Spectroscopic Determination of Two Beta-Blockers – Atenolol and Propanolol by Oxidative Derivatization Using Potassium Permanganate in Alkaline Medium

A simple, rapid, sensitive, cheap and accurate oxidative method for two beta-blockers in pharmaceutical dosage forms was developed and evaluated. The method involved the oxidimetric treatment of atenolol and propanolol with 2 x 10-3 mol L-1 KMnO4 in alkaline medium (pH ≥ 9). Scanned spectra of oxidized complex showed distinctive absorptions at 460, 520, 540 and 570 nm. Arrays of colour changes were observed from violet to blue; blue to bluish-green and yellow. Exhibited colours were due to ligand-metal charge transfer. An indirect spectrophotometric determination of atenolol and propranolol was done after 12-15 minutes at 520 nm. The optimum assay conditions showed linearity ranged from 0 – 15.0 μg mL-1 for both betablockers (R= 0.9997 – 0.9999). Molar absorptivity values were 4.79 x 103 and 4.88 x 103 L mol-1 cm-1 for atenolol and propanolol respectively, with corresponding Sandell’s sensitivity values of 0.056 and 0.053 μg cm-2. Limits of detection and quantification were 0.50 and 1.65 μg mL-1 for atenolol respectively and 0.58 and 1.91 μg mL-1 for propanolol, while relative standard deviation for intraand inter-day precision were < 2.0%. The applicability, accuracy and reliability of the method were demonstrated by the determination of atenolol and propanolol in tablet formulations. The recovery studies ranged from 93.33 103.00% for both beta-blockers and the amounts in brands were from 97.53 ± 2.68 to 100.84 ± 1.82%.

Absorption spectroscopy in the ultraviolet (UV) and visible (VIS) regions of the electromagnetic spectrum has been widely applied in pharmaceutical and biomedical analysis for qualitative and quantitative purposes [20]. It is versatile, robust, simple, quick and cheap for both research and routine analysis; and also found usefulness in the study of drug release from formulations and kinetics of drug degradation. The presences of chromophores in these two β-blockers make them easily assayed in the UV region. In addition, the presence of functional groups such as hydroxyl (-OH), amino (-NH-, -NH 2 ), carbonyl (-C = O), ether (-O -) and unsaturated aromatic ring make them highly susceptible to conjugation on reacting with appropriate chromogens to form derivatized coloured complexes that absorb at higher wavelength in the visible region [20]. Spectrophotometric determinations of derivatized pharmaceuticals through oxidimetric reactions by metal ions precursors such as Fe 3+ , Ce 4+ , Mo 5+ , Cu 2+ and Mn 7+ in acidic or alkaline medium have been reported [21][22][23], while, pharmaceutical organometallics containing Bi, Cu and Co have been reported quantified using synthesized chelating chromogenic compounds [24 -26]. The use of potassium permanganate (KMnO 4 ) to oxidize aminoglycosides [27], 1,4-dihydropyridine drugs [28], metoclopramide [29], ampicillin [30] and in kinetic oxidative degradation and deamination reaction of ATL in alkaline medium [31], have been reported. However, the KMnO 4 oxidimetric quantification and evaluation of atenolol and other β-blockers in alkaline medium by spectrophotometry have not been reported. The proposed method is aimed at developing and evaluating a simple, quick, sensitive, accurate and cheap quality control assessment of atenolol and propranolol in tablet formulations, especially where modern and expensive equipment is not available.

Apparatus
Absorption spectra were measured with JENWAY UV-Vis Spectrophotometer model 6305, while Electric Centrifuge Model 800, manufactured by B BRAN scientific & instrument company, England, was used to enhance the extraction of beta-blockers from solid dosage formulation. Analytical weighing balance (Sartorius) and Micro-pipettors (JENCONS scientific Ltd) were calibrated to be used.

Material and Reagents
The chemicals used were of analytical and spectroscopic grade. The methanol and ethanol were manufactured by Jiahua Chemiclas (JHC), China, while sodium hydroxide and potassium permanganate were from J.T Baker, USA and British Drug House (BDH) of England respectively. The pure atenolol hydrochloride and propanolol hydrochloride reference powder (99.5%) used as reference standards were obtained from Primal Nigeria Ltd, Lagos. Nigeria.

Preparation of reagents
All working reagents were prepared freshly in doubly distilled water.
Potassium permanganate (KMnO 4 ) solution, 2.0 × 10 -3 mol L -1 was prepared by dissolving 0.079 g of KMnO 4 in 10 mL of warm double distilled water in a 250 mL volumetric flask, cooled and made to the mark, while 0.1 mol L -1 NaOH solution was prepared by dissolving 1.0 g of sodium hydroxide pellets in 50 mL distilled water in a 250 mL volumetric flask and then made up to volume.
Stock solutions of 100 µg mL -1 atenolol and propanolol standards, were prepared separately by dissolving 50 mg (equivalent of ATL and PPN) of each with 2 mL methanol in 50 mL calibrated flask and diluting to mark with distilled water (solution A). An aliquot of 1.0 mL solution A was further diluted to 10 mL in a calibrated flask with distilled water to obtain the stock concentration.

General procedure and determination absorption spectra of beta-blockers and reference solution
Into one of two 10 mL volumetric flasks, 0.4 mL standard solution of drugs was transferred, 0.5 mL 0.1 mol L -1 NaOH was added and allowed to stand for 3 minutes, then 1 mL of 0.002 mol L -1 KMnO₄ was added, mixed gently and allowed to stand for 10 minutes screened from direct sunlight. The solutions were made to mark with distilled water and scanned in the visible region from 380 -700 nm. Scanning was done against a reagent blank treated the same way without 0.002 mol L -1 KMnO 4 and drug. To the second volumetric flask, the procedure was repeated without the drugs (reference solution), diluted to mark with distilled water and also scanned.

Calibration and validation 2.5.1 Calibration graphs for ATL and PPN
A six-point calibration graph was prepared by transferring aliquots (0 -0.80 mL) of stock solutions of reference standards into a series of 10 ml volumetric flasks using a micro-pipette to obtain a working concentration range of 0 -8.0 µg mL -1 . To each flask, 0.5 mL mol L -1 NaOH was added, allowed to stand for 3 minutes, then 1 mL of 2 x 10 -3 mol L -1 KMnO₄ was added and allowed to stand for 10 minutes, diluted to mark with distilled water. The absorbance of the resultant solution was measured at 520 nm against a blank.

Validation of proposed methods
In assessing and validating the method performance, criteria such as; precision, accuracy, specific interference, sensitivity, ruggedness and robustness were determined in addition to the calibration curve earlier mentioned. The recovery studies were evaluated for the proposed method using 5 μg mL -1 and 4 μg mL -1 of two brands each of atenolol tablets (ATL 1 and ATL 2 ) and propanalol (PPN 1 and PPN 2 ) respectively -these were spiked at three concentration levels with 2, 4 and 6 μg mL -1 of the reference standards. Percent recoveries of the spiked analytes were calculated, while statistical analyses with respect to data obtained in experimental results were also determined.
Replicate analysis of 5 concentrations -5, 10, 15, 20 and 25 μg mL -1 for atenolol and 4, 8, 12, 16 and 20 μg mL -1 for propranolol were evaluated to determine the intra-day and inter-day precision. Both drugs under investigation were replicated thrice at each concentration on the same day, while using the same concentrations the inter-day assay was done on 3 days within a week.
The linearity of ATL and PPN under optimum experimental conditions and within working concentrations were ascertained from calibration curves while using the least-squares method the regression equations and other key parameters were derived from the data obtained.
The limit of detection (LOD) and the limit of quantification (LOQ) were used to measure the sensitivity of the method. The LOD and LOQ were determined by evaluating the lowest concentrations of each beta-blocker can be detected and measured, respectively. They were obtained using the expressions, LOD =3.3S a /b; LOQ = 10 S a /b (where S a is the standard deviation of the intercept of regression line, and ' b' is the slope of the calibration curve) [32,33].

Procedure for application of method to pharmaceutical preparations
An amount equivalent to 50 mg atenolol and propanolol were weighed from finely powdered tablets (20) of each and transferred into 25 mL of methanol in 50 mL volumetric flask. This was shaken gently and intermittently for 2 -3 minutes and made to mark with the same solvent. Mixtures were filtered through a whatman no. 42 filter paper and the first 10ml portion of the filtrate was discarded. The filtrate, 1 mL was diluted to 10 mL with water (solution B). To 0.4 mL aliquot of solution B, 0.5 mL 0.1 mol L -1 NaOH was added, allowed to stand for 3 minutes, then 1 mL of 2 x10 -3 mol L -1 KMnO₄ was added, allowed to stand for another 10 minutes, diluted to mark with distilled water. The absorbance of the resultant solution was measured at 520 nm against a reagent blank. Figure 2, shows the spectra of oxidized drugs complex by KMnO 4 in alkaline medium (pH ≥ 9). The absorption spectra showed four distinctive peaks at 460, 520, 540 and 570 nm. The peaks at 520 and 540 nm were the most predominant, with the former being the maximum. The reference solution spectrum (i.e., 2 x10 -3 mol L -1 KMnO 4 , without drugs) showed the same spectra trend as the oxidized drugs, however, the absorbance of the predominant peaks were significantly higher when compared to those of the KMnO 4 -drug complex. The spectra were similar to those reported by Askal et al., [28] for determination 1, 4-dihydropyridine drugs using KMnO 4 in an acidic medium. This implied that the spectra for both β-blockers were due to the unreacted (or excess) KMnO 4 solution after the formation of the complex [29,30,34]. Figure 3, confirms this trend as there were a significant green to greenish-yellow and to yellow after 10 minutes -which was relatively stable. In the alkaline medium KMnO 4 was spontaneously reduced to form the blue/ bluish-green coloured K 2 MnO 4 with Mn (+6) oxidation state [30,34], with a life span of 2 -4 minutes, while the green/yellowish-green coloured complex emerges thereafter for a duration of 4 -6 minutes. The reaction time for the formation of the green coloured complex agrees with previous spectrophotometric studies of β-blocker using KMnO 4 [35]. The exhibition of these colours may be due the presences of manganese (Mn) -a transitional metal that is capable of existing in different oxidation states [36] [ Figure 4a]. In potassium permanganate, manganese exist in (+7) oxidation state, which is violet/purple in aqueous solution. The formation of Mn (+6) was evident by the decrease in the absorbance at 520 nm as the reaction progresses [30]. In addition, the deep purple/violet colour exhibited by KMnO 4 solution makes it absorbs the green or bluishgreen and greenish-yellow colour between 500 -550 nm [36]. It is pertinent to mention that these colours exhibited by manganese are not due to d→d electronic transition but due to ligand-to-metal charge transfer (LMCT), since it electronic configuration has an empty d-orbital (4d o ). decline in the absorbance of KMnO 4 -Drug complex as the concentration of beta-blocker increases from 0 -8 μg mL -1 . This decrease was used as a measure for the quantification of beta-blocker drugs in their respective solutions.

Principle and chromogenic property
During the reaction three distinctive transitional colour changes were observed for drug-KMnO 4 complex; from violet to blue; blue to bluish-green, then to green;

Reaction time.
Figure 4b, shows the various colour changes and absorbance with respect to time. Prior to application of the proposed method for quantification of drugs, the time duration for the reaction to go to completion was determined, while absorbance of the formed complex were measured at 7 time amplitudes (0, 2.5, 5, 7.5, 10, 12.5 and 15 min) at 520 nm.

Reaction mechanism
The suggested equations for the oxidimetric reactions are presented in scheme 1, while the proposed reaction mechanism is shown in scheme 2. The stoichiometric equation for the reactions between each β-blocker and Mn (VII) was 1:2 molar ratio. This agrees with the previous report on the kinetic oxidative degradation and deamination of atenolol by aqueous alkaline permanganate [31].

Method validation
The proposed alkaline KMnO 4 oxidative spectrophotometric method was validated in accordance with ICH guidelines [37]. Table 1, shows the experimental conditions and analytical characteristics associated with the proposed method. The calibration plots for ATL and PPN reference standards were found to be linear for concentrations ranging from 0 -15.0 μg mL -1 ( Figure 5). The regression equations were obtained with the aid of Microsoft Excel, using the least-squares method. The intercept and slope for both β-blockers were 0.571 and 0.082 respectively for ATL, with corresponding values for PPN being 0.556 and 0.084 for five concentrations of Beer's plot against absorbance (n = 5). The correlation coefficients (R) ranged were 0.9996 to 0.9999 for PPN and ATL respectively. These values implied high sensitivity of the proposed method. The degree of accuracy was further ascertained by obtaining the Sandell's sensitivity optimum concentrations for both β-blockers, with ATL and PPN being 0.056 and 0.053 μg cm -2 respectively. The LOD were 0.50 and 0.58 μg mL -1 for ATL and PPN respectively, with corresponding LOQ as 1.65 and 1.91 μg mL -1 . Both LOD and LOQ values simply confirmed the repeatability and reliability of the proposed methods.

Selectivity and interference
This study showed that the selectivity and interference in the proposed method were enhanced and negligible respectively. Firstly, drug samples are in tablet formulations, hence co-extractive chromogenic interferences from methanol soluble substances in tablets are expected to be absent. The presence of such co-extractives would contribute to the amount of the beta-blockers reacting with KMnO 4 , thus decreasing further the residual KMnO 4 measured by spectrophotometry. Most solid dosage formulations are compressed with excipients that are mainly inorganics, which are also highly insoluble in organic solvents such as alcohols. In tablet formulations, the excipients and additives used are; sodium lauryl sulfate, magnesium stearate, starch sodium glycolate, lactose spray dried, carboxymethylcellulose (CMC), talc, titanium dioxide, microcrystalline cellulose, hydroxypropylcellulose and pre-gelanitizated starch [38]. These substances are alcohol-insoluble and non-chromogenic. The absence of interference, therefore, implied high selectivity and applicability of the method for routine analysis in pure and solid dosage forms.

Reproducibility, precision and accuracy
The intra-and inter-day assay to ascertain the precision and accuracy of the KMnO 4 oxidative method at specified concentrations are shown in Table 2. The relative standard deviations (RSD) for both β-blockers ranged from 0.2 -1.95 % for inter-and intra-day assays, with corresponding standard errors ≤ 0.12 for all runs. These values potrayed high reproducibility and satisfactory precision and accuracy of the method. Table 3, shows the assessment of the ruggedness of the proposed method under-recovery studies. This was done by studying the effect of minor changes on some experimental conditions using both β-blockers. The aforementioned study provided a measure of the reliability of the proposed method during routine work. Values Table 2 Precision and accuracy studies of proposed oxidative KMnO 4 spectrophotometric methods

Application to pharmaceutical formulations
The proposed oxidative procedure was applied to determine the drug contents in different formulated brands of two beta-blockers (ATL and PPN) of five brands each. The brands were ATL 1 , ATL 2 , ATL 3 , ATL 4 and ATL 5 containing atenolol, while for propranolol the brands were -PPN 1 , PPN 2 , PPN 3 , PPN 4, and PPN 5 .
The proposed method showed the inclination of being free from interferences likely to be caused by the aforementioned excipients and/or common degradation products.

Conclusion
The present method is based on a redox reaction using KMnO 4 as an oxidizing agent and chromogen in inducing a simple ligand-to-metal charge transfer (LMCT) process in an aqueous alkaline medium (pH ≥ 9). The method has demonstrated that β-blockers can be assayed in bulk drugs and tablet formulations in the visible region of the spectrophotometer. The reagents used are cheap and readily available, while the instrument is affordable. In addition, the methodology is time-saving and not tedious. The method has also shown high precision, accuracy, selectivity -devoid of interferences and with high sensitivity in terms of molar absorptivities. These advantages enhance the application of this method in routine quality assessment of β-blockers in the pharmaceutical industry.