Application of Densitometry for Determination of Beta-Adrenergic-Blocking Agents in Pharmaceutical Preparations

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1 Application of Densitometry for Determination of Beta-Adrenergic-Blocking Agents in Pharmaceutical Preparations Jan Krzek* and Anna Kwiecień Key Words: Beta blockers TLC determination Densitometry Drug analysis Summary A chromatographic densitometric method has been established for identification and quantitation of selected beta-adrenergic-blocking agents in pharmaceutical preparations. Retention factors, R F, and characteristic absorption spectra of 11 drugs chromatographed on silica gel 60 F 254 HPTLC plates with six mobile phases were used for identification. Quantitation and validation of the method was performed for atenolol, acebutolol, propranolol, and bisoprolol using chloroform methanol ammonia, (v/v), as mobile phase. UV densitometric measurements were performed at the wavelength of maximum absorption. Pharmaceutical preparations used in medicine and from a variety of manufacturers were analyzed and the method shown to be sufficiently sensitive for analysis of these samples. The limits of detection and determination ranged from 30 to 400 ng and recovery was from to %. The precision of the method, described by the equation y = x mean ±2S, is good and the range of linearity is wide from to 0.250% for individual constituents. Beta-adrenergic-blocking agents are a relatively new generation of drugs, with a wide range of pharmacological applications, which are used to treat ischemic heart disease, heart failure, arrhythmia, and arterial hypertension [1, 2]. Currently betablockers include approximately fifty therapeutic agents with chemical structures based on aryloxypropanolamine derivatives and 1-aryl-2-alkyloaminoethanol bonds [3, 4]. The wide structural variety in this group of drugs leads to very different chemical and physical properties, for example fat and water solubility, which thus substantially affect their pharmacological activity and toxicity [5 7]. A variety of analytical techniques, for example HPLC [8 10], gas chromatography, often combined with mass spectroscopy [11], capillary electrophoresis [12], and thin-layer chromatography [13 15], have been used for qualitative and quantitative analyses of β-adrenergic blocking-agents both in pharmaceutical formulations and in body fluids. The aim of the work discussed in this paper was to develop a chromatographic densitometric method for identification and quantitation of selected beta-blockers in pharmaceutical preparations to be used as an alternative to HPLC. For quantitative analysis the absorption spectra recorded directly from chromatograms were used with, typically, comparison of retention times. Quantitation was performed by densitometry in the UV at selected wavelengths directly after dissolving weighed amounts of the medications in methanol. The reason for undertaking these studies was lack of relevant information in the available literature. 1 Introduction J. Krzek and A. Kwiecieñ, Jagiellonian University, Collegium Medicum, Department of Inorganic and Analytical Chemistry, Medyczna 9, Kraków, Poland. 2 Experimental 2.1 Chemicals and Standards Methanol was from Merck (Darmstadt, Germany), chloroform from Chempur (Poland), 25% ammonia from Zak³ady Azotowe (Tarnów, Poland), and glacial acetic acid from Odczynniki (Lublin, Poland). All reagents were of analytical purity. Standard solutions were prepared by dissolving appropriate amounts of standard substances in methanol to give concentrations in the range % (w/v). Ten tablets, corresponding to approximately 25.0 mg active substance were powdered and weighed to an accuracy of 0.1 mg. Methanol (20.0 ml) was added and the mixture was 308 VOL. 18. JULY/AUGUST 2005 Journal of Planar Chromatography DOI: /JPC

2 Table 1 Analytical wavelengths for selected beta-adrenergic blocking agents and R F values measured after chromatography with different mobile phases. Drug and Methanol Ammonia Methanol Chloroform Chloroform Chloroform ammonia methanol glacial methanol glacial methanol glacial methanol wavelength chloroform, acetic acid, acetic acid, acetic acid, ammonia, [nm] a) Acebutolol , 325 Atenolol , 270 Betaxolol , 275 Bisoprolol , 275 Labetalol Metoprolol , 280 Oxprenolol Pindolol , 278 Propranolol , 289 Sotalol , 270 Timolol a) The analytical wavelength does not depend on mobile phase composition shaken for 20 min, filtered, and the filtrate was diluted to 25.0 ml with methanol. 2.2 Analytical Procedure Figure 1 Densitogram obtained from a mixture of beta blockers: 1, atenolol; 2, sotalol; 3, acebutolol; 4, pindolol; 5, propranolol; 6, timolol. Mobile phase chloroform methanol ammonia, (v/v). Chromatography was performed on 10 cm 10 cm aluminumbacked silica gel 60 F 254 HPTLC plates (Merck, Darmstadt, Germany; # ). Samples and standard solutions (0.1%, from 2 to 5 µl) were applied to the plates as 10 mm bands by means of Camag (Muttenz, Switzerland) Linomat IV or Desaga (Germany) AS 30 sample applicators. Plates were developed to a distance of 95 mm in 18 cm 8 cm 15 cm chromatographic chambers (Sigma Aldrich) with chloroform methanol ammonia, (v/v), as mobile phase. The plates were then dried at room temperature. Densitometric scanning and analysis at selected wavelengths (λ = 270 nm for atenolol, λ = 240 nm for acebutolol, λ = 289 nm for propranolol, and λ = 220 nm for bisoprolol) was performed with a Camag TLC- Scanner 3 densitometer with CATS 4 software. For identification purposes, retention data R F were determined and spectra of reference substances and drug solutions, which should be identical, were recorded. The concentration of the active constituents were calculated by comparing peak areas from appropriate standard solutions with those from sample solutions prepared for the drugs under investigation. Journal of Planar Chromatography VOL. 18. JULY/AUGUST

3 Figure 2 Densitograms and absorption spectra obtained from acebutolol (1), atenolol (2), bisoprolol (3), and propranolol (4) in pharmaceutical preparations. 2.3 Validation of the Method For quantitation purposes the test conditions were validated by determining robustness, limits of detection and quantitation, accuracy, precision, and the relationship between peak area and concentration. Validation was performed for atenolol, acebutolol, bisoprolol, and propranolol, i.e. the drugs selected for quantitation [16] Robustness Solutions of the appropriate reference substances at concentrations of 0.05, 0.075, 0.1, 0.2, and 0.5% were used. To establish conditions for the identification of drugs under investigation, standard solutions and sample solution (5 µl) were applied as bands, with an applicator, to 10 cm 14 cm plates. Chromatograms were developed, at 22 C, in chromatographic cham- 310 VOL. 18. JULY/AUGUST 2005 Journal of Planar Chromatography

4 2.3.2 Limits of Detection and Quantitation Sample drug solutions containing decreasing concentrations of the individual constituents were prepared. To initial solutions containing 0.5 mg drug in 1 ml appropriate reference substances (0.5 mg) were added. The solutions were analyzed both before and after addition of the reference substances. The limits of detection and quantitation were derived from peak areas recorded for the chromatograms. The limits of detection and quantitation were defined as the peak area equal to or greater than four times the peak area corresponding to sample solutions containing none of the constituent under examination Accuracy The accuracy of the method was measured by determining the recovery [%],from the preparations under examination, of analytes added in amounts from 80 to 120% of the expected levels of the substances. The recovery (R [%]) was computed from the formula: R [%] = [(A A 1 )/A 1 ] 100% where A is the peak area [mm 2 ] obtained for the sample solution after adding a specified amount of analyte and A 1 is the peak area [mm 2 ] obtained before the analyte was added Precision The precision was expressed as the consistency of results from repeated analyses. Tests were performed with a model mixture containing the 0.1% of the four compounds under investigation. The amount applied to the plates varied 3 µl for determination of precision for bisoprolol, 4 µl for atenolol, and 5 µl for acebutolol and propranolol. Peak areas were used to evaluate method precision. The scatter of results was described as the standard deviation (S), also a measure of the repeatability of the method, and as the dispersion of the results around the mean at a probability of 95%. Figure 3 Absorption spectra of: (A) acebutolol (1), atenolol (2), labetalol (3), metoprolol (4); (B) oxprenolol (5), pindolol (6), propranolol (7), sotalol (8); (C) timolol (9), bisoprolol (10), betaxolol (11). bers, to a distance of 8 10 cm with the mobile phases listed in Table 1. After drying at room temperature the chromatograms were analyzed densitometrically. A typical chromatogram obtained from six drugs is shown in Figure 1 and individual chromatograms for the four substances under investigation are shown in Figure 2. UV absorption spectra were then recorded, directly from chromatograms, both for the drugs under investigation (Figure 2) and for other beta-blockers (Figure 3). The analytical wavelengths were determined and the retention times for the individual compounds were calculated for use of different mobile phases (Table 1) Linearity To examine linearity a series of solutions was prepared covering the relevant concentration ranges for the individual compounds under investigation and relationships between peak area and analyte concentration were calculated (Table 2). The regression plot, its regression equation, and the correlation coefficients are indicative of the linearity. Results from validation of the method for determination of atenolol, acebutolol, propranolol, and bisoprolol are shown in Table 2. Conditions for qualitative and quantitative analysis of drugs under investigation were established on the basis of the results obtained from these investigations. 3 Results and Discussion A new chromatographic densitometric method has been established for identification and quantitation of beta-adrenergicblocking agents and has been applied to pharmaceutical prepa- Journal of Planar Chromatography VOL. 18. JULY/AUGUST

5 Table 2 Validation data for determination of atenolol, acebutolol, propranolol and bisoprolol. Atenolol Acebutolol Propranolol Bisoprolol λ [nm] R F Limit of detection and quantitation 80 ng 40 ng 30 ng 200 ng 400 ng Recovery [%] RSD [%] Precision, y = x sr ±2S ± ± ± ± ± Linearity range [%] rations. Qualitative analysis was performed on eleven betaadrenergic blocking agents and quantitation was performed on thirteen pharmaceutical preparations containing atenolol, acebutolol, bisoprolol, and propranolol. Silica gel 60F 254 HPTLC plates and six different mobile phases found experimentally (Table 1) are proposed for identification of the substances under investigation. Most of the quantitative determinations were performed with chloroform methanol 25% ammonia, (v/v), because this mobile phase resulted in compact spots which provided peaks suitable for densitometric measurements. The other mobile phases listed can be used for identification of the drugs if use of other mobile phases results in R F values which are close to each other. To detect the presence of individual constituents on densitograms, direct UV densitometric measurements are sufficient. On chromatograms recorded for sample solutions, only peaks of the active substances were observed, indicating that no degradation is observed under the conditions established for the analysis. It is also apparent from the chromatograms are presented in Figure 2 that there was no interference from matrix constituents. The absorption spectra of the drugs, recorded directly from chromatograms, were sufficiently different to enable identification, even when their R F values were similar. The absorption spectra in Figure 2 are very similar to those recorded for standard solutions (Figure 3). Similar retention coefficients, R F, also confirm the identity of these substances. This method enables rapid qualitative and quantitative analysis because the chromatogram development distance is short up to 95 mm (development time up to 50 min). Under the conditions specified low concentrations ranging from 30 to 400 ng of the drugs can be analyzed. Because of this high sensitivity, the method enables determination when only a small amount of analyte is available, for example in toxicology (Table 2). The range of concentrations determined is sufficiently wide for quantitative analysis, and good precision, and sufficient recovery guarantee correct results from quantitation. Deviations in the results obtained from determination of the active substance are within the acceptable ranges stipulated by the pharmacopoeias [17]. When individual drugs from different manufacturers were analyzed no significant differences were found for concentrations of active substances, as confirmed by the results obtained and statistical analysis (Table 3). The results are characterized by good repeatability and a narrow confidence interval. The relative standard deviation (RSD), expressed as a percentage for individual determinations, did not exceed 4%. The analytical procedures described above require no special sample treatment and consist solely of dissolving an appropriate amount of the drug in a solvent, i.e. methanol. In addition to quantitative analysis, the specified conditions enable the simultaneous identification required in drug-quality assessment. 4 Conclusion The results presented above indicate that the conditions established enable simultaneous identification and quantitation of beta blockers in pharmaceutical preparations. Retention data and absorption spectra recorded directly from chromatograms can be used for identification of the drugs. This newly developed HPTLC method can be regarded an alternative to the more widely used HPLC, because sample preparation is simpler and because of the possibility of multi-sample analysis, which reduces analysis cost and time for individual samples. References [1] D. Liszewska-Pfejfer and W. Kostowski, Leki beta-adrenolityczne w farmakoterapii, PZWL, Warsaw, [2] R. Gryglewski and E. Tr¹bka-Kostka, Leki beta-adrenolityczne, PZWL, Warsaw, VOL. 18. JULY/AUGUST 2005 Journal of Planar Chromatography

6 Table 3 Amounts of atenolol, acebutolol, propranolol, and bisoprolol in pharmaceutical preparations. Preparation Active Content [g] X mean Standard Standard Confidence Relative and declared content substance (range from to) [g] deviation, S deviation of interval, standard arithmetic mean, µ (P = 95%) deviation, S x RSD [%] Normocard Polfa, g Atenolol ± Atenolol Polpharma, g Atenolol ± Atenolol Sanofi- Synthelabo, g Atenolol ± Acebutolol Sanofi Biocom, g Acebutolol ± Sectral Polfa, g Acebutolol ± Acecor SPA, g Acebutolol ± Avlocardyl Astra Zeneca, g Propranolol ± Propranolol Polfa (1), g Propranolol ± Propranolol Polfa (2), g Propranolol ± Propranolol Polfa, g Propranolol ± Bisocard Polfa, g Bisoprolol ± Bisohexal Hexal, g Bisoprolol ± Concor Merck, g Bisoprolol ± [3] C.J. Davies, J. Chromatogr. Biomed. Appl. 531 (1990) [4] A. Zejc and M. Gorczyca, Chemia leków, PZWL, Warsaw, [5] R. Kaliszan, A. Nasal, and A. Buciñski, Eur. J. Med. Chem. 29 (1994) [6] R. Kaliszan, A. Nasal, and M. Turowski, Biomed. Chromatogr. 9 (1995) [7] V. Martinez, M.I. Maguregui, R.M. Jimenez, and R.M. Alonzo, J. Pharm. Biomed. Anal. 23(2/3) [8] G. Lamprecht, T. Kreushofer, K. Stoschitzky, and W. Lindner, J. Chromatogr. B 740 (2000) [9] V. Andrisano, R. Gotti, A. Leoni, and V. Cavrini, J. Pharm. Biomed. Anal. 21 (1999) [10] A. El-Gindy, A. Ashour, L. Abel-Fattah, and M. Shabana, J. Pharm. Biomed. Anal. 24(4) (2001) [11] H. Siren, M. Saarinen, and S. Hainari, J. Chromatogr. 632(1/2) (1993) [12] R. Arias, R.M. Jimenez, R.M. Alonso, and M. Telez, J. Chromatogr. A 916 (1/2) [13] A.M. Tivert and A. Backman, J. Planar. Chromatogr. 6 (1993) [14] Z. Pawlak and B.J. Clark, J. Pharm. Biomed. Anal. 10(5) 1992) [15] A.P. Argekar and S.G. Powar, J. Pharm. Biomed. Anal. 21 (2000) [16] Validation of Analytical Procedures: Methodology, ICH Harmonised Tripartite Guideline, [17] European Pharmacopoeia, 3rd edn, Strasbourg, 1997, and supplements 1999, 2000, 2001, Ms received: January 7, 2005 Accepted by SN: March 3, 2005 Journal of Planar Chromatography VOL. 18. JULY/AUGUST