CS242808B1 - A method for determining insulin composition and activity - Google Patents

Abstract

The analyzed sample is separated under suitable chromatographic conditions into bovine and porcine insulin and their monodesamido derivatives and their percentages are calculated from the respective elution curves. The biological activity of the analyzed insulin sample is determined by comparison with a suitable standard. The method is also suitable for the operational determination of insulin during production, starting from the first concentration of the alcoholic extract.

Description

The invention relates to a method for determining the composition and activity of insulin by high-performance liquid chromatography.

One of the hitherto used technological processes in the production of insulin is its isolation from animal pancreas, most often of bovine or pork origin. Depending on the biological material taken into production, either a monocomponent, e.g. bovine or porcine insulin, or a mixed insulin containing both types of insulin can be obtained. The percentage determination of the bovine or porcine insulin content and the corresponding monodesamidoderivatives produced during the production as decomposition products is of great importance in monitoring the production efficiency of different biological materials as well as in evaluating the qualitative composition and effectiveness of the insulin substances obtained and their pharmaceutical forms.

The current method of determining insulin, especially in substances and dosage forms, is based on biological tests in live rabbits or mice. This method of evaluating insulin preparations is material and time-consuming (one assay requires experiments on at least 40 rabbits and lasts 7 days; for mouse tests, 96 mice are required for one assay), laborious, inaccurate (± 10 deviation allowed) %), it does not provide any information on the qualitative composition of the samples and cannot be used for operative quality assurance of the insulin being produced.

These disadvantages are overcome by a method of determining insulin composition and activity by high performance liquid chromatography using a mobile phase consisting of a mixture of acetonitrile-aqueous buffer solvents at a column temperature of 35 ° C to 45 ° C and a mobile phase flow rate of 0.3 to 0.5 ml / min. characterized in that a buffer prepared from a primary amine of 6 to 12 carbon atoms in an amount of 0.15 to 2 ml / 1000 ml of water is added to the mobile phase, and the resulting aqueous suspension is dissolved

-2242 808 by adjusting the pH to 2, G to 3.0 by addition of phosphoric acid and determining by comparing the measured areas of the elution curves of the divided insulin components of the test sample with the standard.

The process according to the invention is further characterized in that an aqueous buffer prepared from 1.6 ml of 2-ethylhexylamine in 1000 ml of water, after adjustment to pH 2.2 with phosphoric acid, is added to the mobile phase in a ratio of 72.8% by volume. The assay was carried out by ultraviolet detection at a cherry length of 280 nm.

According to the method of the invention, the insulin sample of interest is chromatographically separated into its components. This separation is possible by the presence of a 2-ethylhexylamine buffer of pH 2.2 in the mobile phase. The areas of elution curves of the divided insulin components correspond to their percentage by mass in the analyzed sample and their percentage content can be calculated. By comparing the sum of the elution curves of all separate components of the test insulin to that measured with an insulin sample of known biological activity (e.g., a national standard), the biological activity of the insulin preparation being measured can be determined.

The greatest advantage of the method of the invention is that the determination of insulin composition and activity is not dependent on biological factor. Thus, the method according to the invention brings a great time saving and allows for the operative determination of insulin within 1 hour, not to mention the cost savings in the otherwise necessary breeding of rabbits and mice for biological tests. No less important is the fact that the method according to the invention achieves accurate results by a simple method (average deviation of the assay was 13%) / and thus allows accurate and rapid in-process control of substance activity prior to production of insulin injection forms. 40 et al in 1 ml solution for injection. The ability to detect the composition of insulin substances, including breakdown products, is also of great importance in the purchase or sale of insulin. The process of the invention has also been successfully used to control a thickened extract of biological material and other technological steps in the production of insulin; thus, production losses can be monitored and the technological processes hitherto used in insulin production can be optimized.

The method according to the invention is explained in more detail in the accompanying drawings. 1 shows the separation

- 3,242,808 of a standard insulin solution prepared according to point under the conditions of point E / depending on the timeline t (in minutes). Figure 2 shows an example of chromatographic separation of a sample of insulin injection solution prepared as described in Example 2 versus timeline t (in minutes) and Figure 3 shows an example of separation of an alcoholic extract sample from the production of insulin prepared as described in Example 3 again depending on the timeline t (in minutes).

In these figures, the peaks of the elution curves mean:

- bovine insulin

- desamide bovine insulin

- pork insulin

- desamide pig insulin

- preservative

The following examples illustrate the process according to the invention, but do not limit it in any way.

Example 1

A / Preparation of 2-ethylhexylamine buffer

Measure about 950 ml of distilled water into a 1000 ml beaker, add 1.6 ml of 2-ethylhexylamine from the graduated pipette and add a dilute phosphoric acid solution while stirring and checking the pH to a final pH of 2.2. Make up to 100 ml volume with distilled water.

B / Preparation of a standard insulin solution (for example from a national standard)

Weigh several mg of insulin standard into a small sealable vial on a microbalance and add a volume of 20% acidified acetonitrile from a precision microburette to a final concentration of 1 mg of standard in 1 ml of solution. After complete dissolution, add 25 µl of solution to the chromatography column.

C / Sample preparation of test insulin (substance)

Prepare the sample according to the previous procedure B and also add 25 µl of solution to the chromatography column.

D / Preparation of a 20% acetonitrile solution

Make up to 20 ml with acetonitrile in a 100 ml graduated flask

242 808 ~ 4 brand 0.1% phosphoric acid solution in distilled water.

E / Chromatography

Liquid chromatograph with a 25 x 0,3 cm or 30 x 0,4 cm column packed with 5 or 10 µm particle size modified octadecyl residues and a column temperature of 40 ° C. A solution of 27.2% v / v acetonitrile and 72.8% v / v aqueous 2-ethylhexylamine buffer was used as the mobile phase. UV photometric detection was performed at 280 nm and a sensitivity of 0.01 absorbance unit. The evaluation of the chromatographic record was performed by integrator and manual triangulation. A 25 µL insulin solution was loaded onto the column (see Fig. 1).

Calculation of substance activity (IU / mg):

sample activity = -9 i Y Í §-S du du ardu_x_suma_Bloch _- sample _- _ sum of standard areas

Example 2

Sample preparation of test insulin (injection)

Transfer the entire contents of the vial quantitatively to a sealable glass vial, add 10.0 ml of an acidified solution of 20% acetonitrile (prepared according to Example 1) and shake well. Load 25 µl per column, chromatography conditions as in Example 1, graph see Fig. 2.

Calculation of the activity of the solution for injection (IU / ml):

standard activity x sum of sample areas activity inj. solution --------------------------------------- x 2 sum of standard areas

Example 3

Sample preparation of alcoholic extract from insulin production

Filter the sample of the concentrated alcoholic pancreas extract through a G2 glass frit and add 25 µl of filtrate to the column. The activity of the sample in IU / ml of this solution is measured by an external standard method. Chromatography conditions as in Example 1, graph see Fig. 3.

Claims (2)

SUBJECT OF THE INVENTION 242 808 A method for determining the composition and activity of insulin by liquid chromatography using a mobile phase consisting of a mixture of acetonitrile type solvents and an aqueous buffer at a column temperature of 35 ° to 45 ° G and a mobile phase flow rate of 0.3 to 0.5 ml / min, by adding to the mobile phase an aqueous buffer prepared from a primary amine type of 6 to 12 carbon atoms in an amount of 0.15 to 2 ml / 1000 ml of water, the resulting aqueous suspension being dissolved by adjusting the pH to 2.0 »2. 3.0 by the addition of phosphoric acid and determined by comparison. measured areas of elution curves of the divided insulin components of the sample and the standard. 2. The method of claim 1, wherein an aqueous buffer prepared from 1.6 ml of 2-ethylhexylamine in 1000 ml of water, after adjusting to pH 2.2 with phosphoric acid, is added to the mobile phase at a ratio of 72.8. % by volume: 27.2% by volume of acetonitrile and determination is by ultraviolet photometric detection at 280 nm.