Резюме
Яремчук Ан.А., Хішова О.М., Половко Н. П. Розробка та валідація методики спільного кількісного визначення бензалконію хлориду, левоміцетину і декспантенолу в мазі.
У статті представлені дослідження з розробки методики спільного кількісного визначення декспантенолу, хлорамфеніколу і бензалконію хлориду в комбінованій мазі «Комбісепт» для лікування гнійних ран у I фазі ранового процесу методом високоефективної рідинної хроматографії.
Також наведені дані про проведення валідації запропонованої методики та встановлені критеріїв її прийнятності. На підставі проведеної валідації доведено, що методика спільного кількісного визначення хлорамфеніколу, декспантенолу і бензалконію хлориду є специфічною, лінійної в діапазоні її застосування, правильною і прецизійної.
Розроблена валідована методика спільного кількісного визначення декспантенолу, хлорамфеніколу і бензалконію хлориду включена в проект фармакопейної статті виробника.
Ключові слова: валідація, декспантенол, хлорамфенікол, бензалконію хлорид, високоефективна рідинна хроматографія.

Резюме
Яремчук Ан.А., Хишова О.М., Половко Н.П.* Разработка и валидация методики совместного количественного определения бензалкония хлорида, левомицетина и декспантенола в мази.
В статье представлены исследования по разработке методики совместного количественного определения декспантенола, хлорамфеникола и бензалкония хлорида в комбинированной мази «Комбисепт» для лечения гнойных ран в I фазе раневого процесса методом высокоэффективной жидкостной хроматографии.
Также приведены данные о проведении валидации предложенной методики и установлении критериев её приемлемости. На основании проведённой валидации доказано, что методика совместного количественного определения хлорамфеникола, декспантенола и бензалкония хлорида является специфичной, линейной в диапазоне её применения, правильной и прецизионной.
Разработанная валидированная методика совместного количественного определения декспантенола, хлорамфеникола и бензалкония хлорида включена в проект фармакопейной статьи производителя.

Ключевые слова: валидация, декспантенол, хлорамфеникол, бензалкония хлорид, высокоэффективная жидкостная хроматография.

Summary
Yaremchuk An.A., Khishova O.M., Polovko N. P. Development and validation of the method of simultaneous determination of benzalkonium chloride, laevomycetin, and dexpanthenol in the ointment.
The article describes studies on development of the method of simultaneous determination of Dexpanthenol, Chloramphenicol, and Benzalkonium Chloride in the co-formulated ointment Combisept for treatment of septic wounds in phase I of wound process by high-efficiency liquid chromatography.
It also contains data on validation of the offered method and determination of its acceptance criteria. On the basis of the validation performed it was proved that the method of simultaneous determination of Chloramphenicol, Dexpanthenol and Benzalkonium Chloride is specific, linear in the range of its application, correct, and precise.
The developed validated method of simultaneous determination of Dexpanthenol, Chloramphenicol, and Benzalkonium Chloride is included in the project of the manufacturer’s pharmacopoeial monograph.
Key words: validation, Dexpanthenol, Chloramphenicol, Benzalkonium Chloride, high-efficiency liquid chromatography.

Рецензент: д.фарм.н. О.А. Євтіфєєва

УДК 615.454.1:54.061/.062

Витебский медицинский университет (Витебск, Республика Беларусь)

Vitebsk Medical University (Vitebsk, Belarus)

hp4510s@i.ua

*Национальный фармацевтический университет (Харьков)

61002, г. Харьков, ул. Пушкинская, 53

National University of Pharmacy (Kharkov)

Ukraine, 61002, Kharkov, 53, str. Pushkinska

polovko.nat@mail.ru

Introduction. Ointment Combisept is a new co-formulated medicinal agent (MA) for external use indicated for treatment of pyoinflammatory diseases of various etiologies in phase I of the wound process. Ointment Combisept contains substances which refer to different classes of chemical compounds and have different physical and chemical properties: 50.0 mg of Dexpanthenol in 1g of ointment; 7.5 mg of Chloramphenicol in 1g of ointment; 5.0 mg of Benzalkonium Chloride (BC) in 1g of ointment.

Dexpanthenol ((2R)-2,4-dihydroxy-N-(3-hydroxypropyl) 3,3-dimethylbutanamide) is a derivative of pantothenic acid. In its appearance Dexpanthenol is a colourless or slightly yellowish liquid or white or almost white crystalline powder, very soluble in water. Dexpanthenol is freely soluble in ethanol 96 per cent [1]. Boiling and decomposition temperature is 119 ˚С [2].

Among all contemporary physical and chemical methods of analysis used for assay of Dexpanthenol in the MA, the method of reverse phase liquid chromatography is used most frequently and is described in the modern scientific literature [3, 4, 5].

Laevomycetin (Chloramphenicol) (2,2-dichloro-N-[1R,2R)-2-hydroxy-1-(hydroxymethyl)-2-(4‑nitrophenyl)ethyl]acetamide) is derived from nitrobenzene. In its appearance Chloramphenicol is a white, grayish-white or yellowish-white, fine, crystalline powder or fine crystals, needles or elongated plates, bitter in taste. Chloramphenicol is slightly soluble in water (2.5 mg/ml at 25˚С), freely soluble in ethanol and in propylene glycol (150.8 mg/ml at 25˚С). Melting temperature is 149˚С [6]. Assay of Chloramphenicol and its decomposition products is often performed by high-efficiency liquid chromatography method (HPLC) [7, 8, 9].

BC (alkyldimethyl(phenylmethyl)ammonium chloride) is quaternary ammonium compound salt and it is a mixture of alkylbenzyldimethylammonium chlorides, alkyl group of which consists of C₈ – C₁₈ chain. In its appearance BC is a white or yellowish-white amorphous powder or gelatinous yellowish-white fragments, soapy by touch. BC is very soluble in water, 96 per cent ethanol, acetone; slightly soluble in benzene; almost insoluble in ether [10]. The method which is most frequently used for assay of BC in MA is reverse phase liquid chromatography with chromatographic columns with polar bonded phase [11, 12, 13] or chromatographic columns with non-polar bonded phase, but with the use of ion-pair reagent [14].

The purpose of this work is development and validation of the uniform method by HPLC which enables simultaneous determination of Dexpanthenol, Chloramphenicol, and BC in ointment Combisept.

In course of development of the test method for assay it was necessary to develop the procedure of ointment sample preparation, which would meet the physical and chemical properties of APIs, and develop chromatographic conditions, which would enable separation of active ingredients in the ointment, and determine them quantitatively with high degree of reproducibility and accuracy.

Materials and Methods of the Study

The objects of research were samples of the ointment containing APIs registered in the Ministry of Health of the Republic of Belarus and satisfying the requirements of European Pharmacopeia: Dexpanthenol (Normative Documentation of the Republic of Belarus 0103 С-2010), Chloramphenicol (Normative Documentation of the Republic of Belarus 1023 С-2011); BC (Normative Documentation of the Republic of Belarus 0980 С-2011). The ointment base consisted of the following components: Macrogol-400 (State Pharmacopoeia of the Republic of Belarus, volume 2, p. 192); Macrogol-1500 (State Pharmacopoeia of the Republic of Belarus, volume 2, p. 192).

The assay by HPLC was performed on Agilent-1100 chromatograph in the following completeness: Agilent four-channel reciprocating pump (up to 400 bar) with flow rate 0.001-10 ml/min with the bay for solvents and adjustment pitch of solvent supply of 0.001 ml/min; Agilent vacuum degasifier with 4 channels and capacity up to 10 ml/min; Agilent automatic sample introduction unit with injection volume from 0.1 μl to 100 μl and with pitch of 0.03 μl. APIs identification was performed with the help of Agilent diode array spectrophotometric detector.

Purified water for chromatography was received with the help of water purification system Sartorius, which ensured receipt of ultra-clean water with specific resistance not less than 18.2 MOhm·cm.

The following system was used as a solvent: water for chromatography – acetonitrile for chromatography in the ratio (50:50) v/v.

For preparation of the test solution corresponding weight of the MA was taken and dissolved in the solvent in order to receive the final concentration of Dexpanthenol, Chloramphenicol, and BC: 0.5 mg/ml, 0.075 mg/ml, and 0.05 mg/ml, correspondingly.

A mixture of working standards (WS) of substances under analysis was used as reference solution. It was prepared similarly to the test solution, and the contents of Dexpanthenol, Chloramphenicol, and BC was 0.5 mg/ml, 0.075 mg/ml, and 0.05 mg/ml, correspondingly.

Validation. With the aim to prove suitability of the developed method for APIs assay and confirm receipt of expected and reproducible results, the method was validated in accordance with the set requirements [15, 16, 17, 18] on control samples and model mixtures received in laboratory conditions from the MA components.

The following characteristics were chosen for validation: selectivity, correctness, application range, linearity, and accuracy (repeatability and intralaboratory precision).

To confirm selectivity (specificity) in 6 replicas, the analysis of solution of final MA (test solution) and solution of the “base” containing all components of the MA except for Dexpanthenol, Chloramphenicol, and BC. Acceptance criteria: the chromatogram of the solution of the “base” should not contain peaks with retention times corresponding to the retention time of peaks of Dexpanthenol, Chloramphenicol, and BC homologues.

To determine linearity of the test method within the application range (80% ÷ 120% of the stated contents of APIs), the analysis was performed on 5 concentration levels with 6 replicas for each sample: 40.00 mg/g, 45.00 mg/g, 50.00 mg/g, 55.00 mg/g, 60.00 mg/g for Dexpanthenol; 6.00 mg/g, 6.75 mg/g, 7.50 mg/g, 8.25 mg/g, 9.00 mg/g for Chloramphenicol; 4.00 mg/g, 4.50 mg/g, 5.00 mg/g, 5.50 mg/g, 6.00 mg/g for BC.

For number of measurements n = 6 the most trustworthy results were received for Q (P, n) – the test, in which table value of Q-coefficient (P, n) was used as a critical value. If test statistics exceeded the critical value (Q_n > Q (95 %, 6) > 0.56), such value was considered an error and was excluded from the data series.

Linearity of the assay test method was determined by calculation of experimentally amended y-values for given x-values with the help of the least squares method. Linear relation correlation coefficient (r) was used as acceptance criteria, its value should be not less than 0.99.

To confirm repeatability of the test method one chemist subsequently analyzed six samples of one batch of MA in one day and using the same equipment. To confirm intralaboratory precision two different analysts made subsequent analyses of 6 samples each of one batch of MA in different days and using different equipment.

The following acceptance criteria were set: relative standard deviation (RSD, %) calculated for quantitative determination of Dexpanthenol, Chloramphenicol, and BC received for repeatability should not exceed 2.0%; difference between dispersion values S₁² and S₂² for average results of two samplings received for intralaboratory precision during determination of Dexpanthenol, Chloramphenicol, and BC in identical samples of MA should be statistically valid.

The correctness of test method for determination of Dexpanthenol, Chloramphenicol, and BC was determined on model samples with known content of APIs in three concentrations within the method application range: 45.00 mg/g, 50.00 mg/g, 55.00 mg/g (90%; 100% and 110% of  label claim), each concentration of Dexpanthenol was introduced 6 times; 6.75 mg/g, 7.50 mg/g, 8.25 mg/g (90%; 100% and 110% of  label claim), each concentration of Chloramphenicol was introduced 6 times; 4.50 mg/g, 5.00 mg/g, 5.50 mg/g (90%; 100% and 110% of  label claim), each concentration of BC was introduced 6 times.

Test results were checked for uniformity of sampling, data with gross errors were excluded from the sampling.

Correctness of data received was confirmed by calculation of bias |х_ср – m| and verification of significance in the difference between the random value х_ср and the constant m (accepted reference value).

Study results and their discussion. The studies performed enabled justification of the test method for determination of Dexpanthenol, Chloramphenicol, and BC in case of their co-existence in the MA and adaptation of the following sample preparation technique: 20.0 ml of the solvent added to 5.0000 g of the MA under analysis, mixed intensively to dissolve the ointment, made up the volume to 100.0 ml with the same solvent. 5.0 ml of this solution dissolved to 25.0 ml with the same solvent and mixed. The received solution was filtered through the filter with pores diameter 0.2 μm.

Sample preparation of WS was carried out as follows: 0.0250 g of BC, 0.0375 g of Chloramphenicol, 0.2500 g of Dexpanthenol dissolved in 50 ml of solvent, made up the volume to 100.0 ml with the same solvent, and mixed. 5.0 ml of this solution made up to the volume of 25.0 ml with the mixture of water for chromatography and Acetonitrile for chromatography in ratio (50:50) v/v. The solution was not filtered. Water content (w) in BC, in per cent, was determined with the help of semimicromethod by K. Fischer on 0.300 g of sample immediately before the test.

On the basis of analysis of literature data, results of studies performed we developed optimum conditions for chromatography of a complex mixture of APIs which ensures receipt of satisfactory separation of all ointment components: chromatographic column (250 × 4.6) mm filled with cyanosilyl silica gel P for chromatography with particle size 5.0 μm (for example Waters Spherisorb СNRP or similar, which satisfies the requirements of the Chromatographic System Suitability Test); column temperature: 30 °C; mobile phase A: Potassium Dihydrogen Phosphate solution with concentration of 3.0 g/l, pH level of which should be adjusted to 2.8 ± 0.05 with Phosphoric Acid; mobile phase B; Acetonitrile for chromatography; isocratic elution with mobile phase A and mobile phase B ratio (50:50) v/v was used; mobile phase rate – 2.0 ml/min; spectrophotometric detector: wave length for registration was 195 nm for BC and 210 nm for Chloramphenicol and Dexpanthenol; sample injection volume – 10.0 μl.

On test solution chromatogram (picture 1) all peaks are separated; separation between the peaks of Chloramphenicol and Dexpanthenol was 3.42, which is more than the specified value of 1.5. Relative standard deviation calculated according to retention times of BC homologues peaks, retention times of Chloramphenicol and Dexpanthenol peaks for six repetitions of the analysis was 0.834%, 0.939%, and 0.936%, correspondingly. Efficacy of the chromatographic column (number of theoretical plates) and peaks asymmetry factors were 4,384 and 0.833 correspondingly for BC C-12 peaks; 4,211 and 0.846 correspondingly for BC C-14 peaks; 4,795 and 0.939 correspondingly for Dexpanthenol peaks; 6,084 and 0.9355 correspondingly for Chloramphenicol peaks.

Assay of APIs was carried out by comparison of peak areas of Chloramphenicol, Dexpanthenol, and BC on the chromatogram of test solution (picture 1) with the corresponding peaks on the chromatogram of reference solution (picture  2). Test sample weight, WS weight taken for analysis, and percentage of Chloramphenicol, Dexpanthenol, and BC on dry basis were taken into account during calculation of the APIs assay in the ointment.

The developed method was validated. The method was proved to be specific: it was observed that in test conditions the solvent used, mobile phase or placebo components do not interfere with the results of APIs assay.

The following criteria of chromatographic system suitability were set:

• Resolution between Chloramphenicol and Dexpanthenol peaks observed on the chromatogram of test solution (picture 1) should be not less than 1.5;
• Efficacy of chromatographic column calculated for BC C-12 and C-14 peaks should be at least 2,000 and 2,500 theoretical plates, correspondingly;
• Efficacy of chromatographic column calculated for Chloramphenicol peak should be at least 5,000 theoretical plates;
• Efficacy of chromatographic column calculated for Dexpanthenol peak should be at least 4,000 theoretical plates;
• Relative standard deviation calculated for the sum of BC C-12 and C-14, Dexpanthenol and Chloramphenicol peak areas according to the results of five repetitions of the analysis should be not more than 2.0%;
• Peak asymmetry coefficient calculated for peaks of BC C-12 and C-14, Chloramphenicol and Dexpanthenol should be not less than 0.5 and not more than 1.5.

In course of statistical analysis of linear dependence of the received values of Dexpanthenol, Chloramphenicol and BC contents from the label claim, linear regression chart correlation coefficient was 0.9999; 0.9993 and 0.9996 for Dexpanthenol, Chloramphenicol and BC, correspondingly.

Repeatability of the method of determination of Dexpanthenol, Chloramphenicol and BC was established. Relative standard deviation (RSD, %) calculated for the contents of Dexpanthenol, Chloramphenicol and BC and received under repeatability conditions did not exceed 2.0% and was 0.348%, 0.986% and 1.486%, correspondingly.

Intralaboratory precision of the test method for qualitative determination of Dexpanthenol, Chloramphenicol and BC was proved: Fischer’s coefficient F (P, f₁, f₂) does not exceed its table value F_tab (0.95; 5; 5).

Correctness of the method of qualitative determination of Dexpanthenol, Chloramphenicol and BC was proved by corresponding tests on model samples for three concentrations within the test method application range in 6 repetitions. The received results of determination are not burdened by systematic error. Average recovery percent for 18 measurements was 99.75%; 99.64% and 99.76% for Dexpanthenol, Chloramphenicol and BC, correspondingly.

Metrological results of APIs assay received with the help of the developed method are presented in Table 1.

Table 1 – Metrological results of APIs assay

Summary:

1. The most acceptable sample preparation conditions of Combisept and its analysis conditions were determined on the basis of analysis of data on physical and chemical properties of Benzalkonium Chloride, Chloramphenicol, and Dexpanthenol.
2. A uniform method was developed, which enables simultaneous identification and determination of Dexpanthenol, Chloramphenicol, and Benzalkonium Chloride by HPLC on the basis of the chromatographic column (250 × 4.6) mm filled with cyanosilyl silica gel for chromatography with particle size 5.0 μm, with the diode array detector and the two-component mobile phase as eluent.
3. Effective concentrations of APIs in the test solution and reference solution under analysis were determined.
4. Optimum chromatographic conditions were determined, which enable eluation of the substances under determination within a short period of time and ensure express form of the analysis (run time is about 7 minutes).
5. The developed test method was validated and proved to be specific, linear in the range of its application, correct and precise.
6. The developed method of determination of Benzalkonium Chloride, Chloramphenicol, and Dexpanthenol is included in the Manufacturer’s Pharmacopoeial Monograph of the Republic of Belarus for the medicine Combisept.

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