RU2700167C1 - Method for quantitative determination of aminocaproic acid in its simultaneous presence with a copolymer of 2-methyl-5-vinylpyridine and n-vinylpyrrolidone by the method of hplc - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry and provides a method for quantitative determination of aminocaproic acid (ACA) when combined with copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone (CP) in various ready dosage forms, such as, for example, nasal drops or sprays. That is ensured by preparing calibration solutions of ACA of pharmacopoeial quality with concentrations the values of which are at the limits of tolerable deviations, as well as in the area of impermissible deviations. Accumulation of ACA is carried out by reversed-phase HPLC in an ion-pair version on a column with length of 250 mm and an inner diameter of 4.6 mm, filled with a fixed phase formed by spherical particles of silicon dioxide with size of 5 mcm, modified octadecylsilane in a gradient mode, mobile phase A contains (1 % H₃PO₄+10 mmol PSNa+15 mmol K₂NRA₄ in H₂O): methanol in ratio of 70:30, and mobile phase B is methanol. ACA detection is carried out spectrophotometrically at wavelength of 210 nm and calculating arithmetic mean values of area under ACA curve (AUC) with subsequent approximation of linear function to AUC=kc_ACA to calculate coefficient k. Drug solution is diluted with mobile phase A to 0.5–5 mg/ml, chromatographed at least 3 times. After the samples are analyzed, a conclusion is made on the compliance of the preparation with the requirements for the content of ACA.

EFFECT: invention provides rapidness of analysis, as well as reduced labor and cost of quantitative determination of ACA.

1 cl, 5 dwg, 2 tbl, 2 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of analysis of drugs, dietary supplements, cosmetic and food products. More specifically, the invention provides a method for the quantitative determination of aminocaproic acid (ACA) when it is combined with a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone (SP) by high performance liquid chromatography (HPLC) in various formulations, such as, for example, nasal drops or sprays and solutions for injection or infusion.

State of the art

Aminocaproic acid (ACC, 6-aminohexanoic acid) is a synthetic analogue of the natural amino acid lysine and is capable of exerting a systemic hemostatic effect in bleeding caused by increased plasma fibrinolytic activity due to inhibition of profibrinolysin activators and inhibition of its conversion to fibrinolysin. It also has a direct inhibitory effect on fibrinolysin. In addition, it reduces the permeability of capillaries, has antiallergic activity, and improves the antitoxic function of the liver.

Due to its properties, ACC is used in surgical practice to prevent bleeding during surgical interventions on organs rich in fibrinolysis activators (brain and spinal cord, lungs, heart, blood vessels, thyroid and pancreas, prostate gland) and in case of complicated abortion. It is also indicated for diseases of internal organs with hemorrhagic syndrome.

The use of ACC as an antiviral agent is also known. The drug AKK is used to prevent and treat influenza and acute respiratory viral infections, creating a barrier to viruses, effectively blocking the penetration of pathogens into the cells of the nasopharynx and lungs (RU No. UA / 11103/01/01 of 10.24.2014. Order No. 771 of 10.24.2014. g., Ukraine).

It is known that for the quantitative determination of ACC by chromatographic methods in complex matrices, its preliminary derivatization is carried out. In an article by Nagy A. Farid. Fluorescamine Use in High-Performance Liquid Chromatographic Determination of Aminocaproic Acid in Serum // Journal of Pharmaceutical Sciences. Vol. 68, No. 2 (1979) Pp. 249-252, it has been proposed to use fluorescamine (also known as fluram or 4'-phenylspiro [2-benzofuran-3,2'-furan] -1,3'-dione), which does not possess fluorescence but quickly reacts with primary amines with the formation of intensely fluorescent pyrrolinone compounds with an absorption maximum of about 390 nm and a maximum of fluorescence in the region of 475 nm.

The authors of the article indicate that model solutions (50-600 mg / L) were prepared to determine the ACC, for which a linear dependence of the analytical signals (both absorption and fluorescence) on the concentration in the isocratic elution mode was observed. The eluent contained 44% acetonitrile in 0.5 mm H ₃ PO ₄ . In five studies of a human blood serum sample (400 mg / L AKK), the AKK content of 403.6 ± 4.4 mg / L was experimentally found, which confirms the correctness of the method when applied to real matrices.

Another reagent often used to derivatize primary amines before chromatographic determination is dansyl chloride (5- (dimethylamino) naphthalene-1-sulfonyl chloride, DNS-Cl). So in the publication S.A. Lau-Cam & RW Roos (1993) Assay of Aminocaproic Acid in Dosage Forms by Reversed Phase High Performance Liquid Chromatography with Dansylation // Journal of Liquid Chromatography. Vol. 16 No. 2 (1993). Pp. 403-419 describes a method for determining ACC in tablets (500 mg), injection solutions (250 mg / ml) and syrups (0.25 mg / ml) comprising treating the sample with excess DNS-Cl and removing unreacted DNS-Cl by adding monoethanolamine. Chromatography with spectrophotometric detection of the analytical signal is carried out in isocratic mode (mobile phase: methanol / water / acetic acid / triethylamine 60: 38: 1.5: 0.5) on a column 150 mm long and 4.6 mm inner diameter with a stationary phase Econosphere C ₁₈ (5 μm). The main disadvantage of the method is its low expressivity, since the ACA reaction with DNS-Cl is completed in about 30 minutes, removal of excess DNS-Cl takes about 15 minutes, while it takes about 5 minutes to elute the derivatized ACC. In addition, the use of DNS-Cl increases the cost of analysis.

Thus, there is a need for a more rapid and less costly method of quantifying ACA in a particular matrix formed by a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone and commonly used pharmaceutically acceptable excipients.

Disclosure of the invention

The object for the chromatographic analysis of which the present invention is intended is a pharmaceutical composition for the prophylaxis of influenza virus and SARS in the form of a solution for topical use, containing aminocaproic acid and a copolymer of 2-methyl-5-vinylpyridine with N-vinylpyrrolidone (SP), phosphate buffered components solution, preservative and purified water, in the following ratios of components (wt.%):

ACC 0.1-5 Joint venture 0.1-1 components of phosphate buffered saline 0.5-1.5 preservative 0.05-1.5 purified water up to 100.

Sodium benzoate or benzalkonium chloride can act as a preservative. The composition is presented in the form of two variants of a complex nasal spray containing 1% (mass.) Or 3% (mass.) ACC and 0.1% (mass.) SP.

In accordance with the present invention, there is provided a method for the quantitative determination of aminocaproic acid (ACA) by high performance liquid chromatography (HPLC) with the combined presence of ACA with a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone (SP) by the calibration function, for the implementation of which:

a) prepare calibration solutions from ACC of pharmacopoeial quality with concentrations whose values are equally spaced from the nominal ACC concentration in the preparation and are at the borders of permissible deviations, as well as in the region of unacceptable deviations;

b) prepare the mobile phase A (PF A) of 1% H ₃ PO ₄ + 10 mmol PSNa + 15 mmol K ₂ NRA ₄ in H ₂ O / methanol 70:30, degass it and treat it with ultrasound;

c) chromatography of calibration solutions of reverse-phase HPLC in the ion-coupled version in gradient mode is performed by the following program:

where the mobile phase B (PF B) is methanol, at least 3 times for each sample on a column with a length of 250 mm and an inner diameter of 4.6 mm, filled with a stationary phase formed by spherical particles of silicon dioxide with a size of 5 μm, modified with octadecylsilane, with spectrophotometric detecting an analytical signal in the form of absorption at a wavelength of 210 nm;

d) determine the ACC retention time, calculate the arithmetic mean values of the areas under the ACC output curves (AUC) for all concentrations, approximate the obtained AUC values by a linear function of the form

AUC = AUC ₀ + kc _AKK ,

confirm the insignificance of the term AUC ₀ , and then re-approximate the obtained AUC values by a linear function of the form

AUC = kc _AKK

and determine the value of k;

d) prepare a solution of the drug for research, diluting, if necessary, the initial drug with the mobile phase PF A to 0.5-5 mg / ml, assuming that the drug meets the requirements for the content of ACC;

f) chromatograph the solution of the preparation for research under the conditions specified in subparagraph c) at least 3 times and identify the ACC on the output curve by the retention time;

g) calculate the arithmetic mean of the area under the output ACC curves (AUC) for all chromatographic studies and calculate with _AKK as

h) formulate a conclusion on the compliance of the drug with the requirements for the content of ACC.

Preferably, in said method, the stationary phase is the NUCLEODUR C ₁₈ Pyramid phase.

The technical result of the invention is to increase the expressness of the analysis, i.e. in reducing the time required to carry out one determination. Achieving this result will increase the useful life of the chromatograph. In addition, additional advantages of the invention are to reduce the complexity and cost of quantitative determination of ACC.

Brief Description of the Drawings

In FIG. 1 presents a diagram of the preparation of calibration solutions.

In FIG. Figure 2 shows a general view of the HPLC output curve for a sample obtained by tenfold dilution of an aliquot of a 3% complex nasal spray based on AKK and SP.

In FIG. Figure 3 shows an enlarged scale view of the ACC output curve for a calibration ACC sample with a concentration of 3 mg / ml.

In FIG. Figure 4 gives a graphical and analytical representation of the HPLC calibration function with the values of the parameter k, the correlation coefficient R, and the residual sum of squares S _D for the analysis of samples of the 3% complex nasal spray based on ACC and SP.

In FIG. Figure 5 gives graphical and analytical representations of the HPLC calibration function with the values of the parameter k, the correlation coefficient R, and the residual sum of squares S _D for the analysis of samples of the 1% complex nasal spray based on ACC and SP.

The implementation of the invention

As a result of extensive research, the authors of the present invention unexpectedly found that carrying out chromatographic separation in a gradient mode allows to achieve complete separation of the analyte (ACC) and the matrix (SP + excipients) without prior derivatization, for example, treatment with dansyl chloride.

Thus, in accordance with the invention, a method based on the separation of the ACC and the copolymer of the joint venture and further determination of the concentration of the ACC of reverse phase HPLC in the ion-coupled version in a gradient mode with spectrophotometric detection of the analytical signal, which is the absorption at a wavelength of 210 nm, is proposed. The working concentration of SP in the samples after sample preparation is at the level of 1 mg / ml (20 μg / input), which under the conditions of HPLC analysis ensures complete elution of the main amount of the copolymer from the column and ensures a long service life of the separation element.

The method includes dissolving a weighed portion of the test drug in the mobile phase (PF), separating aminocaproic acid and a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone on a chromatographic column, and further determining the ACC concentration by the equation of the calibration function AUC = ƒ (c _AKK ), where AUC represents the area under the ACC output curve for its various concentrations corresponding to unacceptable and maximum permissible concentration limits, as well as the concentration of ACC in the preparation that fully complies with ebovaniyam.

All components of the mobile phases have a purity of “pure for HPLC”. As the mobile phase A (PF A), a mixture of the aqueous part (aqueous solution of phosphoric acid 1%, 10 mmol of sodium pentyl sulfonate and 15 mmol of K ₂ NRA ₄ in H ₂ O) with methanol in the ratio of 70:30 is used, as the mobile phase B use methanol.

To implement the method, at least 5 calibration solutions are prepared according to the scheme shown in FIG. one.

Further, the possibility of carrying out the invention with the achievement of technical results will be shown in non-limiting examples.

Example 1. Preparation of the mobile phase (PF), standard solutions and their chromatographic analysis of the drug "Complex nasal spray 3%"

1.1. Preparation of the mobile phase

To obtain PF A, its aqueous part is first prepared, for which 10 mmol of sodium pentyl sulfonate (PSNa) and 15 mmol of potassium hydrogen phosphate are added to a 1% aqueous solution of phosphoric acid, setting the pH to 2.0-2.2, and then added to it methanol as will be described below.

In a 1000 ml volumetric flask, 1740 ± 0.5 mg of sodium pentyl sulfonate and 4560 ± 0.5 mg of K ₂ NRA ₄ ⋅ 3H ₂ O are added, 800 ml of distilled water are added, approximately 10 ml of concentrated phosphoric acid are added, mixed thoroughly and the value is set pH 2.0-2.2 by the addition of a small volume of phosphoric acid. The volume of the solution in the flask was adjusted to 1000 ml with distilled water and again thoroughly mixed.

In a measuring cylinder with a capacity of 1000 ml with a division value of 10 ml, 700 ml of the resulting solution is placed; in a measuring cylinder with a volume of 500 ml with a division price of 10 ml, 300 ml of methanol is placed. Measured volumes of solutions are combined in a vessel of suitable capacity and mixed thoroughly. The mobile phase A [(1% H ₃ PO ₄ + 10 mmol PSNa + 15 mmol K ₂ NRA ₄ in H ₂ O) / methanol 70:30] is degassed by passing through a 0.45 μm membrane nylon filter and sonicated within 5 minutes.

Phase B. Methanol (HPLC Gradient Grade).

1.2. Preparation of calibration solutions of aminocaproic acid

Calibration solutions are prepared from pharmacopoeial aminocaproic acid, dissolving weights of 25.0 mg, 50.0 mg, 75.0 mg, 100.0 mg and 125.0 mg of aminocaproic acid, taken with an accuracy of ± 0.1 mg in separate volumetric flasks with a capacity of 25 ml, add 20 ml of PF A, placed in an ultrasonic bath for 3-5 minutes until the acid is completely dissolved and the volume of the solution in the flask is adjusted to the mark by adding PF A. Thus, calibration solutions with AKA contents equal to 1 are obtained; 2; 3; 4 and 5 mg / ml.

1.3. Determination of the parameters of the calibration function

To determine the parameters of the calibration function, each of the calibration solutions is chromatographed at least three times with spectrophotometric detection at a wavelength of 210 nm on a NUCLEODUR C ₁₈ Pyramid column (Macherey-Nagel GmbH, Germany, length 250 mm, internal diameter 4.6 mm) filled with spherical particles of 5 microns in size. They work in gradient mode according to the following program:

Numerically integrate the areas under the curves of the dependence of the analytical signal on the elution time and obtain the values of AUC _i (i = 1, 2, 3 ...). The arithmetic average of the AUC for each solution is calculated. AUC values for various with _ACC are given in table 1.

Approximation of the obtained AUC values by a linear function of the form

AUC = AUC ₀ + kc _AKK

using the least squares method gives the following values of its parameters:

AUC ₀ = 0.1291 ± 0.2546

k = 4.5106 ± 0.0767,

wherein the correlation coefficient R is 0.9996, and the residual sum of squares S _D is 0.2427.

From a comparison of the calculated values of the parameters and their errors, it follows that the term AUC ₀ is insignificant, that is, there is no systematic error in the method for determining the concentration of ACC. Given this and taking AUC ₀ identically equal to zero, the refined value of k is determined to be 4.5448 ± 0.1472, while the correlation coefficient R is 0.9991, and the residual sum of squares S _D is 0.2445.

1.4 the Preparation of a solution of the drug "Complex nasal spray 3%" for research

An aliquot of the test drug with a volume of 5.0 ml is placed in a 50 ml volumetric flask, 30 ml of PF A are added, closed with a lid and mixed thoroughly, if necessary, the pH is adjusted to 2.0 with 1 M phosphoric acid solution, the volume of the solution in the flask is adjusted to the mark by adding PF A and re-mixed thoroughly.

1.5 Chromatographic quantification of ACC in the preparation of "Complex nasal spray 3%"

A sample of the test drug solution is chromatographed 3 times with spectrophotometric detection at a wavelength of 210 nm on a NUCLEODUR C ₁₈ Pyramid column (length 250 mm, internal diameter 4.6 mm) filled with spherical particles 5 microns in size. They work in gradient mode according to the following program:

By retention time of 3.51 min. the chromatograms identify ACC signals and determine their area. Quantitative determination of ACC is carried out by the calibration function. The result of chromatographic analysis is the arithmetic mean of the concentrations. The ACA content in the test solution is 3.02 ± 0.03 mg / ml, and the ACA content in the drug solution, taking into account the dilution, is 3.02% (mass.). Thus, the drug exactly meets the requirements for the content of ACC.

Example 2. Preparation of the mobile phase (PF), standard solutions and their chromatographic analysis of the drug "Complex nasal spray 1%"

2.1. Preparation of the mobile phase

The mobile phase is prepared as described in section 1.1 of example 1.

2.2. Preparation of calibration solutions of aminocaproic acid

Calibration solutions are prepared from pharmacopoeial aminocaproic acid, dissolving weighed portions of 20.0 mg, 35.0 mg, 50.0 mg, 65.0 mg and 80.0 mg of aminocaproic acid, taken with an accuracy of ± 0.1 mg in separate volumetric flasks with a capacity of 50 ml, add 40 ml of PF A, place in an ultrasonic bath for 3-5 minutes until the acid is completely dissolved and adjust the volume of the solution in the flask to the mark by adding PF A. Thus, calibration solutions with AKA contents of 0.4 are obtained; 0.7; 1.0; 1.3 and 1.6 mg / ml.

2.3. Determination of the parameters of the calibration function

The parameters of the calibration function are calculated according to the results of chromatographic analysis of calibration samples prepared as described in section 2.2, as described in section 1.3 of Example 1. The AUC values for various with _ACC are shown in Table 2.

Approximation of the obtained AUC values by a linear function of the form

AUC = AUC ₀ + kc _AKK

using the least squares method gives the following values of its parameters:

AUC ₀ = 0.1430 ± 0.2904

k = 5.0113 ± 0.2674,

wherein the correlation coefficient R is 0.9959, and the residual sum of squares S _D is 0.2536.

From a comparison of the calculated values of the parameters and their errors, it follows that the term AUC ₀ is insignificant, that is, there is no systematic error in the method for determining the concentration of ACC. Given this and taking AUC ₀ identically equal to zero, the refined value of k is determined to be 5.1325 ± 0.0940, while the correlation coefficient R is 0.9910, and the residual sum of squares S _D is 0.3147.

2.4 Preparation of a solution of the drug "Complex nasal spray 1%" for research

A study drug solution is prepared as described in section 1.4 of Example 1.

2.5 Chromatographic quantification of ACC in the preparation of "Complex nasal spray 1%"

A sample of the test drug solution is chromatographed 3 times as disclosed in section 1.5 of Example 1. At a retention time of 3.49 minutes. the chromatograms identify ACC signals and determine their area. Quantitative determination of ACC is carried out by the calibration function. The result of chromatographic analysis is the arithmetic mean of the concentrations. The ACC content in the test solution is 1.04 ± 0.05 mg / ml, and the ACC content in the drug solution, taking into account the dilution, is 1.04% (mass.). Thus, the content of AKA in the drug is higher than the nominal, but, nevertheless, is in the acceptable range. In other words, the drug meets the requirements for the content of ACC.

Claims (17)

1. The method for the quantitative determination of aminocaproic acid (ACC) by high performance liquid chromatography (HPLC) in the joint presence of ACC with a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone (SP) by the calibration function, characterized in that for its implementation: a) prepare calibration solutions from ACC of pharmacopoeial quality with concentrations whose values are equally spaced from the nominal ACC concentration in the preparation and are at the borders of permissible deviations, as well as in the region of unacceptable deviations; b) prepare the mobile phase A (PF A) of the composition (1% H ₃ PO ₄ + 10 mmol PSNa + 15 mmol K ₂ NRA ₄ in H ₂ O) / methanol 70:30, degass it and treat it with ultrasound; c) chromatography of calibration solutions of reverse-phase HPLC in the ion-coupled version in gradient mode is performed by the following program:

where the mobile phase B (PF B) is methanol, at least 3 times for each sample on a column with a length of 250 mm and an inner diameter of 4.6 mm, filled with a stationary phase formed by spherical particles of silicon dioxide with a size of 5 μm, modified with octadecylsilane, with spectrophotometric detecting an analytical signal in the form of absorption at a wavelength of 210 nm; d) determine the ACC retention time, calculate the arithmetic mean values of the areas under the ACC output curves (AUC) for all concentrations, approximate the obtained AUC values by a linear function of the form AUC = AUC ₀ + kc _AKK , confirm the insignificance of the term AUC ₀ , and then re-approximate the obtained AUC values by a linear function of the form AUC = kc _AKK , and determine the value of k; d) prepare a solution of the drug for research, diluting, if necessary, the initial drug with the mobile phase PF A to 0.5-5 mg / ml, assuming that the drug meets the requirements for the content of ACC; f) chromatograph the solution of the preparation for research under the conditions specified in subparagraph c) at least 3 times and identify the ACC on the output curve by the retention time; g) calculate the arithmetic mean of the area under the output ACC curves (AUC) for all chromatographic studies and calculate with _ACC as

h) formulate a conclusion on the compliance of the drug with the requirements for the content of ACC. 2. The method according to p. 1, characterized in that the stationary phase is the NUCLEODUR C ₁₈ Pyramid phase.

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