RU2416792C1 - Method of ciprofloxacin evaluation in drug preparations - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: method of ciprofloxacin evaluation in drug preparations by isocratic reverse phase HPLC with using a flow metre detector consists in preparing two samples of the same volume of an investigated diluted solution one of which is added with a standard additive agent of ciprofloxacin, while each solution is added with the reagent -cyclodextrin, analysed in a chromatograph using mixed acetonitrile: ammoniac acetate buffer (43:57 vol./vol., pH 4.3) as a mobile phase, measuring a chromatographic peak area, and comparing the measured values to evaluate the antibiotic concentration by a design formula. ^ EFFECT: invention allows lowering a drug ciprofloxacin detection limit in more than 8 times. ^ 3 tbl

Description

The invention relates to high-performance liquid chromatography (HPLC) with a fluorimetric detector using organized media that allows more than 8-fold lower the detection limit of ciprofloxacin in drugs and can be used in the analysis of multicomponent drugs in the practice of control and analytical laboratories of pharmaceutical enterprises and the pharmacy network .

Ciprofloxacin belongs to the group of fluoroquinolones derived from 4-quinolone-3-carboxylic acid and is widely used in clinical practice. Various methods are known for determining ciprofloxacin and other antibiotics of the fluoroquinolone group: microbiological, for example, diffusion into agar, spectrophotometric, chromatographic, for example high performance liquid chromatography.

Pharmacopoeial method [Firsov A.A., Alekseeva M.E., Kuleshov S.U. // Chem.-farm. journal 1995. No3. C.24] definitions based on a microbiological test, including diffusion of the antibiotic into agar (culture medium) and comparison of growth inhibition of the test microorganism by certain concentrations of the test drug with antibiotic standards. The minimum detectable concentration is 0.05 μg / ml. Microbiological methods are widespread, simple and do not require expensive equipment. In addition, they provide satisfactory sensitivity and accuracy at therapeutic concentrations of fluoroquinolones in the test media.

However, they are characterized by the duration of the analysis, the dependence of the analytical signal on the properties of the antibiotic (its solubility, molar mass, etc.), not related to its activity, the sensitivity of the test cultures to the quality of the agar used in the method.

The spectrophotometric determination method is based on the formation of complexes with Cu ²⁺ or Fe ³⁺ ions [Kapetanovic V., Milovanovic I., Erceg M. / Talanta. 1996. V. 43. R.2123 .; Mathur SC, Kumar Y., Murugesan N. // Indian Drugs. 1992. V.29. P.376], as well as ionic associates with organic reagents of the sulfophthalein series [Tosunoglu S., Savci N. // Acta Pharm. Turc. 1993. V.35. P.1; Anal. Abstr. 1994. V. 56. 3G 49]. This method determines from 3 to 25 mg / ml of ciprofloxacin, low sensitivity and low selectivity are a drawback of this method, and in the extraction version it is necessary to use toxic organic solvents.

Closest to the proposed technical solution is the method of high performance liquid chromatography (HPLC) for the determination of ciprofloxacin in cow's milk [Tyczkowska K.L., Voyksner R. D., Anderson K. L. //J.Chromatogr. B. 1994. V.658. P.341] using a photodiode detector.

The disadvantages of the method include the duration of the determination (separation time 15-20 minutes), as well as the use of highly toxic methanol as the mobile phase.

The objective of the invention is to lower the detection limit and expand the spectrum of the studied objects - a mixture of antibiotics of the group of fluoroquinolones, quinolones and tetracyclines, for example, in dosage forms, as well as the functionality of the method due to the use of organized cyclodextrins, which modify the mobile phase and increase the intensity of the analytical signal of the antibiotic when the formation of the inclusion complex of the antibiotic composition: γ-cyclodextrin 1: 1.

The technical result consists in increasing the detection sensitivity while reducing the time required for analysis, and reducing toxicity.

The problem is solved in that in the method for the quantitative determination of ciprofloxacin in drugs, which consists in preparing two identical volumes of samples of a diluted test solution, γ-cyclodextrin is added and one of the samples is injected with a standard additive of ciprofloxacin, analyzed sequentially on a liquid chromatograph with a fluorescent detector and reversed-phase chromatographic column in isocratic mode using acetonitrile: acetate-amm as the mobile phase egg buffer (43:57 v / v, pH 4.3). Ciprofloxacin retention time 2 min. Calculation of the concentration produced by the formula

C _x = C _st · S ₁ / S ₂ -S ₁ ,

where C _st is the standard supplement of ciprofloxacin mg / ml;

S ₁ is the peak area of ciprofloxacin in the test solution;

S ₂ is the peak area of ciprofloxacin in the test solution with a standard additive;

C _x is the desired concentration of ciprofloxacin in the drug mg / ml.

Adding the indicated reagent causes the formation of an inclusion complex of the composition antibiotic: γ-cyclodexirin 1: 1. This lowers the detection limit of the desired substance.

The initial solution of ciprofloxacin 0.25 mg / ml was prepared by dissolving 0.0125 g of its powder, with a basic substance content of at least 98% in 50 ml of distilled water. A solution of γ-cyclodexirin with a concentration of 1 · 10 ^-2 M was prepared by dissolving 0.0324 g of dry matter in at least 98% of the main component in 25 ml of distilled water. The reaction is carried out in an acetate-ammonia buffer solution in a slightly acidic medium with pH 4-5. To build a grading plot, 1 ml of ammonium acetate buffer solution was added to each of the eight tubes. 0.25 ml of 5 · 10 ^-5 M γ-cyclodexirin, then various volumes of the ciprofloxacin initial solution in the concentration range from 0.10-100 μg / ml were diluted with buffer solution to a total volume of 5 ml and an aliquot of 50 μl was taken from each tube, which was introduced into the chromatograph . A mixture of acetonitrile: acetate-ammonia buffer (43:57 v / v, pH 4.3) was used as the mobile phase, ciprofloxacin retention time 2 min. The calibration graph was built in coordinates of the peak area - the concentration of ciprofloxacin. Table 1 presents some metrological characteristics of the determination of ciprofloxacin.

Table 1
Metrological characteristics of the determination of ciprofloxacin in the absence and presence of γ-cyclodextrin Component to be defined Reagent The range of determined concentrations, μg / ml Pro, μg / ml Calibration Graph Equation r ² Ciprofloxacin - 2,5-100 1.7 y = 17.6x + 80.6 0,998 γ-cyclodextrin 0.25-75 0.2 y = 28.5x + 64.5 0,997 Ciprofloxacin with antibiotics - 2,5-100 1.4 y = 15.4x + 220 0,996 γ-cyclodextrin 0.25-75 0.2 y = 26.6x + 64.5 0,997

As can be seen from the table, the detection limit of ciprofloxacin in the presence of γ-cyclodextrin decreases by more than 8 times.

Retention time is 2 minutes, which reduces analysis time. In addition, a less toxic acetonitrile is used instead of a toxic methanol solvent.

Determination of CF in drugs. The developed method has been tested on dosed medicines containing ciprofloxacin - eye drops of various production: Tsiprolet, Dr. Reddy's laboratories (India), Tsiprofloxacin AKOS Synthesis (Kurgan), Tsipromed Promed Exports (India). In addition to the determined ciprofloxacin, the drug contains auxiliary substances: ethylenediaminetetraacetic acid, mannitol, sodium acetate, acetic acid.

Method of determination.

The test solution of eye drops is diluted 25 times. An aliquot of 0.2 ml was taken from the resulting solution, 0.25 ml of 5 · 10 ^-3 M γ-cyclodextrin was added and diluted to a total volume of 5 ml with ammonium acetate buffer solution, pH 4.3. An aliquot of the resulting solution of 50 μl is introduced into the chromatograph, the area of the chromatographic peak of ciprofloxacin is measured, and the ciprofloxacin content of the drugs is determined from the calibration graph.

A second implementation option is possible. The test solution of eye drops is diluted 25 times. Aliquots of 0.2 ml are taken in two tubes from the resulting solution, 5 · 10 ^-3 M γ-cyclodextrin is added to each 0.25 ml and a standard ciprofloxacin additive (20-30 μg / ml) is added to one of them, the solutions are diluted to a total volume of 5 ml acetate-ammonia buffer solution pH 4. Aliquots of the resulting solutions are added 50 μl sequentially to the chromatograph, the areas of chromatographic peaks of ciprofloxacin for both samples are measured, and the ciprofloxacin content in the drugs is determined by the calculation formula

C _x = 25 · C _st · S ₁ / S ₂ -S ₁ ,

where C _st is the standard supplement of ciprofloxacin mg / ml;

S ₁ is the peak area of ciprofloxacin in the test solution;

S ₂ is the peak area of ciprofloxacin in the test solution with a standard additive;

C _x is the desired concentration of ciprofloxacin in the drug mg / ml.

The results of the determination are presented in table 2.

table 2 The results of the determination of ciprofloxacin in drug preparations of eye drops * (n = 5, P 0.95) A drug Found, mg / ml S _r Tsiprolet, India 2.62 ± 0.07 0.03 Ciprofloxacin, Kurgan 2.49 ± 0.15 0.06 Tsipromed, India 2.29 ± 0.05 0.02

where S _r is the relative standard deviation, S _r S / χ, where S is the standard deviation equal to S = ∑ (χ _i -χ) ² / n-1) ^Ѕ , χ _i is the unit determination result, χ is the average result, ^* - the content of CF specified by the manufacturer is 3 mg / ml.

Control of the correctness of the determination was carried out by the method of standard additives.

The error of determination does not exceed 0.06.

The proposed method can be used to determine ciprofloxacin in the presence of other antibiotics - oxytetracycline, tetracycline, nalidixic acid and flumequin. The results of the determination, the retention time of the provided antibiotics and the characteristics of their chromatographic separation are presented in table 3.

Table 3
Quantitative chromatographic characteristics of antibiotics in the presence of γ-CD Antibiotic Retention time, min Peak area R _s α TO' N Fluoroquinolones CF 2.0 810.0 2,4 1.7 1,0 6.30 · 10 ³ FL 12.8 47.25 11.9 11.6 5.3 1.22 · 10 ³ TETRACYCLINES TT 2.7 6.90 2,4 1.7 1.7 1.13 · 10 ³ OTT 6.2 642.7 5.2 5.3 2,8 2.9610 ³ Quinolones NK 3.8 534.3 4.8 2,8 12 1.54 · 10 ³

R _S is the resolution of the peaks, R _S = (t _R2 -t _R1 ) / {(w ₁ + w ₂ ) / 2}, where t _R2 , t _R1 is the retention time of two adjacent peaks, w ₁ , w ₂ is the width of the peaks measured at the bases;

α is the selectivity coefficient, α = (t _R -t _m ) ₁ / (t _R -t _m ) ₂ , where t _R is the antibiotic retention time, t _m is the antibiotic residence time in the mobile phase;

K 'is the retention factor, (t _R -t _m ) / t _m ), where t _R is the antibiotic retention time, t _m is the antibiotic residence time in the mobile phase;

N is the number of theoretical plates, N = 16 (t _R / W) ² , t _R - retention, W - peak width at the base.

As the mobile phase, the mobile phase is used - a mixture of acetonitrile: acetate-ammonium buffer in the ratio of 43:57 vol./about., PH = 4.3.

The correctness of the methodology was evaluated by the results of the analysis of model solutions containing a mixture of antibiotics, according to the method of "entered-found". To prepare model solutions, 1 ml of acetate-ammonia buffer solution with pH 4.3 was added to each of the ten tubes, then 0.25 ml of 10 ^-3 M γ-CD, 0.6-2.1 ml of CF with a concentration of 0.10-100 μg / ml were added sequentially , then antibiotics were added at a concentration of 50 μg / ml and diluted with buffer solution to a total volume of 5 ml. An aliquot of 50 μl was taken from each solution and introduced into the chromatograph. The chromatographic separation characteristics of antibiotics are presented in Table 3. As can be seen from the table, the detection limit of ciprofloxacin against other antibiotics in the presence of γ-cyclodextrin decreases by 6.8 times. The error of determination is not more than 0.06.

Claims (1)

The method for determining ciprofloxacin in drugs by reversed-phase HPLC in isocratic mode with a fluorimetric detector, which consists in preparing two samples of the same volume of a diluted test solution, in one of which a standard ciprofloxacin additive is introduced, characterized in that γ- reagent is added to each solution cyclodextrin, analyzed on a chromatograph using anetonitrile: ammonium acetate buffer (43:57 vol./about., pH 4.3) as the mobile phase aficheskih peaks and by comparing the measured parameters of the calculation formula determined concentration of antibiotic.