Nano-level detection of certain beta-blockers based on surface plasmon resonance band of silver nanoparticles; Application to content uniformity test

Sayed M Derayea¹*, Mahmoud A Omar ¹, Mohamed Aboel-Kasem Abdel-Lateef²

¹Department of Analytical Chemistry, Faculty of Pharmacy, Minia University, Minia 61519, Egypt

²Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut branch, Assiut 71524, Egypt

*Corresponding Author E-mail: sayed.derayea@gmail.com

ABSTRACT:

A simple, sensitive and cost effective method was developed and validated for the determination of some β-adrenergic blocking agents namely; carvedilol, labetalol hydrochloride, nebivolol hydrochloride and sotalol hydrochloride. The method was based on the ability of the cited drugs to induce the formation of silver nanoparticles (Ag-NPs) in the presence of polyvinyl pyrrolidone as a stabilizer. The intense surface plasmon resonance of the formed nanoparticles appeared at 420 nm, was utilized for the quantitative spectrophotometric determination of the beta-blockers. The linear concentration ranges were 0.05 – 1.6, 0.16 – 3.0, 0.2 – 2.5 and 0.10 – 2.0 μg mL^-1 for carvedilol, labetalol hydrochloride, nebivolol hydrochloride and sotalol hydrochloride respectively with limits of detection of 30, 53, 62 and 34 ng mL^-1, respectively. The commercial dosage forms containing the studied drugs were analyzed by the proposed method and the results were compared statistically with those obtained by the reported methods. The comparison revealed high precision and accuracy of the suggested method. Moreover, the method was extended to the content uniformity testing of commercial tablets dosage forms.

KEYWORDS: Surface plasmon resonance; b-adrenergic blockers; Silver nanoparticles; content uniformity test; Spectrophotometry.

1. INTRODUCTION:

β-adrenergic antagonists (also called β-Blockers) are widely used in the managements of several cardiovascular disorders such as; cardiac arrhythmias, arterial hypertension in addition to angina pectoris. Some members of these group are prescribed for the treatment of glaucoma or anxiety [1]. These drugs can exert their therapeutic effects through blocking the beta-adrenergic receptors, preventing the endogenous agonists noradrenaline and adrenaline from accessing this receptors. It also was reported that an extent of the beneficial cardiovascular effect of some β-adrenergic drugs was due to their antioxidant properties. In this work, the following β-adrenergic blockers were investigated; carvedilol (CRV), labetalol hydrochloride (LBT), nebivolol hydrochloride (NBV) and sotalol hydrochloride (SOT) [2-5]. The chemical structures of the investigated drugs are shown in Fig. 1.

Fig. 1: Chemical structures of the studied ß-adrenergic blockers.

Several methods for determination of the investigated β- blockers have been reported. Among these methods are spectrophotometry [6-14], spectrofluorimetry [15-21] liquid chromatography (HPLC) [4, 5, 22-27], gas chromatography (GC) [28-30], electrochemical [31-35] and capillary electrophoresis [36-38] methods.

Spectrophotometric methods based on colorimetric assay still one of the most commonly used analytical techniques due to their convenience of visual observation, high reproducibility and inherent simplicity. Recently several metal nanomaterials were applied for the colorimetric determination of different analytes. Gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) were commonly used for this purpose because of their unique optical and electric properties [39-42]. Both of them have strong localized surface plasmon resonance absorption with extremely high extinction coefficients. There are different approaches in using metal nanoparticles in colorimetric analysis. One of the most common and simple way is that based on the formation of metal nanoparticles through reduction of the metal ions under the effect of the analvtes. In case of the reduction of gold/silver ions to AuNPs/AgNPs, solutions, the process is associated with the appearance of distinctive colors and surface plasmon resonance bands of metal nanoparticles located in the visible region dependent on the size, shape and composition of the formed a nanoparticles [43]. It should be noted that, the reduction of silver ions is weaker than gold ions which will limits the compounds that can reduce silver ions to AgNPs. Therefore, AgNPs have higher selectivity than that of gold ions. Another advantage of silver nanomaterials are their cost-effectiveness. The AgNPs-based sensor has been applied to determine many substances such as metal ions[44, 45], pharmaceutical compounds [46-49], pesticide[50, 51], small molecules[52], and proteins[53, 54].

In this work, silver nanoparticles were applied for the development and validation of of a simple, sensitive, effective and validated procedure for the determination the cited drugs. The suggested method was applied for the analysis of these drugs either in bulk powders or in their pharmaceutical dosage forms..

2. EXPERIMENTAL:

2.1. Instrumentation

All absorbance measurements were carried out using Shimadzu UV and visible recording spectrophotometer (UV 260) with matched 10 mm. quartz cell.

2.2. Materials and reagents

All chemicals used were of analytical reagent grade. Carvedilol (99.0%) was kindly provided by Sigma Pharmaceutical Industries, Cairo, Egypt. Labetalol hydrochloride of (99.8%), was kindly provided by DBK Pharmaceutical Company, El Obour City, Cairo, Egypt. Nebivolol hydrochloride, of (99.6), was kindly provided by Marcyrl Pharmaceutical Industries, El Obour City, Cairo, Egypt.Sotalol hydrochloride (99.5) was kindly provided by Amoun Pharmaceutical Company S.A.E, El Obour City, Cairo, Egypt.

Silver nitrate (Sigma–Aldrich, Steinheim, Germany) was prepared as 15 mM aqueous solution. Polyvinylpyrrolidone with an average molecular weight of10,000 (Acros, Geel, Belgium), was prepared as 0.2% aqueous solution, Sodium hydroxide (El Nasr. Co. Egypt) was prepared as 2.5 mM aqueous solution.

Distilled water was prepared by water distiller (Tyumen-Midi-A0-25 MO, Russia).

2.3. Pharmaceutical formulations:

Betacor® tablets (Amoun Pharmaceutical Company S.A.E., Cairo, Egypt.) labelled to contain 80 mg sotalol hydrochloride per tablet. Karvex® tablets (Sigma Pharmaceutical industries, Cairo, Egypt) labelled to contain 6.25 mg carvedilol per tablet. Labetalol® tablets (Debaky Pharmaceutical Company, El Obour City, Cairo, Egypt) labelled to contain 200 mg labetalol hydrochloride per tablet. Nevilob® tablets (Marcyrl Pharmaceutical Industries, El Obour City, Cairo, Egypt) labelled to contain 2.5 mg nebivolol hydrochloride per tablet.

2.4. Preparation of standard drug solutions

Stock solutions of labetalol hydrochloride and sotalol hydrochloride were prepared by dissolving 100.0 mg of each drug in distilled water. Carvedilol stock solution was prepared by dissolving 100.0 mg of carvedilol powder in methanol. Nebivolol hydrochloride was prepared by dissolving 100.0 mg of nebivolol hydrochloride powder in 30 ml methanol and further diluted distilled water. Working standard solutions having the required concentrations of the cited drugs were prepared by further dilution of the stock solutions with distilled water. The standard solutions were stable for 7 days when kept in refrigerator.

2.5. General Analytical Procedure:

In stopper test tube, appropriate amounts of silver nitrate, PVP, different concentrations of the cited drugs and appropriate amounts of NaOH were added, 4 ml of double distilled water were added, heated in the water bath at 80°C for appropriate times, quantitatively transfer the contents to 10 ml volumetric flask and then completed to 10 ml with double distilled water. The absorbance of the formed nanoparticles was measured at the wavelength mentioned in Table 1, against reagent blank treated similarly.

Table 1: Optimum conditions for the development of parameters for determination of the studied ß-adrenergic blockers through silver nanoparticles formation.

Parameter	Carvedilol	Labetalol HCl	Nebivolol HCl	Sotalol HCl
Volume of Silver nitrate (0.015M)	1.0 ml	0.8 ml	1.0 ml	0.6 ml
Volume of PVP (0.2%)	0.8 ml	0.6 ml	0.6 ml	0.8 ml
Volume of NaOH (2.5 mM)	0.8 ml	0.6 ml	0.8 ml	0.8 ml
Temperature	80^oC	80^oC	80^oC	80^oC
Time of reaction	25 min.	35 min.	30 min.	35 min.

2.6. Procedure for analysis of tablets:

Twenty tablets from the local market were weighed, finely powder. An amount of the powder equivalent to 50.0 mg of drugs as hydrochloric salts was transferred into a 100-mL separating funnel. The powder shacked with about 20 mL distilled water. The free base of the drug was librated by the drop wise addition of 33% w/v aqueous ammonia and extracted with three successive potions of 20 mL methylene chloride. The combined methylene chloride extracts were filtered through anhydrous sodium sulfate. The organic solvent was evaporated to dryness under reduced pressure. The residue was dissolved in 0.01 M hydrochloride, quantitatively transferred into 100 ml volumetric flask and completed to volume with double distilled water. Further dilution of the resultant solution was performed to obtain solution containing 10 μg mL^-1 of the drug. The general analytical procedure was applied on aliquot volume of the final solution and the drug content was obtained by using the corresponding regression equation.

2.7. Procedure for content uniformity testing for studied drugs:

Tablets contents uniformity was carried out by following the official USP guidelines [55] (Chapter 905: Uniformity of Dosage Units). The drug contents of ten individual tablets were estimated by applying the same procedure for the analysis of tablets.

3. RESULTS AND DISCUSSION:

In this study, the system for the production of silver nanoparticle includes an aqueous solution of silver nitrate in the presence of an alkaline medium and polyvinylpyrrolidone (PVP) as stabilizing agent. The presence of secondary amino and secondary alcoholic groups in the studied beta-blockers (carvedilol, labetalol hydrochloride, nebivolol hydrochloride and sotalol hydrochloride) make these drugs as effective reducing agents which promote the transformation of silver ions into Ag NPs in a seedless process. It should be found that, no absorption band appeared in the visible region (380 – 700 nm) in the absence of cited drugs, However, the addition of b-blockers to the reaction mixture, silver nanoparticles were formed and their surface plasmon resonance bands were detected as shown in Fig 2.

Fig. 2: Absorption spectra of the formed silver nanoparticles using 1.0 µg mL^-1of the studied drugs except labetalol (2.0 µg mL^-1).

3.1. Optimization of experimental factors

3.1.1. Effect of the type and concentration of the stabilizer

The presence of surfactant is essential for the formation of nanoparticles since it prevent their aggregation. PVP and SDS were examined for their stabilizing effect during the preparation of Ag-NPs. It was found that the use of PVP as stabilizer is more preferable since it gave the highest sensitivity and wider linear range compared to SDS. The explanation of this observation can be as follow; the reaction of analytes with silver nitrate results in the formation of protons, as a result, the removal of these protons can enhance the formation of Ag-NPs. PVP can stabilize the formed Ag-NPs by removal of the produced protons through the formation of H(PVP)⁺. In the same time, PVP can also stabilize silver ions by formation of Ag(PVP)⁺ complexes. Removal pf protons will enhance the formation of Ag-NPs while, the stabilization of silver ions reduces its rate of formation. However, the protons removal through formation of H(PVP)⁺ is stronger than the other one consequently the reaction is moved to the forward direction [56]. It was found that, maximum reading were observed when using 0.4 ml of 0.2 % PVP solutions in case of labetalol hydrochloride and nebivolol hydrochloride, while 0.6 ml of the reagent in case of carvedilol, and sotalol hydrochloride. Higher reagent volumes did not any change if the absorbance. Therefore, 0.6 and 0.8 ml of 0.2 % PVP solutions for the two set of drugs respectively (Fig. 3).

Fig. 3: Effect of the volume of 0.2 % PVP solution on silver nanoparticles formation with 1.0 µg mL^-1of the studied drugs

3.1.2. Effect of sodium hydroxide concentration

It was explained previously that, the removal of protons can enhance the formation of Ag-NPs. For that reason, it was important to examine the effect of the alkalinity of the reaction solution by changing the volumes of added 2.5 mM NaOH solution (Fig. 4). By increasing the amount of NaOH, the absorbance increased up to a certain concentration of NaOH. Higher concentration decreased the absorbance with the formation of black precipitate due to the formation of Ag₂O. Thus, 0.8, 0.6, 0.8 and 0.8 ml of 25 mM NaOH was selected as the optimum NaOH volume for carvedilol, labetalol hydrochloride nebivolol hydrochloride and sotalol hydrochloride, respectively.

Fig. 4: Effect of the volume of 2.5 mM NaOH solution on silver nanoparticles formation in the presence of the studied drugs (1.0 µg mL^-1).

3.1.3. Effect of Silver nitrate concentration

It was found that the absorbance of formed Ag-NPs increased by increasing the volume of 15 mM silver nitrate solution. Maximum absorbance values were obtained using 0.8, 0.6, 0.8 and 0.4 ml for carvedilol, labetalol hydrochloride, nebivolol hydrochloride and sotalol hydrochloride respectively (Fig. 5). Further increase in the reagent volume did not produce any significant change in the absorbance. Therefore, the optimum volumes of 15 mM silver nitrate were 1.0, 0.8, 1.0 and 0.6 ml for the studied drugs respectively.

Fig. 5: Effect of the volume of 15 mM Silver nitrate solution on silver nanoparticles formation with the studied drugs (1.0 µg mL^-1).

3.1.4. Effect of temperature and time of heating

Heating of the reaction vessel was important to accelerate the rate of formation of the Ag NPs. It was observed that the highest results were obtained upon heating the solution in water bath at 80 °C for 20, 30, 25 and 30 min for carvedilol, labetalol hydrochloride, nebivolol hydrochloride and sotalol hydrochloride respectively (Fig. 6). The absorbance values were remained unchanged upon heating for longer times. Consequently, 25, 35, 30 and 35 min were the sufficient heating times for the investigated drugs respectively to produce maximum absorption intensities.

Fig. 6: Effect of the reaction time on the formation of the silver nanoparticles with the studied drugs (1.0 µg mL^-1).

3.2. Method validation

3.2.1. Linearity and range

Under the optimum experimental conditions, calibration curves were constructed for carvedilol, labetalol hydrochloride, nebivolol hydrochloride and sotalol hydrochloride by plotting the absorbance versus the corresponding drug concentration in μg mL^-1. The data were statistical evaluated and the linear regression equations were estimated. The linear concentration range, correlation coefficient, intercept and slope for the calibration curve of each drug were calculated. In addition, the standard deviations of the residuals (S_y_/x), intercept (S_a) and slope (S_b) were also calculated (Table 2). The proposed method has a good linearity as indicated by the high correlation coefficient which was in the range of 0.9992 - 0.9998.(Table 2).

Table 2: Summary of quantitative parameters and statistical data for the determination of the studied drugs with the proposed method.

Parameter	Carvedilol	Labetalol HCl	Nebivolol HCl	Sotalol HCl
linear range (μg/ml)	0.05 - 1.6	0.16 - 3.0	0.2 - 2.5	0.10 - 2.0
Slope	0.4468	0.2689	0.4277	0.4568
SD of slope (S_b)	0.0045	0.0023	0.0057	0.0038
Intercept	0.0323	0.0137	-0.0073	0.0197
SD of intercept (S_a)	0.0041	0.0043	0.0080	0.0047
r² ^a	0.9992	0.9996	0.9984	0.9994
r ^a	0.9996	0.9998	0.9992	0.9997
S _y,x ^b	0.0075	0.0046	0.0121	0.0069
LOD (μg/ml)	0.030	0.053	0.062	0.034
LOQ (μg/ml)	0.092	0.161	0.187	0.103

^a r² and r are the correlation and determination coefficients, respectively and number of determinations is eight.

^b S_x,y is the standard deviation of residual.

Table 3: Evaluation of accuracy of the investigated analytical procedure at three concentration levels within the specified range.

Drug	% Recovery^*± SD
	0.5 μg mL^-1	1 μg mL^-1	1.5 μg mL^-1
Carvedilol	100.82 ± 1.43	99.63 ± 0.74	100.02 ± 1.37
Labetalol HCl	99.22 ± 1.35	100.24 ± 0.67	100.73 ± 1.17
Nebivolol HCl	99.57 ± 1.10	1.37 ± 1.34	99.94 ± 1.21
Sotalol HCl	100.89 ± 1.25	100.14 ± 0.93	100.79 ± 1.24

^* The value is the mean of six replicates.

Table 4: Analysis of the bulk powders of the studied drugs with the reported [11-14] methods and the proposed silver nanoparticles method.

Drug	% Recovery ^a ± SD		t- value ^b	F- Value ^b
	Proposed method	Reported method
Carvedilol	99.75 ± 0.76	100.34 ± 0.98	1.317	1.638
Labetalol HCl	100.07 ± 0.89	101.16 ± 1.32	1.522	2.222
Nebivolol HCl	99.54 ± 1.13	99.81 ± 0.95	0.4033	1.402
Sotalol HCl	99.93 ± 1.61	98.5 ± 0.83	1.774	3.758

^a The value is the mean of five determinations for both the reported and proposed methods.

^b Tabulated value at 95% confidence limit; F=6.338 and t =2.306.

Table 5: Intra- and Inter-day precisions the determination of the studied drugs with proposed method.

Conce. Level	% Recovery ±S.D ^a
	Carvedilol	Labetalol HCl	Nebivolol HCl	Sotalol HCl
Intra-day precision
0.5 (μg/mL)	99.83 ± 1.15	98.91 ± 1.50	99.57 ± 1.28	100.24 ± 1.57
1.0 (μg/mL)	99.48 ± 0.82	100.48 ± 0.99	100.25 ± 1.69	100.47 ± 1.76
1.5 (μg/mL)	100.07 ± 1.38	101.69 ± 1.86	99.03 ± 1.31	100.93 ± 1.51
Mean ±S.D	99.79 ± 0.30	100.36 ± 1.39	99.62 ± 0.61	100.55 ± 0.35
Inter-day precision
0.5 (μg/mL)	101.81 ± 0.95	98.79 ± 1.31	99.89 ± 1.95	100.96 ± 1.53
1.0 (μg/mL)	99.78 ± 0.80	99.08 ± 1.12	101.21 ± 1.37	99.24 ± 1.36
1.5 (μg/mL)	99.96 ± 1.67	101.35 ± 0.79	99.41 ± 0.89	99.97 ± 1.45
Mean ±S.D	100.52 ± 1.12	99.74 ± 1.40	100.17 ± 0.93	100.06 ± 0.86

^aThe value is the mean of three determinations.

Table 6: Analysis of the investigated drugs in their pharmaceutical dosage forms using the proposed spectrophotometric and reported methods.

Dosage forms	% Recovery ^a ± SD		t- value ^b	F- Value ^b
	Proposed method	Reported method
Karvex® tablets	99.07 ± 0.74	100.28 ± 1.57	1.557	4.437
Labetalol® tablets	100.00 ± 1.5	98.84 ± 0.84	1.505	3.172
Nevilob® tablets	99.01 ± 0.75	100.00 ± 1.30	1.471	3.007
Betacor® tablets	100.15 ± 1.10	99.05 ± 0.54	1.997	4.143

^a The value is the mean of five determinations for both the reported and proposed methods.

^b Tabulated value at 95% confidence limit; F=6.338 and t =2.306.

Table 7: Results of content uniformity testing of the studied β- blockers tablets using the proposed method.

Dosage form	% of the label claim	% RSD ^*	% error	AV ^#
Karvex® 6.25 mg tablets	99.75	1.57	0.50	3.77
Nevilob® 2.5 mg tablets	100.14	1.46	0.46	3.51

^*RSD is the relative standard deviation

^# AV is the acceptance value