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Application of the KINETIC ENZymATICmethod for benzalkonium chloride determination in aerosol preparation

Application of the KINETIC ENZymATICmethod for benzalkonium chloride determination in aerosol preparationApplication of the KINETIC ENZymATICmethod for benzalkonium chloride determination in aerosol preparationApplication of the KINETIC ENZymATICmethod for benzalkonium chloride determination in aerosol preparation
Елена Ковальская, ph.d. фармацевтических наук

Блажеевский Николай , профессор, доктор фармацевтических наук, профессор

Харьковский национальный фармацевтический университет, Украина

Участник первенства: Национальное первенство по научной аналитике - "Украина";

Открытое Европейско-Азиатское первенство по научной аналитике;

UDC543.395: 543.23: 543.422.7

A new kinetic photometric method for BAC determination based on an inhibition of the enzymatic (cholinesterase) reaction was proposed. The reaction rate was detected at unhydrolised acetylcholine residue, which is determined by the amount of peracetic acid, producted during the impact of H2O2 on it. Indicator reaction is a reaction of peracetic acid with 4-ethoxyaniline interaction that leads to the formation of azoxyphenetole with λmax = 358 nm (lgε =4.18). The measurement velocity of changing of light absorption vs. time (∆А/∆t, min-1)give a chanceto quantitatively determination of BAC. The results confirmed that the method is linear at concentrations ranging from 1.0×10-6mol/L to 5.0 ×10 -6 mol/L. Depending calibration equation ΔA/Δt, min-1 from the village BAC has the form: tgα = 5093 c + 0.0075 (r = 0,999). LOD was 0.4× 10-6mol/L. The method was satisfactorily applied to the determination of BAC in nasal pharmaceutical preparation «Aqua-rinosol». The recovery was 99.9%, RSD = 2.7 % (n=5, P=0.95).This method is beneficial because high sensitivity and selectivity can be achieved using relatively simple technical equipment.

Keywords: Benzalkonium chloride,kinetic photometric method, determination,aerosol preparation

Introduction

«Aqua-rinosol» is a medicine for treatment dryness inside the nose (nasal passages). It helps add moisture inside the nose to dissolve and soften thick or crusty mucus. In babies and young children with stuffy noses who cannot blow their noses, using this product helps to make the mucus easier to remove with a nasal bulb syringe. This helps relieve stuffiness and makes breathing easier. This product contains a purified gentle salt solution (also called saline or sodium chloride solution) and Benzalkonium chloride (BAC) as preservative(w=0,015 %). It is a mixture of alkylbenzyldimethylammonium chlorides of various even numbered alkyl chain lengths(R=С8, С10, С12, С14, С18). This product is a nitrogenous cationic surface-acting agent belonging to the quaternary ammonium group, especially inhibitors of acetylcholinesterase.

Considering widespread use in pharmaceutical formulation the determination of low concentration preservative constitutes a challenging problem in current pharmaceutical analysis and is a topic of global interest. The standard method for determination BAC in pharmaceutical compositions is reversed-phase high performance liquid chromatographic [1-6].

Experemantal section

Instrumantal and apparatus

All the absorbence spectral measurements were made using “photoelectric concentration colorimeter (CPC-2) (Zagorsky Optical & Mechanical plant, Russia).

pH value was measuremented at ionomer I – 160M laboratory (Belarus) by using EGL 43-07 pH glass electrode together with auxiliary choride silver electrode of EAL-1m3.1 type, standard with potassium chloride.

Materials and reagents

All chemicals and reagents used were of analytical grade:Pharmacopoeial acetylcholine chloride medication – 0.2 g per amp/5 ml (manufactured by "VECTOR" – State Science Center of virology and biotechnology in Russian Federation" (Russia); dry protein drug of cholinesterase from horse serum–8 mg/ L (IV class) 22AE / mg (SMU “Biomed”, Russia).  N - phenetidine (4 - ethoxyaniline - 98%) (SIGMA - ALDRICH); Series: A0281408 s, New Jersey, USA; "Stabilized Hydrogen Peroxide 30-40%" (LLC "Inter - Synthes", Boryslav, Ukraine)( the content of hydrogen peroxide was determined by SPU according to the monograph "High-test hydrogen peroxide solution 27.5-31.0%).

 High purity double distilled water was used throughout.

Preparetion Standart stock solution

Standart stock solution  was prepared in double distilled water by taking 0.09910g of standart solution Benzalconium chloride (50% ) in a 500ml volumetric flask and it was diluted up to the mark  x (ppm). 0.1 ml of this solution was taken in a 10 ml volumetric flask and was diluted up to 10 ml.  From the standart stock solution, a standart solution was prepared containing 2.12·10-5mol/L. Alternatively, deriveve the corresponding regration equation.

Preparetion solution of Cholinesterase (ChE)

It was prepared in double distilled water by taking 80 mg of dry cholinesterase drug in volumetric flask and it was shaking diluted up to the mark. After that it was termostate at termostate 10 inutes at 38 ºC above Zero [7].

Phosphate buffer solution (pH 8.35 )preparetion

It was prepared  by dissolve pour 35.75 g of sodium hydrogen phosphate in 500 ml flask, add 300 ml double-distilled water, dissolve it, add 19 ml of 0.1 mol/L solution of hydrochloric acid, stir and dilute double distilled water to 500.0 ml. The ready solution pH is potentiometrically controlled.

Hydrogen peroxide solution 10%.

It is prepared by the appropriate high-test hydrogen peroxide dilution with double-distilled water. The exact hydrogen peroxide content in ready 10% solution is determined permanganatometrically.

p-phenetidine hydrochloride solution preparation 1%.

N- phenetidine hydrochloride (Ph), extracted from the base by hydrogen chloride precipitation in the chloroform solution. Dissolve 1.00g p-phenetidine hydrochloride in 80 ml of double-distilled water into 100 ml capacity measuring bottle and dilute it to volume.

The enzyme substrate of acetylcholine chloride solution (ACh) preparation.

The ampoule’s content of pharmacopoeia drug acetylcholine chloride 0.2 g is in 200 ml of double-distilled water dissolved. For that end, open an ampoule, add 4.0 ml of water with pipette, and shake until acetylcholine is completely dissolved. Then pour the acetylcholine solution into 200 ml capacity measuring bottle and dilute double-distilled water to the volume.

General recommended procedure.

10.0 ml 0.2 mol/L of phosphate buffer solution (pH = 8.3) in 20 ml capacity graduated test tube with ground plug consistently were transferred. The accurate volumes of   test solution (BAC or “Agua-rinosol”)over the concentration range of x mol/L into a standard flask was added.

After that, add 2.0 ml of cholinesterase while stirring, switch a timer, shake up each solution thoroughly and thermostate  for 15 min, then quickly add 1.0 mL of 1% acetylcholine solution, switch on timer, shake thoroughly and thermostate 10 min again, then add 2.0 ml of 10% hydrogen peroxide solution, keep for 10 min in thermostat and add 1.0 ml of 1% p-phenetidine solution (Ph), dilute distilled water to volume at 20 ml. 5 ml solution eye drop was transferred to a 100 ml volumetric flask and made up to the mark with water. Suitable dilution was made to fit the applicable concentration range and the above described procedures were followed.

 The nominal concentration of the bottles was calculated either from calibration graph or using the regression equation.

Results and discussion

The proposed method for BAC detection is based on an enzymatic (cholinesterase) reaction. Mechanism of analytical reaction and sensitivity of this method are the same as, or similar to, those in the human body. The conventional enzymatic method is based on the ability of cholinesterase to accelerate hydrolytic decomposition of the neurotransmitter (substrate) acetylcholine to choline and acetic acid. The reaction rate is detected at unhydrolised acetylcholine residue, which is determined by the amount of peracetic acid, producted during the impact of H2O2 on it. Indicator reaction is a reaction of peracetic acid with 4-ethoxyaniline interaction that leads to the formation of azoxyphenetole with λmax = 358 nm (lgε =4.18). The measurement velocity of changing of light absorption vs time (∆А/∆t, min-1)give a chanceto quantitative determination of BAC.

According to the optical - time relations the kinetic curves are plotted and the slope of the first 15 min is found (Fig.1). According to data received a slope - finite analyte concentration calibrated relation is obtained, c, µmol / L.

A calibration curve equation is solved by the least squares method (Linear regression): tgα =bс+a, where a, b are Y-axis intercept and slope, (tgα, min -1) respectively. Switch on timer and every other minute scan photometrically each solution for 15 min on photoelectric colorimeter CPC-2, use colour filter №2 and 1.0 cm cuvette (Fig2).

The results confirmed that themethod is linear at concentrations ranging from 1.0×10-6mol/L to 5.0 ×10 -6 mol/L. Depending calibration equation ΔA / Δt (tgα), min-1 from the village BAC has the form: tgα = 5093c + 0.0075 (r = 0,999). LOD was 0.4×10-6mol /L. The precision and accuracy of the proposed method was studied by performing the experiment five times at three different concentration levels (low, medium and high) of BAC (tabl.1).

 

Fig. 1. Kinetic curves of couple oxidation p-phenetidine by hydrogen peroxide in presence of the system: 1 – ACh+ChE, 2- 5 – ACh+(ChE+BAC), 6 – AСh.  Initial concentrations: pH 8.3 (phosphate buffer), w(Ph) = 0.05% (w/v), c(H2O2) = 1.5· 10-2 mol /L, w(ACh) = 0.05% (w/v),c(ChE=2.2 AE/mL)  t = 37 ± 0.1 °C, λ= 358 nm.

 

Fig. 2. Dependence of the relative reaction rate on BAC concentration. Initial concentrations: pH 8.3 (phosphate buffer), w(Ph) = 0.05% (w/v), c(H2O2) = 1.5· 10-2 mol /L, w(ACh) = 0.05% (w/v),c(ChE=2.2 AE/mL)  t = 37 ± 0.1 °C, λ= 358 nm.

 

Table 1

Accuracy and precision of the proposed method

 

No.

BAC taken*,

mol /L

BAC found,

±ΔX, mol /L

N

RSD

(Р=0.95)

(-μ)∙100% /μ

1.

 

 

1.065∙ 10-6

 

(1.059±0.015)∙10-6

 

5

 

2.52%

 

0.56%

2.

3.195∙ 10-6

(3.179±0.05)∙10-6

 

5

1.49%

 

0.50%

3.

4.26∙ 10-6

(4.244±0.03)∙10-6

 

5

0.63%

 

0.42%

*calculated according to the certificate.

The method was satisfactorily applied to the determination of BAC in nasal pharmaceutical preparation «Aqua-rinosol». The recovery was 99.9%, RSD = 2.7 % (n=5, P=0.95).

Conclusion

The proposed kinetic-spectrophotometric method for the determination of BAC in pharmaceutical samples of “Agua-rinospl”preparation reported in this work is simple, fast, inexpensive, and thus appropriate for routine quality control analysis of the active drug in the laboratories of hospitals, pharmaceutical industries and research institutions. It should also be suitable for developing countries. The validation of the method shows that the results obtained are in good agreement with the standard Pharmacopoeial method.

 

References:

  • 1.      Hashem Al Aani. // J. Appl.Pharm.Sci. – 2016. –V. 6, №5. – P. 80-89.
  • 2.       KostićD E.// J.Chem. – 2012. –V.9, №3.– P.1599-1604.
  • 3.       Gubin M., Pivovarova S. // Pharmacy. – 2012 – V. 4. – P. 9-12.
  • 4.      Kabeer AS, Ashish TP. // J.Trace Anal. Food & Drugs. – 2013. – V.21, № 1.–P. 14-21.
  • 5.       Hanni M. H. at all. // Euras. J. Anal Chem. – 2015. – V. 10, № 1.–P.46-67. 
  • 6.       Metha J., Patidar K., Vyas N. // E-Jornal Chem. –2010. – V. 7, №1.–P. 11-20.
  • 7.      BlazheyevskiyM.Ye, DyadchenkoV.V. // Pharm. J. –- 2004. – V.2. – P. 52-58 (Ukrainian).
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Ваша оценка: Нет Средняя: 9.5 (2 голоса)
Комментарии: 2

Сметанина Екатерина Ивановна

Уважаемые коллеги! Ваша работа - основа поиска новых лекарственных средств, перспективных лекарственных форм, что составляет основу фармации. Успеха Вам в Ваших дальнейших соисканиях!

Симонян Геворг Саркисович

Уважаемые Елена Васильевна и Никола Евстафьевич интересная аналитическая и практическая работа. Использование холинэстеразы - на основе биосенсоров позволяет определять следовые количества поверхностно-активного вещества бензалкония хлорид в образцах "Agua-rinospl". Вами продолжается применение нового чувствительного фермент-кинетического метода для определения катионного поверхностно -активного вещества в лекарственных средствах. Получен удовлетвориютельний воспроизводимость и точность. С уважением к.х.н., доцент Геворг Саркисович.
Комментарии: 2

Сметанина Екатерина Ивановна

Уважаемые коллеги! Ваша работа - основа поиска новых лекарственных средств, перспективных лекарственных форм, что составляет основу фармации. Успеха Вам в Ваших дальнейших соисканиях!

Симонян Геворг Саркисович

Уважаемые Елена Васильевна и Никола Евстафьевич интересная аналитическая и практическая работа. Использование холинэстеразы - на основе биосенсоров позволяет определять следовые количества поверхностно-активного вещества бензалкония хлорид в образцах "Agua-rinospl". Вами продолжается применение нового чувствительного фермент-кинетического метода для определения катионного поверхностно -активного вещества в лекарственных средствах. Получен удовлетвориютельний воспроизводимость и точность. С уважением к.х.н., доцент Геворг Саркисович.
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