A gas chromatography-mass spectrometry (GC-MS) system, including a gas chromatograph and a mass spectrometer, was used to analyze EG and DEG content. An Agilent 7890 gas chromatograph and an Agilent 5975C mass spectrometer (Agilent Technologies, USA) were used in our study.

[0015] Reagents and standard samples. EG (99.99% purity, CDH, India) and DEG (99.53% purity, Macklin, China) were used as standard samples. Absolute ethanol (HPLC grade, Aqua-M, Russia) was used as a solvent. Water was obtained using a Milli-Q purification system (Merck Millipore, Germany).

[0016] Test samples. Commercially available drug samples in two forms were used to develop and validate the method: two oral solutions (Sample 1 and Sample 3) and one syrup (Sample 2), representing real samples. The brand names of these samples are not disclosed due to the risk of conflict of interest.

[0017] The standard sample serves as the starting material for the preparation of the standard solution, the test sample (the drug itself) serves as the starting material for the preparation of the test solution and the test solution with the addition of the standard solution.

[0018] The invention is explained by figures, which show:

[0019] Fig. 1 - The working window of the data processing program, where 1 is the peak retention time, 2 is the peak area, 3 is the peak height, 4 is the signal-to-noise ratio, 5 is the name of the solution, 6 is the type of solution, 7 is the peak integration window.

[0020] Fig. 2 - Calibration graph of EG for linearity assessment.

[0021] Fig. 3 - Calibration graph of DEG for linearity assessment.

[0022] Fig. 4 - Calibration dependence of EG.

[0023] Fig. 5 - Calibration dependence of DEG.

[0024] Fig. 6 - Chromatogram of standard solution (EG).

[0025] Fig. 7 - Chromatogram of the test solution (EG not detected).

[0026] Fig. 8 - Chromatogram of the test solution (EG detected).

[0027] Fig. 9 - Chromatogram of the test solution with the addition of a standard solution (EG).

[0028] Fig. 10 - Chromatogram of standard solution (DEG).

[0029] Fig. 11 - Chromatogram of the test solution (DEG not detected).

[0030] Fig. 12 - Chromatogram of the test solution (DEG detected).

[0031] Fig. 13 - Chromatogram of the test solution with the addition of a standard solution (DEG).

[0032] The present invention provides a method for determining ethylene glycol and diethylene glycol in cough syrups and solutions for internal use.

[0033] The method for determining ethylene glycol and diethylene glycol in dosage forms is implemented as follows.

[0034] Stage 1. Preparation of standard solutions of ethylene glycol (EG) and diethylene glycol (DEG).

[0035] At this stage, initial standard solutions of EG and DEG, intermediate standard solutions of EG and DEG, and a working standard solution are sequentially prepared. These solutions are needed to assess the suitability of the chromatographic system, as well as to qualitatively and quantitatively assess the EG / DEG content in the test samples. The preparation of standard solutions of ethylene glycol and diethylene glycol involves the preparation of initial standard solutions, intermediate standard solutions, and working standard solutions. The solutions are obtained by dissolving ethylene glycol and diethylene glycol in ethanol.

[0036] Stock standard solution of EG. Place approximately 500 mg of the standard sample of EG in a 50 ml volumetric flask, dissolve in ethanol, dilute to the mark with ethanol, and mix. EG concentration: 10 mg / ml.

[0037] DEG stock standard solution. Place approximately 500 mg of the DEG standard sample in a 50 ml volumetric flask, dissolve in ethanol, dilute to the mark, and mix. DEG concentration: 10 mg / ml.

[0038] Intermediate standard solution of EG. Place 1 ml of the stock standard solution of EG in a 10 ml volumetric flask, add ethanol to the mark, and mix. EG concentration: 1 mg / ml = 1000 μg / ml.

[0039] Intermediate standard solution of DEG. Place 1 ml of the stock standard solution of DEG in a 10 ml volumetric flask, dilute to the mark with ethanol, and mix. DEG concentration: 1 mg / ml = 1000 μg / ml.

[0040] Working standard solution. Place 0.5 ml of EG intermediate standard solution and 0.5 ml of DEG intermediate standard solution in a 20 ml volumetric flask, dilute to the mark with ethanol, and mix. The concentration of EG and DEG is 25 μg / ml.

[0041] Stage 2. Preparation of test solutions.

[0042] At this stage, the procedure for preparing test solutions from the test samples is performed. This involves shaking the test sample solution, diluting it with ethanol and water, and then cooling the resulting test solution to stabilize the system and precipitate. Warming the test solution to room temperature and filtering is then performed. Cooling was performed to 3-5°C in a refrigerator. This process is necessary to accelerate the precipitation of water-soluble sugars and syrup components.

[0043] Before preparation, shake the test sample solution. Shaking is necessary to evenly distribute the substances throughout the solution. Place approximately 500 mg of the drug in a 20 ml measuring flask, add approximately 2 ml of ethanol and 2 ml of water, mix, dilute to the mark with ethanol, mix, and refrigerate for 15 minutes. Remove the test solution from the refrigerator and allow it to warm to room temperature (18-25°C), then filter through a 0.45 µm filter.

[0044] Cooling the test solution of the medicinal product in the refrigerator for 15 minutes is necessary to stabilize the resulting system, precipitate any insoluble impurities, and crystallize any unwanted components. This prevents undesirable chemical reactions at elevated temperatures and improves the solubility of some substances. Subsequent warming to room temperature (18-25°C) and filtration through a membrane filter with a 0.45 µm pore diameter ensures the purification of the test solution from mechanical impurities, the removal of insoluble particles, and the production of a homogeneous, transparent liquid. This, together, guarantees the quality, safety, and dosing accuracy of the finished product during subsequent use.

[0045] Dissolution, cooling, and subsequent heating with filtration are necessary to purify the test solution from large sugar crystals and other components of the drug sample that are insoluble in alcohol and precipitate. This precipitate can reduce the sensitivity and accuracy of the method and may also cause equipment failure. Filtration does not affect the determination of EG and DEG impurities.

[0046] Thus, the test solutions with a concentration of 25 mg / ml were kept in the refrigerator for 15 min, brought to room temperature and filtered through 0.45 μm PVDF syringe filters.

[0047] Stage 3. Preparation of the test solution with the addition of a standard solution.

[0048] At this stage, the test solution is prepared with the addition of a standard solution. This procedure is necessary to assess the validity of the method, as different test samples have different matrices. Consequently, chromatographic peaks of the analytes may overlap with those of the drug matrix. This phenomenon can lead to false-positive or false-negative results. The validity of the method is assessed by the degree of recovery, that is, by the difference between the actual concentration of the analyte added to the test solution and the theoretical value. If the actual concentration differs from the theoretical value by no more than ±30%, the method is considered valid and can be used for the qualitative and quantitative assessment of the analyte in the test sample.

[0049] This step involves shaking the test drug sample solution, diluting it with ethanol and water, then adding standard solutions of EG and DEG. Cooling the resulting test solution with the standard solution added to stabilize the system and precipitate it. Warming the test solution with the standard solution added to room temperature followed by filtration was performed. Cooling was performed to 3-5°C in a refrigerator. This process is necessary to accelerate the precipitation of water-soluble sugars and syrup components.

[0050] Test solution with added standard solution. Shake the test sample before preparation. Place approximately 500 mg of the medicinal product in a 20 ml volumetric flask, add approximately 2 ml of ethanol, 2 ml of water, and 0.5 ml each of EG and DEG standard solutions. Mix, dilute to the mark with ethanol, mix, and refrigerate for 15 minutes. Remove the solution from the refrigerator and allow it to warm to room temperature, then filter through a 0.45 μm filter. The concentration of EG and DEG is 25 μg / ml.

[0051] The spiked test solution (also known as the spike solution) is a drug test solution with a specified volume of a standard sample of known concentration added. The preparation of this solution is necessary to assess the recovery rate, a parameter that characterizes the accuracy of the method. This step confirms the reliability of the obtained results, making it an important step in the method for determining ethylene glycol and diethylene glycol in dosage forms. If the chromatographic peaks of the analyte and the drug matrix component overlap, a false-positive or false-negative result may occur.The introduction of a test solution analysis step with a standard solution helps compensate for the effect of the syrup matrix on the response (including ionization suppression in GC-MS and losses during the injection / evaporation steps), leading to increased accuracy and reproducibility of EG / DEG determination, confirmation of specificity, and correct verification of EG / DEG analysis in the matrix. This reduces systematic error and intermatrix variability compared to methods using external calibration alone.

[0052] The method is based on comparing the analyte concentration in a standard sample (known value) with the analyte concentration in a spike solution (calculated value). A deviation of more than 30% between the actual and true values ​​indicates that the method is not working correctly (this may be due to the overlap of the chromatographic peak of the drug component with the chromatographic peak of the analyte, incorrectly selected chromatographic conditions, incorrectly selected detection conditions, etc.). If the actual concentration differs from the true value by less than 30%, the method is working correctly and allows for the reliable determination of the analyte in the drug matrix.

[0053] In this case, specific volumes of EG and DEG standard solutions are added; their concentration in the Spike solution is 25 μg / mL. This solution is analyzed in sequence with the standard and test solutions. The actual concentrations of EG and DEG are 24.28 and 24.44 μg / mL, respectively. The recovery is 97.17% (24.28 / 25*100%) for EG and 97.75% (24.44 / 25*100%) for DEG. These recovery rates confirm the validity of the method.

[0054] Stage 4. Preparation of solutions for testing the sensitivity of the chromatographic system (hereinafter referred to as PCHS).

[0055] Short-term detector signal noise (baseline noise) affects the precision of quantitative determination. The baseline noise solution confirms that under the current conditions (ionization source, liner, solvent, column, matrix), the system "sees" EG and DEG at levels close to the LOQ, and that the selectivity and stability of the response are within tolerances. This protects against false "not detected" results due to water in the solvent, contaminated liner, emission drift, and matrix suppression. This parameter is assessed by the signal-to-noise ratio, which is expressed as the ratio of twice the peak height to the peak-to-peak noise of the chromatographic system (background signal). The standard for this ratio is described in OFS.1.1.0012 "Validation of Analytical Methods" and is at least 10:1. Fulfilling this requirement means that the chromatographic peak is high enough to unambiguously identify analytes at low concentrations.

[0056] At this stage, the initial standard solutions for the EG and DEG PCS, as well as the PCS solution, are prepared. This stage is necessary for determining sensitivity.

[0057] Limit of quantification (LOQ) is the minimum concentration that can be reliably determined using this equipment.

[0058] To determine the sensitivity, the following solutions are prepared: the initial standard solution of ethylene glycol and diethylene glycol for PCHS is obtained by placing the intermediate standard solution of ethylene glycol and diethylene glycol in a volumetric flask with subsequent dilution and stirring, the working solution for PCHS is obtained by adding the initial standard solution of EG for PCHS and the initial standard solution of DEG for PCHS into a volumetric flask with subsequent dilution with ethanol and stirring.

[0059] Stock standard solution of EG for PCHS. Place 1 ml of the intermediate standard solution of EG in a 10 ml volumetric flask, dilute to the mark with ethanol, and mix. EG concentration: 100 μg / ml.

[0060] Initial standard solution of DEG for PCHS. Place 1 ml of the intermediate standard solution of DEG in a 10 ml volumetric flask, dilute to the mark with ethanol, and mix. DEG concentration: 100 μg / ml.

[0061] Solution for PCHS. Place 279 µl of the EG stock standard solution for PCHS and 161 µl of the DEG stock standard solution for PCHS in a 10 ml volumetric flask, dilute to the mark with ethanol, and mix. The EG concentration is 2.79 µg / ml, and the DEG concentration is 1.61 µg / ml.

[0062] Stage 5. Chromatography.

[0063] This step produces chromatograms and mass spectra used for the qualitative and quantitative determination of EG and DEG. First, the solutions enter the liner of a gas chromatograph, where they evaporate. The gaseous sample is then carried by the carrier gas into the chromatographic column, where chromatographic separation occurs. Chromatographic separation involves repeated chemical interactions between the analyte and the column's stationary phase (the sorption-desorption process). The more such interactions occur, the longer the substance will remain in the column and, therefore, the later it will reach the detector. The number of interactions depends on the affinity of the analyte for the column's stationary phase—the higher the affinity, the more strongly the substance is retained in the column. The separation itself is determined by the different chemical structures of the substances that comprise the components of the drug matrix.The separation results in the formation of individual chromatographic peaks, which are displayed on a chromatogram (a chromatogram is the dependence of the detector signal on time as the analyzed mixture passes through the chromatograph column). The chromatogram contains peaks, each of which corresponds to the exit of a specific component of the mixture from the column, and their parameters (height, area, retention time) determine the quantitative and qualitative composition of the sample. After the column, the separated sample enters a mass-selective detector, where a series of sequential processes occur:

[0064] 1) Fragmentation. During this process, analyte molecules are bombarded with electrons, causing them to disintegrate into fragments with specific mass-to-charge ratios. The composition of these fragments is unique for each molecule, increasing the specificity of the method. A unique composition here refers to a kind of "molecular fingerprint" or "barcode" for the substance. It is a characteristic and reproducible mass spectrum that indicates the specific fragments into which the original molecule was disintegrated during ionization. This pattern allows for the unambiguous identification of a substance, even if it exits the chromatograph simultaneously with other components of the mixture.

[0065] 2) Quadrupole Separation. A mass spectrometer quadrupole is a mass analyzer consisting of four parallel electrode rods used to separate ions based on their mass-to-charge ratios. During this separation, only those ions specified by the operator when creating the analysis method pass through the quadrupole (see "Monitored Ions (SIM Mode") below).

[0066] 3) Detection. Ions passing through the quadrupole strike a detector, where their concentration is recorded over time. The data is recorded on a personal computer using specialized software for recording and processing the obtained data.

[0067] The solvent (ethanol) is introduced to evaluate the background pattern of the chromatogram, the solution for checking the sensitivity of the chromatographic system is introduced to evaluate the sensitivity of the detector for analytes, the working standard solution is introduced to evaluate the suitability of the chromatographic system in terms of precision (standard deviation for the areas and retention times of EG / DEG peaks), as well as for the quantitative assessment of the EG / DEG impurity content, the test solution is introduced for the direct determination of the EG / DEG content in the test samples, and the test solution is introduced to evaluate the accuracy of the method in terms of the degree of extraction. The chromatography sequence is strictly defined; deviations from the specified program may lead to erroneous data (due to analyte residues from solutions with higher concentrations and their transition to solutions with lower concentrations - the phenomenon of mass transfer).

[0068] At this stage, the following are sequentially chromatographed: solvent (1 time), solution for checking the sensitivity of the chromatographic system (1 time), working standard solution (6 times), solvent (1 time), test solution No. 1 (3 times), test solution No. 2 (3 times), test solution with the addition of standard solution (3 times).

[0069] Monitored ions (SIM mode): EG: m / z 29, 31*, 33, 43, 62; DEG: m / z 31, 43, 45*, 75, 76 (* - quantitative ions). SIM (Single Ion Monitoring) is an operating mode of a mass spectrometer in which a specific ion is detected. This mode increases the sensitivity of the detector, reduces noise and improves selectivity. Process: after passing through the column, the molecule is bombarded with electrons, which causes it to break up into certain fragments (fragmentation process). Each molecule has its own fragmentation process (m / z ratios and their intensity), which makes each mass spectrum unique. The fragments then enter a quadrupole, where unnecessary ions are filtered out. This means that only specific fragments with the desired m / z ratios (in our case, 29, 31*, 33, 43, 62; m / z 31, 43, 45*, 75, 76 for EG and DEG, respectively) reach the detector. Their intensity is then measured, which is used to construct a chromatographic peak.

[0070] During development, the chromatographic conditions specified in Tables 1 and 2 were selected, while the detection conditions are presented in Tables 3 and 4. The selection of conditions was performed during the method development stage, during which various test conditions and detector settings were tested. The result of the development is a method with ready-made test conditions, so further selection of conditions is not required. The conditions were selected experimentally, taking into account literature data on the separation of alcohols by gas chromatography. The operating parameters of the mass detector and the controlled mass-charge ratios were selected based on literature data and the NIST mass spectral database.

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] It is allowed to change the chromatography conditions in accordance with the requirements of OFS.1.2.1.2.0001 "Chromatography" (Stationary phase: - particle size: maximum decrease by 50%, increase is not allowed (packed columns); - film thickness: from -50% to +100% (capillary columns). Column dimensions: - column length: from -70% to +100%; - column inner diameter: ±50%. Column temperature: ±10%. Temperature program: it is allowed to adjust the temperature as specified above; it is allowed to adjust the rate of temperature change and the time held at a constant temperature up to ±20%. Flow rate: ±50%. Injection volume: can be changed, provided that the system suitability requirements are met. When decreasing the injection volume or increasing the split ratio, special attention is paid to the detection limit and repeatability of the peak area of ​​the substance being determined.Increasing the injection volume or decreasing the split ratio is permitted, provided that response linearity and peak resolution remain satisfactory. However, mass spectrometric detection parameters and controlled mass-to-charge ratios must not be altered.

[0077] Stage 6. Checking the suitability of the chromatographic system.

[0078] This step includes assessing the relative standard deviation of the ethylene glycol and diethylene glycol peak area, the relative deviation of the ethylene glycol and diethylene glycol peak retention time, and the signal-to-noise ratio for the ethylene glycol and diethylene glycol peak in the chromatogram of the solution to test the sensitivity of the chromatographic system by analyzing the peaks in the chromatogram, each of which corresponds to the yield of a specific component of the mixture from the column. Based on their parameters (height, area, retention time), the quantitative and qualitative composition of the sample is determined. The assessment of the parameters is performed using specialized software (a program for the qualitative analysis of mass spectrometry data; Agilent MassHunter Qualitative Analysis was used for the invention), which allows for the processing of chromatograms. An example of the program working window is shown in Fig. 1. In Fig.1 shows the working window of the data processing program, where 1 is the peak retention time, 2 is the peak area, 3 is the peak height, 4 is the signal / noise ratio, 5 is the name of the solution, 6 is the type of solution, 7 is the peak integration window.

[0079] The chromatographic system suitability assessment is in progress. If any of the chromatographic system suitability assessment conditions are not met, the obtained results cannot be considered reliable. For example, a chromatographic peak area standard deviation exceeding 15.0% indicates uneven analyte transport into the system, which may result in false-positive or false-negative results. Similarly, a chromatographic peak retention time standard deviation exceeding 5.0% may result in incorrect identification of analyte peaks, which may also result in false-positive / negative results. Chromatographic system suitability assessment is a mandatory step in chromatography and is included in all chromatographic analysis methods.

[0080] After chromatographing the solutions, it is necessary to ensure that the chromatographic system is functioning correctly. This is accomplished by performing a system suitability check. The chromatographic system is considered suitable if the following conditions are met:

[0081] • The relative standard deviation of the peak area of ​​EG (DEG) based on the results of 6 consecutive injections of the working standard solution should be no more than 15.0%;

[0082] • The relative standard deviation of the retention time of the EG (DEG) peak based on the results of 6 consecutive injections of the working standard solution should be no more than 5.0%;

[0083] • The signal-to-noise ratio for the EG (DEG) peak in the chromatogram of the solution for checking the sensitivity of the chromatographic system should be at least 10.

[0084] If any of the above criteria are not met, the chromatographic system is considered unsuitable, and the EG / DEG determination results are unreliable. The analytical conditions must be verified, adjusted if necessary, and the solutions must be re-injected.

[0085] The standards were described based on laboratory practice and FDA method validation documentation.

[0086] Stage 7. Calculations.

[0087] This step is the final step of the determination method. It involves calculating the concentrations of ethylene glycol and diethylene glycol, as well as calculating the recovery rate for ethylene glycol and diethylene glycol.

[0088] The concentration of EG (DEG) in the test samples (%) is calculated using the formula:

[0089]

[0090] Convert to volume % (ml / ml) (X v / v ) carried out according to the formula (optional / if necessary):

[0091]

[0092] where:

[0093] S Smp - the average area of ​​the EG (DEG) peak on the chromatogram of the test solution;

[0094] S Std - average area of ​​the EG (DEG) peak on the chromatogram of the standard solution;

[0095] a Std - sample of CO EG (DEG), mg;

[0096] a Smp - weight of the tested medicinal product, mg;

[0097] P Std - content of the main substance in SO EG (DEG), %;

[0098] V м.к.Smp . - volume of the measuring flask taken to prepare the standard solution, ml (50 ml);

[0099] V м.к.Smp - the volume of the measuring flask taken to prepare the test solution, ml (20 ml);

[0100] ρ Smp - density of the tested medicinal product, g / ml;

[0101] ρ Std - density of CO EG (DEG), g / ml.

[0102] Note: the formulas were derived manually based on the general formula of the external standard method described in OFS.1.2.1.2.0001 “Chromatography”.

[0103] After processing the chromatograms and performing all the necessary calculations, it is necessary to estimate the degree of extraction for EG and DEG, which should be in the range of 70 - 130%.

[0104] Calculation of the extraction rate:

[0105]

[0106] SI - degree of extraction, %

[0107] C Spike - concentration of EG (DEG) in the test solution, with the addition of a standard solution, μg / ml;

[0108] C Smp - concentration of EG (DEG) in the test solution, μg / ml;

[0109] C std - the concentration of the component being determined in the standard solution that was added to the test sample, μg / ml.

[0110] Note: The formula for calculating the extraction rate was manually derived based on laboratory practice.

[0111] After the methodology was developed, it was validated.

[0112] Validation of the method.

[0113] Validation of the method - assessment of the suitability of the method, was carried out according to the following indicators:

[0114] 1) Specificity.

[0115] Specificity was determined based on the recovery rates of EG and DEG in test solutions supplemented with a standard solution. Satisfactory values ​​confirm that the method allows for the reliable identification of the analyzed impurities and their quantitative assessment.

[0116] Specificity characterizes the ability to unambiguously determine the analyte in the presence of components included in the medicinal product. Specificity was confirmed by the absence of chromatographic peaks with the retention times of EG and DEG in the chromatograms of the test solutions, as well as by the degree of recovery of EG and DEG in the test solutions with the addition of a standard solution. No interfering peaks that interfere with the determination of EG and DEG were detected in the chromatograms of the test solutions, and the degrees of recovery were within the range of 70-130% (80-100% for EG and 72-98% for DEG).

[0117] 2) Linearity.

[0118] Linearity of an analytical procedure is its ability (within a given range) to obtain analytical results that are directly proportional to the concentration (amount) of the substance being determined in the sample.

[0119] The range of an analytical procedure is the interval between the highest and lowest concentrations (quantities) of the analyte in a sample (including these concentrations) for which the analytical procedure has been demonstrated to have an acceptable level of precision, trueness and linearity.

[0120] The calibration graph was plotted in the range of 50–150% of the limit. The calibration graph displays the dependence of the chromatographic peak area (Y-axis) on the analyte concentration (X-axis). Standard solutions with linearly increasing concentrations were introduced into the chromatograph; accordingly, the peak areas also increased linearly.

[0121] Linearity was assessed using the calibration graphs shown in Figs. 2 and 3.

[0122] For Fig. 2:

[0123] EG calibration dependence:

[0124] y = 2812.4⋅x + 8074.8

[0125] where:

[0126] y - area of ​​the chromatographic peak of EG;

[0127] x - EG concentration, µg / ml.

[0128] For Fig. 3:

[0129] DEG calibration curve:

[0130] y = 6344.1⋅x + 3643.7;

[0131] where:

[0132] y - area of ​​the chromatographic peak of DEG;

[0133] x - DEG concentration, µg / ml.

[0134] For EG: regression equation: y = 3066.8⋅x + 843.21, determination coefficient: R 2 = 0.9973;

[0135] For DEG: regression equation: y= 5755.2⋅x + 20285, determination coefficient: R 2 = 0.9991.

[0136] %RSD by points did not exceed 2.72%.

[0137] The correlation coefficients were greater than 0.99, indicating a high level of linearity of the method. The correlation coefficient indicates the degree of linear relationship between the concentration of a substance and its response (chromatographic peak area) in the calibration curve. An ideal value close to 1 indicates a strong positive linear relationship and high accuracy of the calibration curve, which is important for reliable determination of analyte concentration. A value less than 0.95 may require rebuilding the calibration curve.

[0138] 3) Sensitivity.

[0139] Then the limits of detection (LOD) and limits of quantification (LOQ) were calculated using the formulas:

[0140] PO = (3.3⋅S) / b

[0141] PCO = (10⋅S) / b

[0142] where:

[0143] S - standard deviation of the analytical signal;

[0144] b is the sensitivity coefficient, which is the ratio of the analytical signal to the determined value (the slope of the calibration curve). The limit of detection (LOD) and limit of quantification (LOQ) were estimated using calibration graphs. The LOQ was 0.92 μg / mL for EG and 0.53 μg / mL for DEG. The LOQ for EG was 2.79 μg / mL for EG and 1.61 μg / mL for DEG.

[0145] The results are presented in Table 5.

[0146]

[0147] The obtained data were compared with the sensitivity data of a flame ionization detector (which is currently used to determine EG and DEG in glycerol). The results are presented in Table 6.

[0148] The sensitivity of the mass-selective detector (MS) exceeds the sensitivity of the flame ionization detector (FID) by 2-3 times.

[0149] After calculations, the linearity of the method was evaluated over a wider analytical range, which includes the limit of determination (LOD) and the limit of quantification (LOQ).

[0150] The updated calibration curves with description are shown in Fig. 4 and 5.

[0151] For Fig. 4:

[0152] EG calibration dependence:

[0153] y = 3066.8⋅x + 843.21

[0154] where:

[0155] y - area of ​​the chromatographic peak of EG;

[0156] x - EG concentration, µg / ml.

[0157] For Fig. 5:

[0158] DEG calibration curve:

[0159] y = 6344.1⋅x + 3643.7;

[0160] where:

[0161] y - area of ​​the chromatographic peak of DEG;

[0162] x - DEG concentration, μg / ml.

[0163] For EG: regression equation: y = 3066.8⋅x + 843.21, determination coefficient: R 2 = 0.9956;

[0164] For DEG: regression equation: y = 6344.1⋅x + 3643.7, determination coefficient: R2 = 0.9958.

[0165] The correlation coefficients were greater than 0.99, which indicates a high level of linearity of the method in the range from the detection limit (DL) to 150% of the content limit.

[0166] 4) Correctness.

[0167] Accuracy is the closeness of the obtained value to the true (reference) value, which is expressed as the detection rate. It characterizes systematic error.

[0168] Discoverability is the ratio between the obtained mean and true values, taking into account the corresponding confidence intervals.

[0169] The preparation of the solutions is described in step 3. There is no direct correlation between the obtained recovery value, as the samples represent different medicinal products with different compositions and matrices. The reference value is the concentration of the standard solution added to the test solution. The range of satisfactory values ​​is not described in the regulatory documents (since it is impossible to standardize this condition across a wide range of medicinal products and substances). For the determination of impurities using chromatographic methods, laboratories have adopted a range of 70-130%.

[0170] EG: For all three tested samples, the recovery rate was in the range of 74-101%, for DEG - in the range of 70-98%. All values ​​fell within the range of 70-130%, %RSD did not exceed 7.1%.

[0171] Satisfactory recovery rates for EG (74-101%) and DEG (70-98%) in test solutions supplemented with a standard solution fall within the range of 70-130%. Recovery is the ratio of the analyte concentration in the spike solution (if the analyte is present in the test solution, its concentration is subtracted from the obtained value) to the concentration of the standard solution added to the test solution. For clarity, the formula is as follows: where C Spike - concentration of the analyte in the test solution with the addition of a standard solution (Spike solution), C Smp - concentration of the analyte in the test solution, C Ad - the concentration of the standard sample that was added to the test solution (for each sample, its own pair of solutions is prepared - the test sample and a solution with the addition of a standard solution, since the composition and matrices of the original drugs are different).

[0172] 5) Precision.

[0173] Precision is an expression of the closeness (degree of dispersion) of results (values) between measurement series taken from the same homogeneous sample under the conditions specified by the method. The measure of precision is the standard deviation of the result of an individual determination, obtained for a sufficiently large sample. It was estimated based on the standard deviation (%RSD) of the chromatographic peak areas of EG and DEG after sequential six-fold injections of a standard solution. The standard deviation of the peak area for EG is 0.90%, and for DEG, 1.04%. The obtained values ​​satisfy the requirement of "no more than 20.0%."

[0174] 6) Stability of solutions.

[0175] Solution stability is an indicator reflecting the invariance of the chemical composition of samples during storage and analysis. The stability of the test solutions and the standard comparison solution was studied at room temperature. It was assessed as a change in the areas of chromatographic peaks over time. The solutions were sequentially injected into the chromatograph every 4 hours. The injection order was: solvent - standard solution - test solution - test solution with the addition of a standard solution. The relative change in the signal area of ​​the EG and DEG peaks in the standard solution does not exceed 20.0% 8 hours after the first injection. The relative change in the signal area of ​​the EG and DEG peaks in the test solution with the addition of a standard solution does not exceed 20.0% 8 hours after the first injection.

[0176] Validation summary results:

[0177] The method demonstrated a high level of sensitivity, which allows the determination of the specified impurities in trace amounts, and specificity, which allows for reliably confirming the presence / absence of the specified impurities even in complex matrices.

[0178] The sensitivity of the mass-selective detector is 2-3 times higher than that of the flame ionization detector (which is used to determine ethylene glycol and diethylene glycol in glycerol), high selectivity is achieved by controlling the specific mass / charge ratios and retention times characteristic of ethylene glycol and diethylene glycol.

[0179] Testing the method on real samples.

[0180] After development and validation, the method was tested on 20 real drug samples (the names are not provided to maintain confidentiality and avoid conflicts of interest). According to the test results, the extraction rate for ethylene glycol was 75.6 - 127.3% with a norm of 70.0 - 130.0% (average value 98.1%), for diethylene glycol - 80.1 - 121.0% with a norm of 70.0 - 130.0% (average value 101.1%); the signal-to-noise ratio for ethylene glycol was 95.43 - 1564.47 (average value 744.03) with a norm of at least 10, for diethylene glycol - 22.77 - 128.91 (average value 76.1) with a norm of at least 10; The standard deviation for ethylene glycol was 1.3 - 4.5% (average value 3.2%) with a norm of no more than 15.0%, for diethylene glycol - 1.8 - 4.7% (average value 3.5%) with a norm of no more than 15.0%.All obtained values ​​meet the requirements for validation of the chromatographic system, such a spread of signal / noise values ​​is normal for mass spectrometry and does not affect the test performance and interpretation of the results.

[0181] According to the analysis results, ethylene glycol was detected in 14 samples, and diethylene glycol was detected in 11 samples. However, their contents are in the trace range (approximately 0.001-0.002%), which is not toxic or hazardous to the patient. Examples of chromatograms are shown in Figs. 6-13.

[0182] Thus, the advantages of using the claimed method are:

[0183] - High sensitivity allows detection of trace amounts of impurities;

[0184] - Possibility of confirming the presence of ethylene glycol and diethylene glycol impurities not only in glycerin, but also in drugs with a complex matrix;

[0185] - High selectivity achieved by controlling the specific mass / charge ratios and retention times characteristic of ethylene glycol and diethylene glycol;

[0186] - Accuracy of indicators and the absence of false positive and false negative results in the analysis of ethylene glycol and diethylene glycol impurities in medicinal products.

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