Determination and Validation of pKa value of Atropine Sulfate using Spectrophotometry

 

Job Herman Berkhout1, Aswatha Ram H.N.2*

1Faculty of Science, Radboud University, Nijmegen, the Netherlands.

2Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal-576104, India.

*Corresponding Author E-mail: aswatharam@gmail.com

 

ABSTRACT:

The ionization state (pKa) of a compound influences its absorption, distribution, metabolism and excretion. Therefore, proper knowledge of the pKa is crucial in understanding the behavior of a compound behavior inside the body. In this research paper, we validate the pKa of muscarinic acetylcholine receptor antagonist atropine sulfate using Uv-Vis spectroscopy, to validate the use of this method and to re-evaluate the of atropine sulfate. The pKa value from absorption measurements is first estimated using 3rd degree polynomial and then precisely calculated according to the Albert-Sergeant method. We established that the pKa value of Atropine sulfate is 9.94±0.050 which is comparable to the values present in the literature.

 

KEYWORDS: Atropine Sulfate, pKa, Uv-Vis Spetrophotometry.

 

 


INTRODUCTION: 

The measure for the ionization state of a compound is the acid dissociation constant(pKa)1. This state plays a major role in the absorption, distribution, metabolism, and excretion (ADME) profile of a compound2,3. Thus influencing psychochemical properties like: pH dependent aqueous solubility, protein interaction and membrane permeability. compounds with a different pKa are absorbed in different compartments of the digestive tract, since the different compartments contain a different pH (e.g. stomach pH 1-3.5, colon pH 5.5-8, intestine pH 5.5-8 and blood pH 7.4)4. Thus understanding knowing the pKa value of a compound is trivial, in drug development and its behaviour in vivo.

 

Atropine is analkaloid, that occurs naturally in plants from the nightshade family.Atropine terminates the parasympathetic nervous system (“rest and digest”) as itis a competitive antagonist of the muscarinic acetylcholine receptors, which are responsible for G protein-coupled receptor complex formation in certain neuronal cell membranes5.

 

This pharmaceutical effect has various applications including treatments for myopia, treatment of the muscarinic symptoms of nerve agent poisoningsand inhibition of secretions. The most common atropine used in medicine is Atropine Sulfate (figure 1). Due to its usage in medicine, it is important to understand the chemical properties of this compound.

 

Figure 1: The chemical structure of atropine sulfate

 

One of these chemical properties is the acid dissociation constant (pKa). The pKa indicates the ionization state of a compound at a given pH.. The pKa of atropine sulfate has been described in literature, as9.65 and 9.856,7. However, after extensive literature search, the methods used to estimate these values remain unknown. In this research paper, we will validate the given values using Uv-Vis spectroscopy. A well validated method for pKa determination, due to its availability, accuracy, simplicity and reproducibility. As Atropine Sulfate is a weakly basic compound [5], the pKamay be calculated using the Albert-Serjeant formula (1)8. Here, the pH is the value recorded on the pH meter, D is the absorbance of the compound in the selected buffer, AM and AI indicate the absorbance of the unionized and ionized compound respectively. Using this, a rough estimate of pKa is required.As we perform a validation of atropine sulfate, basing our estimation on literature values will create a bias. Thus we approximate thepKa value using polynomial fitting of the 3rd degree to absorbance measurements in a wide pH range

                                AM-A

Pka = pH + log 10 ----------                               ……(1)

                                A- AI                                     

 

From this fit, the inflection point was determined, which was in turn used to consider a narrow pH range. upon which one acidic, one basic and 6 buffer solutions are prepared.

 

The solubility property of salts of acidic or basic drugs depends on how they dissociate into their free acid or base forms easily and on interrelationships of several factors, viz pH, pKa, solubility product and maximum solubility in different dissolution media of varying pH9.

 

The other available methods for the determination of pKaare potentiometry and conductometry.  For example, the pKa of Enalapril Maleate was determined by potentiometric titration and conductometry, and the values were found to be 2.7 and 2.6 respectively10.

 

MATERIAL AND METHODS:

Drugs and Chemicals:

Atropine Sulfate was obtained from Merck Limited, Mumbai – 400018, India, Cat.No. 5908 -99-6. The desired pH value was achieved by increasing or decreasing the buffer pH using 1M HCl or NaOH. For pH 7 a Phosphate buffer (potassium phosphate, Merck, Mumbai – 400018, India, Cat.No MC3M630067) pH8,9&10 an alkaline borate buffer (boric acid, Fisher, Chennai – 600024, India, Cat.No 6005) and for pH11&12 a disodium phosphate buffer (disodium phosphate, HiMedia, Mumbai – 400086, India, Cat.No. RM1257-500G) was used. The pH values were verified using a pH meter and variation was kept within ± 0.05 pH units.

 

For Uv-Vis spectroscopy in the narrow pH range; alkaline borate buffer (boric acid, Fisher, Chennai – 600024, India, Cat.No 6005), pH 8.8-10.0 with steps of 0.2was prepared according to the UPS protocol. The desired pH value were achieved by increasing or decreasing the buffer pH using 1M HCl or NaOH.The pH values were verified using a pH meter and variation was kept within ± 0.02 pH units.

 

Stock solutions:

A stock solution of 0.1 M Atropine Sulfate in pure HPLC graded methanol (Finair limited, Ahmedabad – 382110, India, Cat.No. 67-56-1) was prepared. For measurements, the stock solution was diluted 50 times to a final concentration of 2 mM in the respective solvents.

 

Instruments:

Digital pH meter MK VI, Systronics Limited, Ahmedabad – 380013, India. UV-Vis spectrophotometer of model UV-2450, Shimadzu Analytical, New Delhi – 110082, India, Cat.No. 208-24301-93.

 

UV-Vis measurements:

UV-Vis spectroscopic measurements were performed at in 1ml quartz cuvettes at 30 ±2ºC. Uv-Measurements were carried out in triplicateusing freshly prepared stock solutions. For absorption measurements, 257 nm was selected. The fitting and processing of data was performed using Graphpad – Prism 6 (Graphpad Software, California, San Diego, USA)).

 

RESULTS AND DISCUSSION:

As the pKa value is a crucial component in the ADME properties of a compound, it needs to be well understood. In this paper, we validate the pKa value of atropine sulfate at 30 ±2ºC using Uv-Vis spectroscopy, combined with the Albert-Serjeant method for pKa calculations. To apply the Albert-Serjeant method, an approximation of the pKA value is required, as the relation between pH and absorbance needs to measured in a narrow pH range. A pH titration range from 7 to 12 was chosen, as the pKa of atropine sulfate exists in basic pH range, to determine the narrow pH range. In figure 2A, Uv-vis measurements at a wavelength of 257 nm are displayed. To estimate the pKa of Atropin sulfate, polynomial fitting of the 3rd degree was applied to the absorbance measurements, which returned the following formula (2):

 

y = 0.005755x2 – 0.1648x2 + 1.561x – 4.167….. (2)


 

 

Figure 2: Atropine Sulfate pKa estimation using 3rd degree polynomial curve fit

(a) The absobance of  a tropine sulfate plotted against a buffered pH range from 7 to 12 at a wavelength of 257nm. The black dots represent the average of two separate duplo measurments The red curve respresents the polynomial fit. (B) The residuals of the curve fit. Average avsorption values reative to the fit are displated as black dots

 


Thequality of the fit wasreviewed using theR2 (0.986) and the residuals of the fit(figure 2B).In the residual plot, all values remain within ± 0.01 units variation of the calculated polynomial curve. To ensure the values are evenly distributed and within fitting guidelines, the residuals were linear fitted. The fit (red) showed no deviation from zero, which proves that our fit model is accurate and well suitable for our data.

 

Figure 3: Raw Uv-spectrum of atropine sulfate at different pH.

The x-axis reads wavelength in nm. The y-axis reads the absorbance. A tropine sulfate is ionized in HCI and un-ionized in NaOH. The pH of is indicated by colors according the legend in the top-right corner.

 

Using the formula generated by curve fitting, the inflection point was determined by calculating the 2nd derivative from the fit formula and solve x for y = 0. This resulted in an estimated pKa value of 9.54 from with a narrow titration range of 8.8 till 10 was considered.

 

Atropine sulfate is suitable for pKa determination by UV-spectroscopy sincethe absorption between the unionized and ionized forms differs (figure 3). The un-ionized form has a higher absorbance compared to the ionized form. Furthermore, a clear correlation between pH and pKa, where the absorbance of atropine sulfate increases in proportion to the pH increase. To precisely determine the pKa value of atropine sulfate,Uv-spectroscopic measurements were performed within a narrow pH range of 8.8 to 10.0, with steps of 0.2. Potential outliers were excluded using the Graphpad – Prisms ROUT method, with a strength of Q5% (figure 4). From the average of the measurements the pKa was calculated for each pH separately using the Albert - Serjeant calculation (1) (table 1). The average of all pKacalulations display the pKa of Atropine Sulfate at the temperature of 30±2ºC. Which is 9.94 ± 0.050 (SEM).

 

Our determined atropine sulfate pKa value, is comparable to the previously described values of 9.65 and 9.856,7.

 

Table 1: Calculated pKa value using the Albert-Serjeant formula

NaOH (AM) = 0.912, HCI (AI)= 0.765

pH

pKa

8.8

9.766

9.0

9.803

9.2

9.954

9.4

10.081

9.6

10.122

9.8

9.933

10.0

9.895

Average

9.936

 


Figure 4: Uv-measurements of Atropine sulfate at different pH values at 257nm. The error bars indicate the standard error of the mean (SEM). Using the albert – Srjeant method, the pKa is calculated form these average values.

 


CONCLUSION:

The pKa of atropine sulfate was estimated to be 9.94 at 30±2ºC using the simple, cost-effective, and accurate methodUv-Vis Spectroscopy.

 

ACKNOWLEDGEMENT:

The authors sincerely thank Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, India and Radboud University, the Netherlands for providing the necessary facilities to carry out this work.

 

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Received on 20.01.2021            Modified on 24.02.2023

Accepted on 19.01.2024           © RJPT All right reserved

Research J. Pharm. and Tech. 2024; 17(3):1029-1032.

DOI: 10.52711/0974-360X.2024.00159