Uv-Vis Spectrophotometric Assay Of Metoprolol

FARMACIA, 2008, Vol.LVI, 4 107

UV-VIS SPECTROPHOTOMETRIC ASSAY OF METOPROLOL. NOTE 2. METHOD VALIDATION

MAGDALENA BĂDULESCU1*, DAN BĂLĂLĂU2, IOANA CACOVEAN2, MIHAELA ILIE2, DANIELA LUIZA BACONI2 1National Medicines Agency, 48 Aviator Sănătescu Str., Bucharest 2”Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Toxicology Department, 6 Traian Vuia Str., Bucharest, Romania *corresponding author: [email protected]

Abstract Metoprolol is a widely used beta-blocker mostly administrated in cardiovascular diseases. Due to the highly probable overdosage, a rapid and easy to perform method for the quantitative determination of metoprolol tartrate in biologic matrices would be useful in its therapeutic monitoring as well as in intoxication cases. The paper presents two validated methods for the UV-VIS quantitative assay of metoprolol tartrate in tablets: the UV direct method and the oxidative method with potassium permanganate. The parameters targeted for the validation were: specificity, range, detection and quantification limits, accuracy, and precision.

Rezumat Metoprololul este un beta-blocant cu utilizare frecventă, administrat cu precădere în maladii cardiovasculare. Deoarece există posibilitatea supradozării, este necesară găsirea unor metode rapide de determinare cantitativă a tartratului de metoprolol din matrice biologică, atât pentru monitorizare terapeutică, cât şi pentru dozare în situaţia suspiciunii de intoxicaţie. Prezenta lucrare are ca scop validarea unor metode spectrofotometrice UV-VIS de determinare a tartratului de metoprolol din comprimate: metoda UV directă şi metoda oxidativă cu permanganat de potasiu. Parametrii avuţi în vedere la validare au fost: specificitatea, domeniul de liniaritate, limitele de detecţie şi cuantificare, acurateţea şi precizia.

 metoprolol quantitative assay  UV-VIS absorption  validation

INTRODUCTION Metoprolol tartrate is a selective beta-adrenergic antagonist, which is used in the treatment of cardiovascular disorders such as hypertension, angor pectoris, cardiac arrhythmias, congestive heart failure and myocardial 108 FARMACIA, 2008, Vol.LVI, 4 infarction [1,2,3]. The drug is quite sensitive, even a small dose of the drug giving a sufficient blockade of the beta-adrenergic receptors. Thus the quantitative determination of the drug is important and a simple method such as the UV-VIS absorption for the assay would be of a great interest [4,5,6]. The paper presents the validated UV-VIS methods for the metoprolol tartrate assay in tablets [6]. The validation was performed in respect of the ICH Q2R1 regulations [7] for analytical procedures. The following parameters were taken into account: specificity, linearity, limits of detection and quantification, accuracy, precision.

MATERIALS AND METHODS Reagents: - metoprolol tartrate USP 30 (Sigma) - metoprolol tablets Betaloc® ZOK (AstraZeneca) - sodium hydroxyde (Merck), - potassium permanganate (Chimopar). Device UV-Vis Cary 100 Bio spectrophotometer (Varian Inc.) Procedure a. General procedure 0.1 mg/mL metoprolol tartrate stock solution is prepared in double distilled water to be further dilluted for the standard solutions. Five tablets are analytically weighted (0.1 mg precision), triturated and analytically transferred with 50 mL of double distilled water to a 100 mL Erlenmeyer flask. The content of the flask is filtered through analytical filter paper in a 100 mL volumetric flask, the filter paper is washed with another 40 mL of double distilled water, than the volumetric flask is completed to the mark to give the stock sample solution. The working solution is obtained by 1:100 dilution with double distilled water. b. Direct UV method First, a background correction of the spectrophotometer is performed by using double distilled water. Then the spectra of a series of working standards towards double distilled water, at least five concentrations in the range of 0.5 – 10 μg/mL, in triplicate, is plotted, as well as the spectra of the sample solution, by using the following parameters: spectral range: 190 – 400 nm, spectral bandwidth 0.2 nm, scanning speed 600 nm/min. The absorbance of each solution at 222 nm [6] is measured. FARMACIA, 2008, Vol.LVI, 4 109

c. Oxidative method First, a background correction of the spectrophotometer is performed by using double distilled water. Stock solutions of 0.6 M sodium hydroxyde and 0.015M potassium permanganate are prepared ex temporae in double distilled water. In 10 mL volumetric flasks aliquots of 0.2 to 1 mL of stock standard solution and of 0.1 mL from the sample working solution are added. Similary, the same aliquots of distilled water are added to 10 mL volumetric flasks. To each flask 2 mL of NaOH stock solution and 2 mL of 0.015 M KMnO4 solution are added, then the flasks are completed to the mark with double distilled water. As previous experiments showed that the reaction is completed after 15 minutes, each standard and sample solution is let to stand still for 15 min, than the spectrum of each sample and the corresponding blank solution are plotted in the range 500 – 650 nm, spectral bandwidth 0.2 nm, scanning speed 100 nm/min. The absorbance at 610 nm is measured and the difference between the solutions containing metoprolol tartrate and the corresponding blank solution is computed to be further used. Data analysis For both methods the calibration curve absorbance vs. concentration is plotted, and the concentration of the sample is computed by interpolation on the corresponding calibration curve. The amount of metoprolol tartrate is computed, taking into consideration the original dillution (1:10000). For the calculation of the detection (DL) and quantification (QL) limits the following equations were used [7]: 3.3 DL  A (1) B 10  QL  A (2) B where σA is the standard deviation of the intercept, and B – the slope of the calibration line. The recovery coefficient was computed following the equation: R (%)=100 x (amount found)/(amount known) (3) 110 FARMACIA, 2008, Vol.LVI, 4

RESULTS AND DISCUSSION 1. Direct UV method Specificity In order to demonstrate the specificity of the method, UV spectra of a standard solution and of the sample were recorded. As can be seen in figure 1, no spectral interferences could be noticed from the excipients for the 222 nm band of metoprolol tartrate.

Figure 1 Spectra of a 6 μg/mL metoprolol tartrate and a sample solution

Linearity, DL and QL The linearity of the method was studied in the range of 1-30 μg/mL using the 222 nm absorption band. The results are presented in Table I.

Table I Linearity, limits of detection and quantification for the direct UV method Parameter Values Linearity range 1-30 μg/mL Intercept 0.02604±0.00291 Slope 0.03029±0.0005 Correlation coefficient (R) 0.99873 Probability (p) <0.0001 Detection limit (DL) 0.317 μg/mL Quantification limit (QL) 0.961 μg/mL FARMACIA, 2008, Vol.LVI, 4 111

Accuracy The accuracy was studied using known amounts of metoprolol tartrate in the range of 1 -20 μg/mL, via the recovery coefficient, which has to be in the range of 99-101%. The obtained results are presented in Table II, showing that method is accurate. Table II Accuracy of the UV direct method Concentration of Concentration of the recovered Recovery metoprolol tartrate metoprolol tartrate coefficient taken into analysis (µg/mL) (%) (µg/mL) 2.00 2.00 100.0 6.00 6.06 101.0 10.00 10.8 100.8 14.00 13.96 99.7 20.00 19.88 99.4

Precision We investigated the precision of the method through its repetability and reproducibility. We repeated 5 times the assay of metoprolol tartrate in the same day, by the same analyst, and 5 times the assay of the content of metoprolol tartrate in the tablet by five different analysts in different days, then we computed the relative standard deviation (RSD). The acceptance criteria demands that RSD should be less than 2% . The results are summarised in Table III, and show that the method is precise.

Table III Precision of the UV method Declared Repetability Reproducibility amount Amount found Amount Amount Amount (mg/tablet) (mg/tablet) found (%) found found (%) (mg/tablet) 49.2 98.4 51.0 102 49.7 99.4 50.3 100.6 50.6 101.2 49.0 98 50 50.2 100.4 49.7 99.4 50.2 100.4 50.0 100 50.5 101 50.2 100.4 Mean 50.067 100.13 50.033 100.067 SD 0.5279 1.0558 0.6653 1.3307 RSD (%) 1.05 1.05 1.33 1.33 112 FARMACIA, 2008, Vol.LVI, 4

2. Oxidative method Specificity In order to prove the specificity of the method we registered spectra of a sample, a 4 μg/mL standard and a blank solution (Figure 2) prepared as mentioned above.

Figure 2

VIS absorption spectra of metoprolol tartrate – KMnO4 in alkaline conditions

As it can be seen in figure 2, in the presence of a sample or a standard of metoprolol tartrate, the band at 610 nm is better differentiated that in case of the blank sample, suggesting that in the redox reaction only the active substance (metoprolol tartrate) is involved, not the excipients present in the pharmaceutical formulation. Linearity, DL and QL The linearity of the method was studied in the range of 4-10 μg/mL. The results are presented in Table IV. Table IV Linearity, limits of detection and quantification for the oxidative method Parameter Values Linearity range 4 – 8 μg/mL Intercept -0.102±0.0021 Slope 0.041±0.00036 Correlation coefficient (R) 0.99989 Probability (p) <0.0001 Detection limit (DL) 0.566 μg/mL Quantification limit (QL) 1.714 μg/mL FARMACIA, 2008, Vol.LVI, 4 113

Accuracy The accuracy was studied using known amounts of metoprolol tartrate in the range of 4 -8 μg/mL, via the recovery coefficient, which has to be in the range of 99-101%. The obtained results are presented in Table V, showing that the method is accurate. Table V Accuracy of the oxidative method Concentration of Concentration of the recovered Recovery metoprolol tartrate metoprolol tartrate coefficient taken into analysis (µg/mL) (%) (µg/mL) 4.00 4.03 100.7 6.00 5.99 99.8 8.00 7.96 99.5

Precision We investigated the precision of the method through its repetability and reproducibility. We repeated 5 times the assay of metoprolol tartrate in the same day, by the same analyst, and 5 times the assay of the content of metoprolol tartrate in the tablet by five different analysts in different days, then we computed the relative standard deviation (RSD). The acceptance criteria demands that RSD should be less than 2% . The results are summarised in Table VI, and show that the method is precise.

Table VI Precision of the UV method Declared Repetability Reproducibility amount Amount found Amount Amount found Amount (mg/tablet) (mg/tablet) found (mg/tablet) found (%) (%) 50.31 100.6 51.20 102.4 50.24 100.5 50.71 101.4 50.62 101.2 49.86 99.7 50 49.98 100.0 50.06 100.1 49.96 99.9 51.08 102.1 50.222 100.44 50.582 101.18 Mean 50.222 100.44 50.582 101.18 SD 0.27096 0.52249 0.6000 1.0759 RSD (%) 0.54 0.52 1.19 1.06 114 FARMACIA, 2008, Vol.LVI, 4

CONCLUSIONS The validation of two methods for the quantitative assay of metoprolol tartrate in tablets are presented: a direct UV absorption method at 222 nm and an indirect method, using the redox reaction with potassium permanganate. The methods have been validated according to ICH Q2(R1) recommendations, for selectivity, range, detection and quantification limits, accuracy and precision. While the direct method can be useful in the determination of metoprolol in biological matrices, the oxidative method, due to the fact that the measured physical parameter is the absorption of the manganate ion, cannot be applied as mentioned for the metoprolol assay in biological matrices.

REFERENCES 1. *** Martindale: The Complete Drug Reference. Metoprolol Monograph – The Pharmaceutical Press 2007 2. *** AHFS Drug Information. Metoprolol Tartrate, Metoprolol Succinate Monographs – American Society for Health-System Pharmacists, 2007 3. Brunton LL, Lazo JS, Parker KI (eds.) – Goodman and Gilman’s The Pharmacological Basis of Therapeutics, eleventh edition, McGraw Hill, 2005 4. *** Clarke’s Analysis of Drugs and Poisons – Pharmaceutical Press, 2007 5. Rahman N., Rahman H., Aami S.N.H. – Validated Kinetic Spectrophotometric Method for the Determination of Metoprolol Tartrate in Pharmaceutical Formulations – Chem. Pharm. Bull., 2005, 53(8): 942-948 6. Cacovean Ioana, Ilie Mihaela, Bădulescu Magdalena, Bălălău Dan, Gubandru Miriana – UV-VIS Spectrophotometric Assay of Metoprolol. Note 1. Comparison Between the Direct and the Oxydative Methods – Farmacia, 2007, LV (5): 540-544 7. *** Validation on Analytical Procedures: Text and Methodology – ICH Q2 (R1) – International Conference on Harmonisation, Geneva, Switzerland, 2005