RU2167410C2 - Method of quantitative estimation of aliphatic amino acids - Google Patents

Abstract

FIELD: analytical chemistry, more particularly methods of quantitative estimation of aliphatic amino acids for inspecting quality of products manufactured by pharmaceutical industry and prepared by drugstores. SUBSTANCE: estimation is carried out by treating sample under study with 1% alcoholic solution of ninhydrin in buffer phosphate solution with pH from 6.4 to 7.6 in the presence of 1 mg of ascorbic acid. The resulting colored solution is diluted with water and optical density is measured by spectrophonometer using wave length of 568 nm in cell with layer thickness of 1 cm. EFFECT: higher sensitivity, simpler and cheaper estimation. 2 tbl

Description

 The invention relates to analytical chemistry, namely, to methods for the quantitative determination of aliphatic amino acids used to control the quality of products manufactured by pharmaceutical industries and manufactured by pharmacies.

The closest analogues are methods for the determination of amino acids using alloxanhydrate as a color reagent [A.S. 1191790 USSR, MKI ⁴ G 01 N 21/78; A.S. 731362 USSR, MKI ² G 01 N 21/24], alloxanthin followed by the addition of calcium salt [A.S. 1325335 USSR, MKI ⁴ G 01 N 21/78], perinaftindantrione-1,2,3 with the subsequent introduction of copper sulfate [A. from. 1432392 USSR, MKI ⁵ G 01 N 21/78]. The reaction is carried out in a medium of dimethylformamide, which for one determination consumes from 70 to 250 ml.

Using ninhydrin as a color reagent and carrying out the reaction in a phosphate buffer solution with a pH of 6.4 to 7.6 in the presence of 2 ml of a 0.05% aqueous solution (1 mg) of ascorbic acid can increase the sensitivity and make the determination simpler and cheaper. Ascorbic acid, being a sufficiently strong reducing agent (E ^o +0.18 V), restores part of ninhydrin, which helps to increase the sensitivity of the reaction and the stability of the resulting colored product.

 Example 1. Quantification of aminalon in a substance. An exact sample of the substance, previously dried to constant weight, in the range of 0.035-0.065 g is dissolved in water in a 250 ml volumetric flask, the solution volume is adjusted to the mark with the same solvent and mixed. 1 ml of the resulting solution is placed in a 100 ml volumetric flask, 4 ml of water, 4 ml of a phosphate buffer solution with a pH of 6.8 [5], 2 ml of a 1% solution of ninhydrin in 95% ethanol and 2 ml of 0.05% aqueous are added. ascorbic acid solution. The reaction mass is heated in a boiling water bath for 25 minutes, quickly cooled, the volume of the solution is adjusted to the mark with water and stirred. In parallel, an experiment is carried out with a solution of a working standard sample (RSO) of aminalon (0.05 g in 250 ml of water) and a control. The optical density of the colored solutions was measured against a background of control on a spectrophotometer at 568 nm in a cuvette with a layer thickness of 1 cm.

где A - оптическая плотность анализируемого раствора;
A₀ - оптическая плотность стандартного раствора;
a₀ - навеска РСО аминалона, г;
a - навеска субстанции аминалона, г.The content of aminalon (X), in percent, is calculated by the formula:

where A is the optical density of the analyzed solution;
A ₀ is the optical density of the standard solution;
a ₀ - a sample of the PCO of aminalon, g;
a - sample of aminalon substance, g.

 The results of the quantitative determination of aminalon in the substance are given in table. 2.

 Example 2. Quantitative determination of aminalon in 0.25 g coated tablets. An exact weighed powder of ground tablets containing aminalon in the range of 0.035-0.065 g is placed in a volumetric flask with a capacity of 250 ml, 100 ml of water are added and the contents of the flask are mixed for 10 minutes. The volume of solution in the flask was adjusted to the mark with water, stirred and filtered through a paper filter, discarding the first 10 ml of the filtrate. 1 ml of the filtrate is placed in a volumetric flask with a capacity of 100 ml, 4 ml of water are added, 4 ml of a phosphate buffer solution with a pH of 6.8 are added, and then proceed as described in example 1.

где A, A₀, a₀ - см. пример 1;
a - навеска порошка растертых таблеток, г;
P - средняя масса одной таблетки, г.The content of aminalon (X) in one tablet, in grams, is calculated by the formula:

where A, A ₀ , a ₀ - see example 1;
a - a sample of powder of powdered tablets, g;
P is the average weight of one tablet, g.

 The results of the quantitative determination of aminalon in 0.25 g coated tablets are given in table. 2.

Example 3. Quantitative determination of aminocaproic acid in solution for injection composition:
Aminocaproic acid - 5 g
Sodium chloride - 0.9 g
Water for injection - up to 100 ml
1 ml of the solution is placed in a volumetric flask with a capacity of 100 ml, the volume of the solution is adjusted to the mark with water and mixed. 1 ml of the resulting solution is placed in a volumetric flask with a capacity of 100 ml, 4 ml of water, 4 ml of phosphate buffer solution with a pH of 7.6 [5], 2 ml of a 1% solution of ninhydrin in 95% ethanol and 2 ml of 0.05% aqueous are added. ascorbic acid solution. Then proceed as described in example 1. At the same time, an experiment is carried out with a solution of OCO of aminocaproic acid (0.05 g in 100 ml of water) and a control.

где A, A₀, - см. пример 1;
a₀ - навеска РСО кислоты аминокапроновой, г.The content of aminocaproic acid (X) in 1 ml of solution, in grams, is calculated by the formula:

where A, A ₀ , - see example 1;
a ₀ - weighed portion of the PCO of aminocaproic acid,

 The results of the quantitative determination of aminocaproic acid in solution for injection are given in table. 2.

 Example 4. Quantitative determination of phenibut in tablets of 0.25 g. An exact weighed powder of ground tablets containing phenibut in the range of 0.025-0.055 g is placed in a volumetric flask with a capacity of 100 ml, 50 ml of water are added and the contents of the flask are mixed for 10 minutes. The volume of solution in the flask was adjusted to the mark with water, stirred and filtered through a paper filter, discarding the first 10 ml of the filtrate. 1 ml of the filtrate is placed in a 100 ml volumetric flask, 4 ml of water, 4 ml of a phosphate buffer solution with a pH of 6.4 [5], 2 ml of a 1% solution of ninhydrin in 95% ethanol and 2 ml of a 0.05% aqueous solution are added. ascorbic acid and then proceed as described in example 1. At the same time, an experiment is conducted with a solution of PHO phenibut (0.05 g in 100 ml of water) and a control.

где A, A₀, - см. пример 1;
a, P - см. пример 2;
a₀ - навеска РСО фенибута, г.The content of phenibut (X) in one tablet, in grams, is calculated by the formula:

where A, A ₀ , - see example 1;
a, P - see example 2;
a ₀ - weighed portion of the RNO of phenibut,

 The results of the quantitative determination of phenibut in tablets of 0.25 g are given in table. 2.

 Comparative characteristics of the proposed method with the known are presented in table. 1.

 When comparing the open minima, it is seen that the proposed method is more sensitive (2-4 times). The use of ninhydrin as a color reagent contributes to the accessibility and widespread introduction of the developed method in the practice of laboratories and pharmacies. The use of dimethylformamide is excluded, which makes the method more economical.

Sources of information
1. A.S. 1432392 USSR, MKI ⁵ G 01 N 21/78. A method for determining aminalon / V. D. Yanchuk, V. V. Petrenko, Yu.T. Rotberg (USSR). - N 3974618 / 28-4; Claim 11/10/85; Publ. 10.23.88; Bull. N 39.

2. A.S. 1191790 USSR, MKI ⁴ G 01 N 21/78. The method for determining aminocaproic acid / L.D. Ryabykh, V.V. Trokhimchuk (USSR). - N 3722585 / 23-04; Claim 04/06/84; Publ. 11/15/85; Bull. N 42.

3. A.S. 1325335 USSR, MKI ⁴ G 01 N 21/78. The method for determining glutamic acid / V.D. Yanchuk, V.V. Petrenko, N.A. Klyuev (USSR). - N 3988025 / 28-04; Claim 12/12/85; Publ. 07/23/87; Bull. N 27.

4. A.S. 731362 USSR, MKI ² G 01 N 21/24. Method for the quantitative determination of methionine / B. V. Petrenko (USSR). - N 2557305 / 23-04; Claim 12/05/77; Publ. 04/30/80; Bull. N 16.

 5. State Pharmacopoeia of the USSR: Issue. 1. General methods of analysis / Ministry of Health of the USSR. - 11th ed., Ext. - M .: Medicine, 1987.

 6. Petrenko V.V. Polycarbonyl compounds in the pharmaceutical analysis of nitrogen-containing drugs. Auth. diss. ... doc. farm. sciences. - Kharkov, 1984. - 38 p.

7. FS 42-1768-96. Phenibut Tablets ^® 0,25 g

Claims (1)

 A method for the quantitative determination of aliphatic amino acids by processing the analyzed sample with a color reagent when heated, followed by spectrophotometry of the obtained colored solution, characterized in that the sample is treated with a 1% alcohol solution of ninhydrin in a phosphate buffer solution with pH = 6.4 - 7.6, in the presence of 1 mg of ascorbic acid, dilute the resulting colored solution with water, and the optical density is measured at a wavelength of 568 nm.

Images