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Simultaneous determination of propranolol and pindolol by synchronous spectrofluorimetry
Talanta 45 (1998) 969 – 976
Simultaneous determination of propranolol and pindolol by synchronous spectrofluorimetry Toma´s Pe´rez Ruiz *, Carmen Martı´nez-Lozano, Virginia Toma´s, Jose´ Carpena Department of Analytical Chemistry, Faculty of Chemistry, Uni6ersity of Murcia, 30071, Murcia, Spain Received 27 November 1996; received in revised form 11 March 1997; accepted 30 June 1997
Abstract The simultaneous determination of propranolol and pindolol using synchronous fluorescence spectrometric techniques is described. The method involves measuring the natural fluorescence of these drugs in a 50% (v/v) ethanol–water medium using zero and first derivative synchronous spectrofluorimetry. Under the optimum conditions, the linear determination ranges of propanolol and pindolol are ca. 0.02 – 1.0 and 0.04 – 1.2 mg ml − 1, respectively. The results showed that propranolol and pindolol can be determined simultaneously when the concentration ratio of propranolol to pindolol varies from 1:100 to 50:1 in the mixed sample. The method has been satisfactorily applied to the determination of propranolol and pindolol in urine samples and propranolol in pharmaceutical preparations. © 1998 Elsevier Science B.V. Keywords: Propranolol; Pindolol; Synchronous fluorescence spectrometry
1. Introduction Propranolol (PRO: 1-[isopropylamino-3-[1naphthyloxy]-2-propanol) and pindolol (PIN: [1(isopropylamino)-3 (4-indolyloxy) 2-propanol]) are members of a heterogeneous group of drugs classified as beta-adrenergic receptor blockers and are generally prescribed in the treatment of hypertension, angina pectoris, cardiac arrhythmias and hypertrophic subaortic stenosis [1,2], but are also sometimes used as doping agents in sport. Because of their similar spectral features, the determination of these compounds in mixtures has so far been carried out by using gas chromatogra* Corresponding author. Fax: +34 68 364148.
phy [3–5] and liquid chromatography [6–9].
Several fluorimetric methods have been applied to the individual determination of PIN [10,11] and PRO [12–14] based on their native fluorescence or on derivative reactions. Because the fluorescence spectra of PRO and PIN overlap, conventional spectrofluorimetry does not permit simultaneous determination of these compounds.
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 3 9 - 9 1 4 0 ( 9 7 ) 0 0 2 0 3 - 8
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T. Pe´rez Ruiz et al. / Talanta 45 (1998) 969–976
Synchronous luminescence spectrometry involves the simultaneous scanning of both the excitation and emission monochromators, which are synchronised in such a way that a well-defined relationship is maintained between their wavelengths. Conventionally, this relationship is a constant wavelength difference. The advantages of the synchronous technique are a reduction in spectral complexity, peak bandwidth, Rayleigh scattering and Raman scattering [15] The combination of synchronous scanning fluorimetry and derivatives is more advantageous than differentiation of the conventional emission spectrum in terms of sensitivity, because the amplitude of the derivative signal is inversely proportional to the band width of the original spectrum [16,17] The aim of the present work was to find a new fluorimetric method that is simple, time-saving and accurate for the determination of PRO and PIN. The paper describes two methods to resolve the mixture of these drugs by employing synchronous fluorescence spectrometry, and first derivative synchronous spectrofluorimetry when the molar ratio PRO/PIN is higher than ten or lower than 0.04.
2. Experimental
2.1. Reagents All reagents were of analytical grade and doubly distilled water was used in all experiments. PRO and PIN (both \99.6% pure) were obtained from Sigma (St. Louis, MO) and used for preparing 300 mg ml − 1 stock standard solutions by dissolving the drugs in 50% v/v ethanol–water mixture. Working solutions of lower concentration were freshly prepared by appropriate dilution of the stock standard solution. Britton–Robinson buffer solutions of different pH values were prepared by the addition of 1 M sodium hydroxide solution to a 0.2 M solution of phosphoric, boric and acetic acids. The final pH was checked with a glass electrode.
2.2. Apparatus An Aminco Bowman Series 2 spectrofluorimeter equipped with a xenon lamp and interfaced to an DTK computer was used for fluorescence measurements. The software provides mathematical manipulation of the spectra and calculates first and second derivatives by the simplified leastsquares procedure of Savitzky and Golay [18]. In addition, tridimensional spectra presented in the form of isometric projection may be obtained. The contour plots were calculated using the ‘Galactic’ application program from the data in ASCII format obtained with the spectrofluorimeter. Further, a Colora thermostatic water-bath circulator was used for temperature control of the cell compartment and a Radiometer digital pHmeter with a combined glass-saturated calomel electrode was used to check the pH values.
2.3. Procedures 2.3.1. Determination of PRO and PIN by constant wa6elength synchronous spectrofluorimetry To a 10 ml volumetric flask was added an adequate volume of the PRO/PIN sample to give final concentrations in the range 1.0–0.02 mg ml−1 PRO and 1.2–0.04 mg ml − 1 PIN. Then 5 ml of ethanol was added and the mixture was diluted to the mark with distilled water. Synchronous spectra were recorded (at 25 90.5°C) by scanning both monochromators at a constant wavelength difference Dl = 18 nm (lex = 200–280 nm, lem = 218–298 nm) and a scan speed of 240 nm min − 1. Hereafter all wavelengths referring to synchronous spectra are taken as equal to those of the corresponding emission wavelengths. The fluorescence intensities measured at 305 and 323 nm were directly proportional to PIN and PRO concentrations, respectively. Each analyte in the mixture was evaluated from the corresponding calibration graph, obtained previously under the same conditions as those for the mixture. The synchronous spectrum of the 50% (v/v) ethanol– water was stored in memory as a ‘background spectrum’ and was subtracted from all subsequently obtained spectra.
T. Pe´rez Ruiz et al. / Talanta 45 (1998) 969–976
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2.3.2. Determination of PRO and PIN by first deri6ati6e constant wa6elength synchronous spectra This technique allows the simultaneous determination of PRO and PIN by recording only one synchronous spectrum when drugs are found in the molar ratio PRO/PIN from 1/100 to 50/1. First-derivative measurements were made as the vertical distance from the first-derivative synchronous spectrum at 291 nm to the baseline for PIN and at 341 nm to the base line for PRO.
The filtrate was then diluted with distilled water to 100 ml in a calibrated flask. An appropriately diluted aliquot of this solution was analysed following the synchronous fluorescence spectrometric procedure. The injections were appropriately diluted with 50% (v/v) ethanol–water and analysed following the procedure for tablets.
2.3.3. Determination of PIN and PRO in urine The urine sample was filtered. A suitable aliquot (5 ml) was placed into 25 ml stopped glass tubes in ice. To each tube were added 0.5 ml 2 mol l − 1 sodium hydroxide and 10 ml diethyl ether. PRO and PIN were extracted into the ethereal layer by mechanically vortexing the phases for 2 min. The phases were separated by centrifugation (3 min at 1000 g) and 8 ml of the ethereal extract was transferred to clean 15-ml tapered glass-stoppered tubes containing 2 ml of 0.01 M hydrochloric acid. PRO and PIN were extracted into the aqueous phase by mechanically vortexing the solution for 1 min. After separation of the phases by centrifugation the aqueous phase was frozen by immersion of the tubes in ice and the ethereal phase aspirated. The aqueous phase was then washed with 5 ml of n-heptane by mechanically vortexing the solution for 1 min. After centrifugation and freezing of the aqueous phase, the n-heptane was aspirated and discarded. Accurate aliquots of the aqueous phase were neutralised with 0.1 M sodium hydroxide and analysed following the procedure described for the first-derivative synchronous fluorescence spectrometry.
PRO and PIN are fluorescent in ethanol–water media. The effect of the ethanol content in the medium was investigated. An increase of ethanol content in the medium causes a continuous increase in the fluorescence intensity of PIN while the fluorescence intensity of PRO increases up to a 25% (v/v) ethanol–water, above which it decreases slightly. A 50% (v/v) ethanol–water medium was chosen for further studies. PRO shows two excitation maxima at 232 and 295 nm and exhibits fluorescence maximum at 324 and 337 nm. PIN has its excitation maxima at 223 and 281 nm and exhibits maximum fluorescence at 308 nm. The fluorescence spectra of these drugs overlap considerably and, as a result, the conventional spectrofluorimetric method does not permit the simultaneous determination of both compounds. The extent of the overlap of these compounds was examined by obtaining the total spectrofluorimetric information available in the excitation–emission matrix. In Fig. 1, the three-dimensional spectra of PRO and PIN are represented as an isometric projection, where the emission spectra at stepped increments of the excitation wavelength have been recorded and plotted. In Fig. 1 (low part) the tree dimensional spectra have been transformed into a contour plot en the excitation–emission plane. The contour representation of the fluorescence profile of PRO and PIN offers an easy way of finding the best trajectory to be followed in order to obtain synchronous fluorescence spectra for the complete resolution of overlapping component peaks. The parallel diagonal lines superimposed on the contour plots represent the scan paths through the
2.3.4. Determination of PRO in pharmaceutical preparations The tablets were finely powdered and weighed. An amount of this powder, equivalent to about 50 mg of PRO was accurately weighed and shaken with 50 ml of distilled water in a water-bath at 60–70°C for 10 min. After cooling, 50 ml of ethanol were added and the solution was sonicated in an ultrasonic bath for 5 min before being filtered through a Millipore filter.
3. Results and discussion
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Fig. 1. Three-dimensional and two-dimensional (contour plots) total fluorescence spectra of pindolol (A) and propranolol (B). Other conditions as in figure CPIN = 10 mmol l − 1, CPRO = 1 mmol l − 1.
excitation–emission matrix that would be obtained with synchronous scans at the wavelength interval shown. The optimum path for determining PRO and PIN in mixtures seems to be Dl = 18 nm; it passes near the maximum for PIN but at some distance from the maximum for PRO; however, the determination of the mixture is sufficiently sensitive because PRO is much more fluorescent than PIN.
3.1. Synchronous fluorescence spectrometry The synchronous fluorescence spectra of PRO, PIN and that of their mixture taken at a constant wavelength difference Dl = 18 nm are shown in Fig. 2. The peaks corresponding to PIN (305 nm) and PRO (323 nm) are well resolved and it is thus possible to determine both drugs simultaneously in a mixture.
T. Pe´rez Ruiz et al. / Talanta 45 (1998) 969–976
Chemical variables were studied to obtain the best measurement conditions and maximum fluorescence signals. Thus, the influence of pH on the fluorescence was studied by adding Britton– Robinson buffer solutions of different pH values. Fluorescence intensity of PRO and PIN is not affected by this variable in the range studied (pH between 3.0 and 9.0). A pH value of 6.0 was selected for further fluorescence measurements.
Table 1 Analysis of synthetic mixtures of pindolol and propranolol Added (mg ml−1)
Foundb (mg ml−1)
PIN
PRO
PIN
PRO
SFS
0.900 0.900 0.800 0.300 0.100 0.100
0.050 0.100 0.200 0.800 1.00 2.00
0.898 9 0.008 0.902 9 0.012 0.810 9 0.001 0.305 9 0.006 0.103 9 0.004 0.108 9 0.003
0.051 90.003 0.099 90.003 0.202 90.004 0.806 90.009 0.990 90.008 2.02 90.02
FDSFS
0.02 0.05 0.90 0.90
1.0 1.0 0.02 0.01
0.019 9 0.001 0.048 9 0.001 0.903 90.009 0.892 90.020
1.02 9 0.02 0.997 90.01 0.022 9 0.002 0.0096 9 0.003
Method useda
3.2. Analytical characteristics The concentration of PRO and the fluorescence intensity measured at 323 nm are linearly related over a sample concentration range 0.02–1.0 mg ml − 1. PIN concentration and the fluorescence measured at 305 nm are linearly related over the range 0.04– 1.2 mg ml − 1. The correlation coefficient for the standard calibration graphs were 0.9998 and 0.9994 (n =11) for PRO and PIN, respectively. The detection limits (signal three times the standard deviations of the average blank signal) were 0.003 mg ml − 1 for PRO and 0.007 mg ml − 1 for PIN. The relative standard deviation for a mixture of 0.10 and 0.09 mg ml − 1 PIN (n=6) were 0.29 and 0.36%, respectively.
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a
SFS = Synchronous fluorescence spectrometry, FDSFS = First-derivative synchronous fluorescence spectrometry. b Means 9SD of four determinations.
The proposed method was applied to the simultaneous determination of PRO and PIN in synthetic mixtures containing different ratios of both drugs and the results are given in Table 1. It can be seen that a good recovery was achieved for the two drugs in PRO/PIN ratios between 10 and 0.05.
3.3. First-deri6ati6e synchronous fluorescence spectrometry
Fig. 2. Synchronous fluorescence spectra of pindolol (dotted line), propranolol (dashed line) and their mixture (solid line). Dl= 18 nm. Other conditions as Fig. 1.
The resolution of the mixture PRO/PIN at molar ratios higher than 10 is poor because the PIN band partially overlaps that of PRO. This spectral overlapping can be resolved, however, by differentiation of the synchronous spectra. The use of first and second derivatives was tried and, as expected, a degradation of the signal-to-noise ratio occurred with the higher order differentiation. The most appropriate parameters to register synchronous derivative spectra were selected. A scan speed of 240 nm min − 1 was selected after verifying that this parameter hardly affect the derivative signal obtained.
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Fig. 3 shows first derivative synchronous fluorescence spectra of PRO and PIN and of a mixture of both drugs. By applying the zerocrossing technique to the first derivative synchronous spectrum of the mixture, both analytes can be determined by measuring the vertical distance to the zero line at 291 and 341 nm, which are proportional to PIN and PRO concentrations, respectively. The resolution of the mixture is precise because the heights measured correspond to peaks in the first derivative spectrum. The determination was carried out in a single scan by using the calibration graphs obtained for each component, which covered concentration ranges from 0.04 to 1.2 mg ml − 1 for PIN and from 0.02 to 1.0 mg ml − 1 for PRO. Mixtures of PRO and PIN in ratios up to 1:100 and 50:1 were resolved (see Table 1).
3.4. Interferences In order to assess the possible analytical applications of the synchronous spectrofluorimetric procedure described above, the effect of concomitant species on the determination of PRO and PIN in real samples was studies by analysing synthetic sample solutions containing 0.3 mg ml − 1 of each analyte and various excess amounts of the foreign compound up to 120 mg ml − 1 . The
Fig. 3. Fist-derivative synchronous fluorescence spectra of pindolol (dotted line), propranolol (dashed line) and their mixture (solid line). Other conditions as in figure.
tolerance ratio of each foreign substance was taken as the largest amount yielding an error less than 4% in the analytical signal of PRO or PIN. Glucose, sucrose, maltose, lactose, saccharin, fructose, caffeine, cyclamate, and citrate were tolerated in large amounts (400-fold excess was the maximum tested); a 50-fold excess of acetylsalicylic acid and 20-fold excess of riboflavin and salicylic acid were also tolerated.
3.5. Applications PRO and PIN are almost completely absorbed from the gastro-intestinal tract. Since they are only partially metabolised and are excreted in the urine both unchanged and in the form of metabolytes, the proposed method appears to be useful for the simultaneous determination of these drugs in urine samples. However, human urine is composed of numerous organic substances, some of which are fluorescent [19] and provide a high background fluorescence, thus interfering with the direct determination of the drugs. In this case a prior extraction step is necessary. The pretreatment and procedure described under Experimental were used in each instance. Urine samples spiked with PRO and PIN at different concentrations and at different mass ratios were prepared by adding known amounts of PRO and PIN to drug-free urine samples. The mean analytical recoveries were 969 3 and 97 9 3% for PRO and PIN, respectively as shown in Table 2. As the dosage forms containing PIN were not available in the local market, we prepared our own according to the literature methods. Commercial pharmaceuticals containing PRO were available and some of them were analysed. The data in Table 3 show that the PRO and PIN contents measured by the proposed method were in good statistical agreement with the amounts added and with the values supplied by the manufacturers. Recovery studies were also carried out on samples to which known amounts of PRO and PIN had been added. In all cases quantitative recoveries between 98.6 and 105% were obtained.
T. Pe´rez Ruiz et al. / Talanta 45 (1998) 969–976
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Table 2 Analytical recoveries of pindolol and propranolol added to drug-free urine samples Concentration added (mg ml−1)
Concentration founda (mg ml−1)
Recovery (%) 9SDa
PIN
PRO
PIN
PRO
PIN
PRO
12.0 12.0 12.0 15.0 15.0 20.0
50.0 5.0 0.2 2.0 0.5 1.0
11.5 11.7 11.8 14.5 14.6 19.8
49.7 4.9 0.18 1.92 0.49 0.96
96 94 98 92 98 92 97 9 3 97 93 99 9 2
99 9 2 98 9 3 90 9 6 969 4 98 9 2 96 9 3
Mean 97 93
96 9 3
a
Average of three determinations.
Table 3 Determination of propranolol in pharmaceutical preparations Product (laboratory)a
Tablets I (Home made) Tablets II (Home made) Sumial (Farma) Betadipresan-DIU (Fides) Betadipresan (Fides) Sumial injectable (Farma)
Content (mg tab−1 or ml−1)
Foundb (mg tab−1 or ml−1)
PIN
PRO
PIN
PRO
5 1
5 10 40 100 100 5
5 90.2 1 90.1
4.9 90.3 9.9 9 0.2 40.6 90.6 100.2 90.2 100.1 90.2 4.9 90.3
a
Composition of samples: Tablets I: pindolol, 5 mg, propranolol, 5 mg, lactose, 50 mg, other excipients upto 100 mg. Tablets II: pindolol, 1 mg, propranolol, 10 mg, methyl cellulose upto 50 mg. Sumial 40: propranolol hydrochloride, 40 mg; lactose,125 mg; other excipients up to 200 mg. Betadipresan-DIU: propranolol hydrochloride, 100 mg; hydralazine hydrochloride, 50 mg; bendroflumethiazide, 5 mg; lactose and other excipients. Betadipresan: propranolol hydrochloride, 100 mg; hydralazine hydrochloride, 50 mg; lactose and other excipients. Sumial injectable: propranolol hydrochloride, 5 mg; citric acid and water up to 5 ml. b Means 9SD of three determinations.
4. Conclusions Molecular synchronous spectrofluorimetry resolves binary mixtures of PIN and PRO in a 50% (v/v) ethanol – water medium. Better separation of the compounds is achieved by applying the first derivative to the synchronous fluorescence spectra. Mixtures of PRO and PIN in molar ratios up to 1:100 and 50:1 were resolved. The results obtained in the study of the re-
covery of these analytes testifies to the usefulness of the proposed method to expedite routine analysis of these two drugs.
Acknowledgements The authors gratefully acknowledge financial support from the ‘Direccio´n General de Investigacio´n Cientı´fica y Te´cnica’ and ‘Comunidad Autonoma de Murcia’.
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References [1] O.B. Garfein, Ther. Drug Monit. 4 (1982) 1. [2] S. Wolfson, R.A. Heinle, M.V. Herman, Am. J. Cardiol. 18 (1966) 345. [3] M. Guerret, J. Chromatogr. 221 (1980) 387. [4] H. Maurer, K. Pileger, J. Chromatogr. 382 (1986) 147. [5] C.L. Davies, J. Chromatogr. A 664 (1995) 341. [6] L. Ye, Z. Xiangxi, Anal. Chim. Acta 196 (1987) 255. [7] W.H. Pirkle, J. Chromatogr. 558 (1991) 1. [8] M.T. Saarinen, H. Siren, M.L. Riekkola, J. Chromatogr. A 664 (1995) 341. [9] P. Lukkari, H. Siren, J. Chromatogr. A 717 (1995) 211. [10] M.E. Mohamed, M.S. Tawakkol, H.Y. Aboul-Enein, J. Assoc. Off. Anal. Chem. 66 (1983) 273.
.
[11] C.L. Jennings, A. Bobick, E.T. Fagan, P.I. Korner, Brit. J. Clin. Pharmacol. 7 (1979) 245. [12] J.W. Black, W.A.M. Duncan, R.G. Shanks, Brit. J. Pharmacol. 25 (1965) 577. [13] M. Kraml, W.T. Robinson, Clin. Chim. Acta 24 (1978) 171. [14] H. Migulla, H. Mack, Pharmacie 36 (1981) 443. [15] E.L. Wehry, in: G.G. Guilbault (Ed.), Practical Fluorescence, Marcel Dekker, New York, 1990, pp. 378– 406. [16] G.L. Green, T.C. O’Haver, Anal. Chem. 46 (1974) 2191. [17] P. John, I. Soutar, Anal. Chem. 48 (1976) 520. [18] A. Savitzky, M.J.E. Golay, Anal. Chem. 36 (1964) 1627. [19] M.J.P. Leiner, M.R. Hubmann, O.W. Wolfbeis, Anal. Chim. Acta 198 (1987) 13.
Simultaneous determination of propranolol and pindolol by synchronous spectrofluorimetry Toma´s Pe´rez Ruiz *, Carmen Martı´nez-Lozano, Virginia Toma´s, Jose´ Carpena Department of Analytical Chemistry, Faculty of Chemistry, Uni6ersity of Murcia, 30071, Murcia, Spain Received 27 November 1996; received in revised form 11 March 1997; accepted 30 June 1997
Abstract The simultaneous determination of propranolol and pindolol using synchronous fluorescence spectrometric techniques is described. The method involves measuring the natural fluorescence of these drugs in a 50% (v/v) ethanol–water medium using zero and first derivative synchronous spectrofluorimetry. Under the optimum conditions, the linear determination ranges of propanolol and pindolol are ca. 0.02 – 1.0 and 0.04 – 1.2 mg ml − 1, respectively. The results showed that propranolol and pindolol can be determined simultaneously when the concentration ratio of propranolol to pindolol varies from 1:100 to 50:1 in the mixed sample. The method has been satisfactorily applied to the determination of propranolol and pindolol in urine samples and propranolol in pharmaceutical preparations. © 1998 Elsevier Science B.V. Keywords: Propranolol; Pindolol; Synchronous fluorescence spectrometry
1. Introduction Propranolol (PRO: 1-[isopropylamino-3-[1naphthyloxy]-2-propanol) and pindolol (PIN: [1(isopropylamino)-3 (4-indolyloxy) 2-propanol]) are members of a heterogeneous group of drugs classified as beta-adrenergic receptor blockers and are generally prescribed in the treatment of hypertension, angina pectoris, cardiac arrhythmias and hypertrophic subaortic stenosis [1,2], but are also sometimes used as doping agents in sport. Because of their similar spectral features, the determination of these compounds in mixtures has so far been carried out by using gas chromatogra* Corresponding author. Fax: +34 68 364148.
phy [3–5] and liquid chromatography [6–9].
Several fluorimetric methods have been applied to the individual determination of PIN [10,11] and PRO [12–14] based on their native fluorescence or on derivative reactions. Because the fluorescence spectra of PRO and PIN overlap, conventional spectrofluorimetry does not permit simultaneous determination of these compounds.
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 3 9 - 9 1 4 0 ( 9 7 ) 0 0 2 0 3 - 8
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T. Pe´rez Ruiz et al. / Talanta 45 (1998) 969–976
Synchronous luminescence spectrometry involves the simultaneous scanning of both the excitation and emission monochromators, which are synchronised in such a way that a well-defined relationship is maintained between their wavelengths. Conventionally, this relationship is a constant wavelength difference. The advantages of the synchronous technique are a reduction in spectral complexity, peak bandwidth, Rayleigh scattering and Raman scattering [15] The combination of synchronous scanning fluorimetry and derivatives is more advantageous than differentiation of the conventional emission spectrum in terms of sensitivity, because the amplitude of the derivative signal is inversely proportional to the band width of the original spectrum [16,17] The aim of the present work was to find a new fluorimetric method that is simple, time-saving and accurate for the determination of PRO and PIN. The paper describes two methods to resolve the mixture of these drugs by employing synchronous fluorescence spectrometry, and first derivative synchronous spectrofluorimetry when the molar ratio PRO/PIN is higher than ten or lower than 0.04.
2. Experimental
2.1. Reagents All reagents were of analytical grade and doubly distilled water was used in all experiments. PRO and PIN (both \99.6% pure) were obtained from Sigma (St. Louis, MO) and used for preparing 300 mg ml − 1 stock standard solutions by dissolving the drugs in 50% v/v ethanol–water mixture. Working solutions of lower concentration were freshly prepared by appropriate dilution of the stock standard solution. Britton–Robinson buffer solutions of different pH values were prepared by the addition of 1 M sodium hydroxide solution to a 0.2 M solution of phosphoric, boric and acetic acids. The final pH was checked with a glass electrode.
2.2. Apparatus An Aminco Bowman Series 2 spectrofluorimeter equipped with a xenon lamp and interfaced to an DTK computer was used for fluorescence measurements. The software provides mathematical manipulation of the spectra and calculates first and second derivatives by the simplified leastsquares procedure of Savitzky and Golay [18]. In addition, tridimensional spectra presented in the form of isometric projection may be obtained. The contour plots were calculated using the ‘Galactic’ application program from the data in ASCII format obtained with the spectrofluorimeter. Further, a Colora thermostatic water-bath circulator was used for temperature control of the cell compartment and a Radiometer digital pHmeter with a combined glass-saturated calomel electrode was used to check the pH values.
2.3. Procedures 2.3.1. Determination of PRO and PIN by constant wa6elength synchronous spectrofluorimetry To a 10 ml volumetric flask was added an adequate volume of the PRO/PIN sample to give final concentrations in the range 1.0–0.02 mg ml−1 PRO and 1.2–0.04 mg ml − 1 PIN. Then 5 ml of ethanol was added and the mixture was diluted to the mark with distilled water. Synchronous spectra were recorded (at 25 90.5°C) by scanning both monochromators at a constant wavelength difference Dl = 18 nm (lex = 200–280 nm, lem = 218–298 nm) and a scan speed of 240 nm min − 1. Hereafter all wavelengths referring to synchronous spectra are taken as equal to those of the corresponding emission wavelengths. The fluorescence intensities measured at 305 and 323 nm were directly proportional to PIN and PRO concentrations, respectively. Each analyte in the mixture was evaluated from the corresponding calibration graph, obtained previously under the same conditions as those for the mixture. The synchronous spectrum of the 50% (v/v) ethanol– water was stored in memory as a ‘background spectrum’ and was subtracted from all subsequently obtained spectra.
T. Pe´rez Ruiz et al. / Talanta 45 (1998) 969–976
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2.3.2. Determination of PRO and PIN by first deri6ati6e constant wa6elength synchronous spectra This technique allows the simultaneous determination of PRO and PIN by recording only one synchronous spectrum when drugs are found in the molar ratio PRO/PIN from 1/100 to 50/1. First-derivative measurements were made as the vertical distance from the first-derivative synchronous spectrum at 291 nm to the baseline for PIN and at 341 nm to the base line for PRO.
The filtrate was then diluted with distilled water to 100 ml in a calibrated flask. An appropriately diluted aliquot of this solution was analysed following the synchronous fluorescence spectrometric procedure. The injections were appropriately diluted with 50% (v/v) ethanol–water and analysed following the procedure for tablets.
2.3.3. Determination of PIN and PRO in urine The urine sample was filtered. A suitable aliquot (5 ml) was placed into 25 ml stopped glass tubes in ice. To each tube were added 0.5 ml 2 mol l − 1 sodium hydroxide and 10 ml diethyl ether. PRO and PIN were extracted into the ethereal layer by mechanically vortexing the phases for 2 min. The phases were separated by centrifugation (3 min at 1000 g) and 8 ml of the ethereal extract was transferred to clean 15-ml tapered glass-stoppered tubes containing 2 ml of 0.01 M hydrochloric acid. PRO and PIN were extracted into the aqueous phase by mechanically vortexing the solution for 1 min. After separation of the phases by centrifugation the aqueous phase was frozen by immersion of the tubes in ice and the ethereal phase aspirated. The aqueous phase was then washed with 5 ml of n-heptane by mechanically vortexing the solution for 1 min. After centrifugation and freezing of the aqueous phase, the n-heptane was aspirated and discarded. Accurate aliquots of the aqueous phase were neutralised with 0.1 M sodium hydroxide and analysed following the procedure described for the first-derivative synchronous fluorescence spectrometry.
PRO and PIN are fluorescent in ethanol–water media. The effect of the ethanol content in the medium was investigated. An increase of ethanol content in the medium causes a continuous increase in the fluorescence intensity of PIN while the fluorescence intensity of PRO increases up to a 25% (v/v) ethanol–water, above which it decreases slightly. A 50% (v/v) ethanol–water medium was chosen for further studies. PRO shows two excitation maxima at 232 and 295 nm and exhibits fluorescence maximum at 324 and 337 nm. PIN has its excitation maxima at 223 and 281 nm and exhibits maximum fluorescence at 308 nm. The fluorescence spectra of these drugs overlap considerably and, as a result, the conventional spectrofluorimetric method does not permit the simultaneous determination of both compounds. The extent of the overlap of these compounds was examined by obtaining the total spectrofluorimetric information available in the excitation–emission matrix. In Fig. 1, the three-dimensional spectra of PRO and PIN are represented as an isometric projection, where the emission spectra at stepped increments of the excitation wavelength have been recorded and plotted. In Fig. 1 (low part) the tree dimensional spectra have been transformed into a contour plot en the excitation–emission plane. The contour representation of the fluorescence profile of PRO and PIN offers an easy way of finding the best trajectory to be followed in order to obtain synchronous fluorescence spectra for the complete resolution of overlapping component peaks. The parallel diagonal lines superimposed on the contour plots represent the scan paths through the
2.3.4. Determination of PRO in pharmaceutical preparations The tablets were finely powdered and weighed. An amount of this powder, equivalent to about 50 mg of PRO was accurately weighed and shaken with 50 ml of distilled water in a water-bath at 60–70°C for 10 min. After cooling, 50 ml of ethanol were added and the solution was sonicated in an ultrasonic bath for 5 min before being filtered through a Millipore filter.
3. Results and discussion
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Fig. 1. Three-dimensional and two-dimensional (contour plots) total fluorescence spectra of pindolol (A) and propranolol (B). Other conditions as in figure CPIN = 10 mmol l − 1, CPRO = 1 mmol l − 1.
excitation–emission matrix that would be obtained with synchronous scans at the wavelength interval shown. The optimum path for determining PRO and PIN in mixtures seems to be Dl = 18 nm; it passes near the maximum for PIN but at some distance from the maximum for PRO; however, the determination of the mixture is sufficiently sensitive because PRO is much more fluorescent than PIN.
3.1. Synchronous fluorescence spectrometry The synchronous fluorescence spectra of PRO, PIN and that of their mixture taken at a constant wavelength difference Dl = 18 nm are shown in Fig. 2. The peaks corresponding to PIN (305 nm) and PRO (323 nm) are well resolved and it is thus possible to determine both drugs simultaneously in a mixture.
T. Pe´rez Ruiz et al. / Talanta 45 (1998) 969–976
Chemical variables were studied to obtain the best measurement conditions and maximum fluorescence signals. Thus, the influence of pH on the fluorescence was studied by adding Britton– Robinson buffer solutions of different pH values. Fluorescence intensity of PRO and PIN is not affected by this variable in the range studied (pH between 3.0 and 9.0). A pH value of 6.0 was selected for further fluorescence measurements.
Table 1 Analysis of synthetic mixtures of pindolol and propranolol Added (mg ml−1)
Foundb (mg ml−1)
PIN
PRO
PIN
PRO
SFS
0.900 0.900 0.800 0.300 0.100 0.100
0.050 0.100 0.200 0.800 1.00 2.00
0.898 9 0.008 0.902 9 0.012 0.810 9 0.001 0.305 9 0.006 0.103 9 0.004 0.108 9 0.003
0.051 90.003 0.099 90.003 0.202 90.004 0.806 90.009 0.990 90.008 2.02 90.02
FDSFS
0.02 0.05 0.90 0.90
1.0 1.0 0.02 0.01
0.019 9 0.001 0.048 9 0.001 0.903 90.009 0.892 90.020
1.02 9 0.02 0.997 90.01 0.022 9 0.002 0.0096 9 0.003
Method useda
3.2. Analytical characteristics The concentration of PRO and the fluorescence intensity measured at 323 nm are linearly related over a sample concentration range 0.02–1.0 mg ml − 1. PIN concentration and the fluorescence measured at 305 nm are linearly related over the range 0.04– 1.2 mg ml − 1. The correlation coefficient for the standard calibration graphs were 0.9998 and 0.9994 (n =11) for PRO and PIN, respectively. The detection limits (signal three times the standard deviations of the average blank signal) were 0.003 mg ml − 1 for PRO and 0.007 mg ml − 1 for PIN. The relative standard deviation for a mixture of 0.10 and 0.09 mg ml − 1 PIN (n=6) were 0.29 and 0.36%, respectively.
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a
SFS = Synchronous fluorescence spectrometry, FDSFS = First-derivative synchronous fluorescence spectrometry. b Means 9SD of four determinations.
The proposed method was applied to the simultaneous determination of PRO and PIN in synthetic mixtures containing different ratios of both drugs and the results are given in Table 1. It can be seen that a good recovery was achieved for the two drugs in PRO/PIN ratios between 10 and 0.05.
3.3. First-deri6ati6e synchronous fluorescence spectrometry
Fig. 2. Synchronous fluorescence spectra of pindolol (dotted line), propranolol (dashed line) and their mixture (solid line). Dl= 18 nm. Other conditions as Fig. 1.
The resolution of the mixture PRO/PIN at molar ratios higher than 10 is poor because the PIN band partially overlaps that of PRO. This spectral overlapping can be resolved, however, by differentiation of the synchronous spectra. The use of first and second derivatives was tried and, as expected, a degradation of the signal-to-noise ratio occurred with the higher order differentiation. The most appropriate parameters to register synchronous derivative spectra were selected. A scan speed of 240 nm min − 1 was selected after verifying that this parameter hardly affect the derivative signal obtained.
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Fig. 3 shows first derivative synchronous fluorescence spectra of PRO and PIN and of a mixture of both drugs. By applying the zerocrossing technique to the first derivative synchronous spectrum of the mixture, both analytes can be determined by measuring the vertical distance to the zero line at 291 and 341 nm, which are proportional to PIN and PRO concentrations, respectively. The resolution of the mixture is precise because the heights measured correspond to peaks in the first derivative spectrum. The determination was carried out in a single scan by using the calibration graphs obtained for each component, which covered concentration ranges from 0.04 to 1.2 mg ml − 1 for PIN and from 0.02 to 1.0 mg ml − 1 for PRO. Mixtures of PRO and PIN in ratios up to 1:100 and 50:1 were resolved (see Table 1).
3.4. Interferences In order to assess the possible analytical applications of the synchronous spectrofluorimetric procedure described above, the effect of concomitant species on the determination of PRO and PIN in real samples was studies by analysing synthetic sample solutions containing 0.3 mg ml − 1 of each analyte and various excess amounts of the foreign compound up to 120 mg ml − 1 . The
Fig. 3. Fist-derivative synchronous fluorescence spectra of pindolol (dotted line), propranolol (dashed line) and their mixture (solid line). Other conditions as in figure.
tolerance ratio of each foreign substance was taken as the largest amount yielding an error less than 4% in the analytical signal of PRO or PIN. Glucose, sucrose, maltose, lactose, saccharin, fructose, caffeine, cyclamate, and citrate were tolerated in large amounts (400-fold excess was the maximum tested); a 50-fold excess of acetylsalicylic acid and 20-fold excess of riboflavin and salicylic acid were also tolerated.
3.5. Applications PRO and PIN are almost completely absorbed from the gastro-intestinal tract. Since they are only partially metabolised and are excreted in the urine both unchanged and in the form of metabolytes, the proposed method appears to be useful for the simultaneous determination of these drugs in urine samples. However, human urine is composed of numerous organic substances, some of which are fluorescent [19] and provide a high background fluorescence, thus interfering with the direct determination of the drugs. In this case a prior extraction step is necessary. The pretreatment and procedure described under Experimental were used in each instance. Urine samples spiked with PRO and PIN at different concentrations and at different mass ratios were prepared by adding known amounts of PRO and PIN to drug-free urine samples. The mean analytical recoveries were 969 3 and 97 9 3% for PRO and PIN, respectively as shown in Table 2. As the dosage forms containing PIN were not available in the local market, we prepared our own according to the literature methods. Commercial pharmaceuticals containing PRO were available and some of them were analysed. The data in Table 3 show that the PRO and PIN contents measured by the proposed method were in good statistical agreement with the amounts added and with the values supplied by the manufacturers. Recovery studies were also carried out on samples to which known amounts of PRO and PIN had been added. In all cases quantitative recoveries between 98.6 and 105% were obtained.
T. Pe´rez Ruiz et al. / Talanta 45 (1998) 969–976
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Table 2 Analytical recoveries of pindolol and propranolol added to drug-free urine samples Concentration added (mg ml−1)
Concentration founda (mg ml−1)
Recovery (%) 9SDa
PIN
PRO
PIN
PRO
PIN
PRO
12.0 12.0 12.0 15.0 15.0 20.0
50.0 5.0 0.2 2.0 0.5 1.0
11.5 11.7 11.8 14.5 14.6 19.8
49.7 4.9 0.18 1.92 0.49 0.96
96 94 98 92 98 92 97 9 3 97 93 99 9 2
99 9 2 98 9 3 90 9 6 969 4 98 9 2 96 9 3
Mean 97 93
96 9 3
a
Average of three determinations.
Table 3 Determination of propranolol in pharmaceutical preparations Product (laboratory)a
Tablets I (Home made) Tablets II (Home made) Sumial (Farma) Betadipresan-DIU (Fides) Betadipresan (Fides) Sumial injectable (Farma)
Content (mg tab−1 or ml−1)
Foundb (mg tab−1 or ml−1)
PIN
PRO
PIN
PRO
5 1
5 10 40 100 100 5
5 90.2 1 90.1
4.9 90.3 9.9 9 0.2 40.6 90.6 100.2 90.2 100.1 90.2 4.9 90.3
a
Composition of samples: Tablets I: pindolol, 5 mg, propranolol, 5 mg, lactose, 50 mg, other excipients upto 100 mg. Tablets II: pindolol, 1 mg, propranolol, 10 mg, methyl cellulose upto 50 mg. Sumial 40: propranolol hydrochloride, 40 mg; lactose,125 mg; other excipients up to 200 mg. Betadipresan-DIU: propranolol hydrochloride, 100 mg; hydralazine hydrochloride, 50 mg; bendroflumethiazide, 5 mg; lactose and other excipients. Betadipresan: propranolol hydrochloride, 100 mg; hydralazine hydrochloride, 50 mg; lactose and other excipients. Sumial injectable: propranolol hydrochloride, 5 mg; citric acid and water up to 5 ml. b Means 9SD of three determinations.
4. Conclusions Molecular synchronous spectrofluorimetry resolves binary mixtures of PIN and PRO in a 50% (v/v) ethanol – water medium. Better separation of the compounds is achieved by applying the first derivative to the synchronous fluorescence spectra. Mixtures of PRO and PIN in molar ratios up to 1:100 and 50:1 were resolved. The results obtained in the study of the re-
covery of these analytes testifies to the usefulness of the proposed method to expedite routine analysis of these two drugs.
Acknowledgements The authors gratefully acknowledge financial support from the ‘Direccio´n General de Investigacio´n Cientı´fica y Te´cnica’ and ‘Comunidad Autonoma de Murcia’.
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References [1] O.B. Garfein, Ther. Drug Monit. 4 (1982) 1. [2] S. Wolfson, R.A. Heinle, M.V. Herman, Am. J. Cardiol. 18 (1966) 345. [3] M. Guerret, J. Chromatogr. 221 (1980) 387. [4] H. Maurer, K. Pileger, J. Chromatogr. 382 (1986) 147. [5] C.L. Davies, J. Chromatogr. A 664 (1995) 341. [6] L. Ye, Z. Xiangxi, Anal. Chim. Acta 196 (1987) 255. [7] W.H. Pirkle, J. Chromatogr. 558 (1991) 1. [8] M.T. Saarinen, H. Siren, M.L. Riekkola, J. Chromatogr. A 664 (1995) 341. [9] P. Lukkari, H. Siren, J. Chromatogr. A 717 (1995) 211. [10] M.E. Mohamed, M.S. Tawakkol, H.Y. Aboul-Enein, J. Assoc. Off. Anal. Chem. 66 (1983) 273.
.
[11] C.L. Jennings, A. Bobick, E.T. Fagan, P.I. Korner, Brit. J. Clin. Pharmacol. 7 (1979) 245. [12] J.W. Black, W.A.M. Duncan, R.G. Shanks, Brit. J. Pharmacol. 25 (1965) 577. [13] M. Kraml, W.T. Robinson, Clin. Chim. Acta 24 (1978) 171. [14] H. Migulla, H. Mack, Pharmacie 36 (1981) 443. [15] E.L. Wehry, in: G.G. Guilbault (Ed.), Practical Fluorescence, Marcel Dekker, New York, 1990, pp. 378– 406. [16] G.L. Green, T.C. O’Haver, Anal. Chem. 46 (1974) 2191. [17] P. John, I. Soutar, Anal. Chem. 48 (1976) 520. [18] A. Savitzky, M.J.E. Golay, Anal. Chem. 36 (1964) 1627. [19] M.J.P. Leiner, M.R. Hubmann, O.W. Wolfbeis, Anal. Chim. Acta 198 (1987) 13.