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Studies on properties and application of non-protected room temperature phosphorescence of propranolol
Spectrochimica Acta Part A 58 (2002) 2185– 2191 www.elsevier.com/locate/saa
Studies on properties and application of non-protected room temperature phosphorescence of propranolol Wen-Qing Long 1, Zhong-Xiao Zhang, Long-Di Li * Department of Chemistry, Tsinghua Uni6ersity, Beijing 100084, People’s Republic of China Received 2 October 2001; accepted 17 November 2001
Abstract A direct and simple non-protected room temperature phosphorimetry (NP-RTP) for determine propranolol, which using I− as a heavy atom perturber and sodium sulfite as a deoxygenator, has been developed. The phosphorescence peak wavelength maxima uex/uem =288/494, 522 nm. The analytical curve of propranolol gives a linear dynamic range of 8.0 ×10 − 8 –2.0× 10 − 5 mol l − 1 and a detection limit of 3 × 10 − 8 mol l − 1. The influence of I− concentration on RTP lifetime of propranolol was studied and the luminescence kinetic parameters were calculated. It is found that the relation between I− concentration (x) and RTP lifetime (~) can be expressed as ~= 1.25e − 0.477x and the rate constants of phosphorescence emission kp was 0.800 per ms. The method was applied directly to determination of propranolol in urine and drug tablets with a satisfactory result. The recoveries were 96.6– 97.4% and the relative standard deviation was 2% for the 1.00 ×10 − 6 –4.00×10 − 6 mol l − 1 propranolol in spiked urine sample. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Propranolol; Non-protected room temperature phosphorescence; Iodide; Luminescence kinetic parameters; Urine; Pharmaceuticals
1. Introduction Propranolol, 1-(isopropylamino)-3-(1-naphthyloxy)-4-propanol hydrochloride, C16H21NO2 · HCl, is a kind of beta-blocking drug widely used as standard therapy in the treatment of high blood pressure, arrhythmias and angina pectoris. Besides, when administered chronically, it reduces the mortality due to hypertension and lengthen
* Corresponding author E-mail address: [email protected] (L.-D. Li). 1 Visiting scholar come from Jinggangshan Normal College.
survival in patients with coronary heart disease [1,2]. Different instrumental methods have been proposed in the literature for the determination of this important compound in numerous matrices, such as LC/MS [3], CE [4], HPLC [5], LC [6], indirect AAA [7,8] and SPME/LC/ESI-MS [9] etc. It is well known that luminescence analytical methods have high sensitivity and selectivity, there have been extensive studies on the fluorescence of propranolol and fluorimetric detection was widely used in HPLC, CE etc. methods for determination of propranolol. But till date, only a few investigations have been reported on phosphorescence properties of propranolol, es-
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pecially at room temperature. Phosphorimetry is sensitive and more selective than fluorimetry for the analysis of many compounds, but the conventional low temperature phosphorimetry (LTP) is inconvenient and time-consuming. Recently, several methodologies have been proposed to obtain room temperature phosphorescence (RTP) signals of lumiphor solution. For a long time, a conventional point of view is that a protective medium, such as surfactant micelle, cyclodextrin, emulsion etc, is a necessary condition to induce RTP of a lumiphor in solution. Martinez et al. [10] reported the micelle stabilized room temperature phosphorimetry (MS-RTP) for the determination of propranolol, in which sodium dodecyl sulfate micelle was used as a protecting medium. More recently, the RTP emission phenomenon in a lumiphor solution without any protecting medium was discovered, which is a new kind of RTP phenomenon and was named non-protected fluid room temperature phosphorescence (NP-RTP) [11–22] or heavy atom induced room temperature phosphorescence (HAI-RTP) [23– 26]. NP-RTP has many advantages with respect to other RTP methods and allows one to develop very simple, fast, automatic analytical methods. After a careful review of the methods published in the literature for the analytical determination of propranolol, it is deduced that there are only a few RTP methods have been proposed for this compound and the NP-RTP method for determination of propranolol has not been reported. The aim of this work is the development of the first application of the NP-RTP for the determination of propranolol.
and emission slits were 15 and 20 nm, respectively, the gate time 2.0 ms and delay time 0.1 ms were kept constant.
2.2. Reagents The propranolol was purchased from Wu-Jin pharmaceutical factor (Jiangsu Province, China) and a 1.0× 10 − 3 mol l − 1 aqueous solution was prepared. KI and Na2SO3 (AR grade, Beijing Chemical Plant, China) were used as received. A 1.0 mol l − 1 solution of KI (or solid) and 0.1 mol l − 1 solution of Na2SO3 were used in the experiments. The Na2SO3 solution was prepared when used and kept in a tightly stoppered container. Propranolol drug tablets were purchased from Beijing Second Pharmaceutical Plant. All of the organic solvents used were AR grade, the water used was prepared by twice sub-boiling distillation.
2.3. General procedure A 0.1 ml aliquot of 1.0×10 − 3 mol l − 1 aqueous solution was added to a 10-ml quantitative test tube, a certain amount of KI (used as heavy atom perturber) solution (or solid) and Na2SO3 (used as chemical deoxygenator) solution were then added. The solution was filled to the mark with water. After the solution was shaken, the fluorescence spectra, phosphorescence spectra and lifetime were measured on the LS-50B apparatus with a 1-cm quartz cell.
3. Results and discussion 2. Experiment
2.1. Instrument
3.1. Fluorescence spectra of propranolol in the presence of KI and Na2SO3
All fluorescence and phosphorescence spectra were measured on a Perkin– Elmer LS-50B luminescence spectrometer, the RTP lifetime was made on the LS-50B apparatus with a pulsed xenon lamp and the Obey – Decay application program. Both excitation and emission slits were 3 nm for fluorescence measurements, but for all of the phosphorescence measurements, the excitation
Fig. 1 shows that fluorescence peak wavelengths of propranolol aqueous solution, uex/uem, are 228, 288/339, 353 nm. When Na2SO3 was added into the solution, the fluorescence peak of excitation spectrum at shorter wavelength (near 228 nm) was disappear immediately. If an external heavy atom perturber (HAP), KI, was also added into the luminescent system, the fluorescence in-
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Fig. 1. Fluorescence spectra of propranolol. (1) C = 1× 10 − 5 mol l − 1; (2) C=1 ×10 − 5 mol l − 1 + 0.01 mol l − 1 Na2SO3 (3) C= 1 × 10 − 5 mol l − 1 + 0.01 mol l − 1 Na2SO3 + 0.1 g/10 ml KI; left, excitation spectra; right, emission spectra.
tensity was decreased obviously, which means that KI is a strong quencher for fluorescence of propranolol and a RTP signal may be induced for the system.
3.2. NP-RTP spectra of propranolol The experiment results show that propranolol aqueous solution can emit a strong and stable RTP signal in the absence of a protective medium, only using KI as a HAP and Na2SO3 as a deoxygenator (Fig. 2). The excitation spectrum exhibits a single peak, whereas two peaks were found for emission spectrum, the phosphorescence peak wavelength maxima uex/uem =288/494, 522 nm.
Fig. 2. NP-RTP spectra of propranolol; C =1× 10 − 5 mol l − 1, 1 g/10 ml KI, 0.01 mol l − 1 Na2SO3.
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Fig. 3. Effect of pH of the system on RTP intensity. uex/uem = 288/522 nm, C = 1 ×10 − 5 mol l − 1, 2.5 g/10 ml KI, 0.01 mol l − 1 Na2SO3.
3.3. Optimum conditions for RTP emission 3.3.1. Effects of system pH The effect of system pH on the RTP intensity of propranolol is very significant (see Fig. 3) because propranolol molecular contained a hydroxyl and an amino group, pH of the system will affect not only the deoxygenation efficiency[27], but also the existing forms of the luminescent moiety, which have various luminescence quantum yield. The experiment result shows that when pH of the system was controlled in the range of neutral to week basic (pH 7–8),the phosphorescence intensity is the highest. 3.3.2. Effects of the concentration of Na2SO3 Various volumes of 0.1 mol l − 1 Na2SO3 solution were added to a solution with a fixed amount of propranolol (0.1 ml of 1× 10 − 3 mol l − 1) and KI (2.5 g). As shown in Fig. 4, when the concentration of sodium sulfite in the system was larger than 0.005 mol l − 1, the maximum and stable RTP signal was obtained. A 1.0 ml of 0.1 mol l − 1 Na2SO3 solution was selected for the rest of experimental work. In this case, the concentration of Na2SO3 in the system is 1.0× 10 − 2 mol l − 1 and the pH of the solution falls just in the optimum range of pH described above, a buffer solution is not needed.
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Fig. 4. Effect of the concentration of Na2SO3 on RTP intensity. uex/uem =288/522 nm, C= 1× 10 − 5 mol l − 1, 2.5 g/10 ml KI.
3.4. Effects of concentration of KI on fluorescence and phosphorescence spectra of propranolol 3.4.1. Effects of concentration of KI on fluorescence intensity As described above, when the heavy atom perturber, KI, was added into the luminescent system, the fluorescence was quenched regularly (Fig. 5). The effect of the concentration of KI on the intensity of fluorescence was studied. According to Stern –Volmer equation, I0/I = 1 +Ksvx, here x
Fig. 6. Dependence of I0/I on the concentration of KI for fluorescence. uex/uem =288/353 nm, C =1 ×10 − 5 and 0.01 mol l − 1 Na2SO3.
is the molar concentration of KI, a plot of I0/I against x exhibits a good linear relationship (Fig. 6). The quenching constant calculated for fluorescence is Ksv = 107.2 l mol − 1 (r=0.9992, n=12).
3.4.2. Effects of concentration of KI on NP-RTP intensity and determination of phosphorescence kinetic parameters for propranolol The experiment results show that RTP intensity of the system was increased with every increase of the concentration of KI in a certain range. At the same time, RTP lifetime was decreased. When the concentration of KI was 1.5 mol l − 1 (added 2.5 g KI into 10 ml solution), the RTP intensity of the system comes to the highest (Fig. 7). According to the definition of phosphorescence lifetime, we have: ~0 = ~=
Fig. 5. Effect of the amount of KI on fluorescence intensity of propranolol. uex/uem =288/353 nm, C=1× 10 − 5 and 0.01 mol l − 1 Na2SO3.
1 kp
1 1 = [kp + ki] [kp + ax]
(1) (2)
where kp is a rate constant for phosphorescent decay; ki is a rate constant for triplet state radiationless deactivation from T1 S0; a is a constant that expressing the effect level of the molar concentration of KI to rate constant of radiationless deactivation. From the above relations, we have following relation:
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where x is the concentration of KI (mol l − 1), k is the decreasing rate of lifetime. When ln ~ was plotted as a function of the concentration in KI, we have also got another linear relationship: ln ~= ln ~0 − kCKI
Fig. 7. Effect of the amount of KI on NP– RTP intensity of propranolol. uex/uem =288/522 nm, C=1× 10 − 5 and 0.01 mol l − 1 Na2SO3.
ax ~0 [kp +ax] = 1+ = kp ~ kp 1 1 = +ax ~ ~0
(3)
When 1/~ or ~0 /~ was plotted as a function of the concentration in KI, we have got a linear relationship. On the other hand, as KI can promote the rate of intersystem crossing and radiative processes of triplet state, the phosphorescence lifetime was shortened. Thus: ~ =~0e − kx
(4)
(5)
According to the RTP lifetime obtained experimentally at different concentration of KI, we calculated luminescence kinetic parameters of propranolol based on the above relationships. the RTP lifetimes ~0 obtained by extrapolating curves of 1/~ = 1/~0 + ax and ln ~0 = ln ~0 − kx (ref. Fig. 8) were 1.27 and 1.23 ms, respectively, which were in good agreement with each other. Take an average value ~0 = 1.25 ms, we obtained luminescence kinetic parameters of propranolol, kp = 1/~0 = 0.800 ms − 1, a= 0.560 ms − 1 l − 1mol − 1 and k= 0.477 ms − 1 l − 1 mol − 1, respectively. So the relation between KI concentration (x) and RTP lifetime (~) can be expressed as ~= 1.25e − 0.477x. 4. Analytical parameters According to the experimental procedure and using the optimum conditions described above, the concentration of propranolol solution in the range of 8× 10 − 8 –2× 10 − 5 mol l − 1 has a good linear relationship with RTP intensity r= 0.9994, n =7. The calculated detection limit by methodology of IUPAC [28] based on three times the standard deviation of background was 3×10 − 8 mol l − 1.
Fig. 8. The plot of 1/~ (left) or ln ~ (right) vs. x (concentration of KI) for phosphorescence. C =1 ×10 − 5 and 0.01 mol l − 1 Na2SO3.
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Table 1 Standard recovery of propranolol in urine samples Added (mmol l−1)
Found (mmol l−1)
Average (mmol l−1)
Recovery (%)
R.S.D. (%)
1.00 2.00 4.00
0.952 0.986 0.962 1.984 1.944 1.912 3.812 3.952 3.824
0.967 1.947 3.863
96.7 97.4 96.6
1.8 1.9 2.0
5. Analysis for propranolol in real sample
5.1. Determination of propranolol in urine sample In order to mimic a real sample situation and eliminate the matrix effect, the quantitative analysis of trace propranolol in spiked urine samples has been carried out by standard additions method. The preparation and pretreatment of spiked urine sample is as follows: 1 ml urine sample, a various amount of propranolol standard solution and 0.2 ml of 0.5 mol l − 1 NaOH solution was added in turn to a 10 ml quantitative test tube, shaken for 10 min, then 5 ml dichloromethane was added and shaken for 10 min again, then the mixture was centrifuged (3000 rpm) for 15 min. The upper aqueous layer was discarded and 3 ml of transparent solution of down layer was pipetted into another 10 ml quantitative test tube and evaporated to dryness at 50 9 1 °C to move dichloromethane. The dry residue was redissolved in 0.5 ml of water (or acetonitrile) and the recovery experiment of the propranolol standard in the spiked urine sample was performed based on the general procedure described above. At the same time, the analytical curves in the presence and absence of 5%(v/v) acetonitrile were made for the real sample. The regression equations of the analytical curves were Ip = 0.2633+3.544× 106 C (mol l − 1), r =0.9992, n= 7 for without acetonitrile system; and Ip = 0.08300+ 4.116×106 C (mol l − 1), r = 0.9998, n= 7 for the system in the presence of 5% acetonitrile, respectively. As shown in Table 1, the recovery and precision are good. The results show that a litter of acetonitrile (5%) is suitable for determination of propranolol (Fig. 9). The slope of the analytical curve in the presence of 5% (v/v) acetonitrile was higher than that of in absence of
acetonitrile, and the intercept was just contrast. We think, maybe it is relative with the weekly self-association property of propranolol in aqueous-electrolyte solution [29], because the concentration of KI in the luminescent system is high (2.5 g/10 ml is about 1.5 mol l − 1). The presence of acetonitrile is suitable for dissociation of associated molecule of propranolol.
5.2. Determination of propranolol in drug tablet Ten drug tablets were ground to powder in an agate mortar, a certain quantity of powder sample (66.5 mg) was dissolved in water and was filled to 100 ml with water. The mixture was filtered with a piece of dry filter-paper to remove insoluble excipient in the drug tablet. Finally, a certain quantity of filtrate was pipetted into a 10 ml quantitative test tube and the general procedure described above was applied to the solution. The results show that the content of propranolol in
Fig. 9. The NP-RTP analytical curves of propranolol for urine sample in the presence and absence of acetonitrile. uex/uem = 288/522 nm, 0.01 mol l − 1 Na2SO3, 2.5 g/10 ml KI.
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drug tablet was 10.8, 11.0, 11.0 and 10.8 mg per tablet, respectively, for four determinations, which are in good agreement with the results of UV method [30]. The relative standard deviation (R.S.D.) of 1.06%, indicates also good precision.
6. Conclusions The NP-RTP method developed in this paper for the determination of propranolol is high sensitive and is simple compared with other analytical methods proposed for the determination of this compound. As no protected media is needed, the luminescent system is completely transparent, no precipitation and no foam problems exist as in cyclodextrin-induced RTP and in MS-RTP methods. This simplification improves the analytical characteristics, which means that NP-RTP has good potential uses with other analytical techniques such as HPLC and FIA etc. On the other hand, this paper demonstrated that the lifetime method based on the heavy atom effect can be used for the determination of the luminescence kinetic parameters.
Acknowledgements This work was supported by the National Natural Science Foundation of China.
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[6] A.J. Braza, P. Modamio, E.L. Marino, J. Chromatogr. B 738 (2000) 225. [7] S. Khalil, N. Borham, J. Pharm. Biomed. Anal. 22 (2000) 235. [8] S. Khalil, M.M. Elrabiehi, J. Pharm. Biomed. Anal. 22 (2000) 7. [9] H. Kataoka, S. Narimatsu, H.L. Lord, J. Pawliszyn, Anal. Chem. 71 (1999) 4237. [10] I.R. Martinez, R.M.V. Camanas, M.C. Garciaalvarezcoque, Analyst 119 (1994) 1093. [11] L.D. Li, Y.L. Chen, A.J. Tong, Huaxue Tongbao (Chemistry) 6 (1996) 3. [12] L.D. Li, Y.L. Chen, Y. Zhao, A.J. Tong, Anal. Chim. Acta 341 (1997) 241. [13] L.D. Li, Y. Zhao, Y.G. Wu, A.J. Tong, Talanta 46 (1998) 1147. [14] Y.P. Zhu, L.D. Li, A.J. Tong, Chinese J. Anal. Chem. 26 (1998) 383. [15] L. Mou, X.K. Chen, Y.G. Wu, L.D. Li, Chinese J. Anal. Chem. 26 (1998) 1504. [16] L. Mou, X.K. Chen, L.D. Li, Chem. J. Chinese Universities 20 (1999) 214. [17] L. Mou, X.K. Chen, L.D. Li, Chinese J. Anal. Chem. 27 (1999) 509. [18] X.K. Chen, L. Mou, L.D. Li, Chem. J. Chinese Universities 20 (1999) 1052. [19] L.D. Li, X.K. Chen, L. Mou, W.Q. Long, A.J. Tong, Anal. Chim. Acta 424 (2000) 177. [20] L.D. Li, L. Mou, X.K. Chen, Chem. J. Chinese Universities 21 (2000) 1040. [21] L.D. Li, Z.X. Zhang, W.Q. Long, A.J. Tong, Spectrochimica Acta Part A 57 (2001) 385. [22] J.A.M. Pulgarin, A.A. Molina, Fresenius’ J. Anal. Chem. 368 (2000) 505. [23] A.S. Carretero, C.C. Blanco, B. Canabate Diaz, et al., Anal. Chim. Acta 417 (2000) 19. [24] A.S. Carretero, C.C. Blanco, A.F. Gutierrer, J. Agric. Food Chem. 46 (1998) 3683. [25] A.S. Carretero, C.C. Blanco, B.C. Diaz, et al., Anal. Chim. Acta 361 (1998) 217. [26] A.F. Gutierrer, A.S. Carretero, B.C. Diaz, et al., Appl. Spectroscopy 53 (1999) 741. [27] W.J. Jin, Shanxi University (China), Master Dissertation, 1990. [28] IUPAC, Pure Appl. Chem., 45 (1976) 101. [29] J.M. Ruso, P. Taboada, D. Attwood, V. Mosquera, F. Sarmiento, Phys. Chem. Chemical Phys. 2 (2000) 1261. [30] Editorial Committee of Pharmacopoeia of People’s Republic of China, Pharmacopoeia of People’s Republic of China, Section 2, People’s Hygiene Press, Beijing, 1990, pp. 542 – 544.
Studies on properties and application of non-protected room temperature phosphorescence of propranolol Wen-Qing Long 1, Zhong-Xiao Zhang, Long-Di Li * Department of Chemistry, Tsinghua Uni6ersity, Beijing 100084, People’s Republic of China Received 2 October 2001; accepted 17 November 2001
Abstract A direct and simple non-protected room temperature phosphorimetry (NP-RTP) for determine propranolol, which using I− as a heavy atom perturber and sodium sulfite as a deoxygenator, has been developed. The phosphorescence peak wavelength maxima uex/uem =288/494, 522 nm. The analytical curve of propranolol gives a linear dynamic range of 8.0 ×10 − 8 –2.0× 10 − 5 mol l − 1 and a detection limit of 3 × 10 − 8 mol l − 1. The influence of I− concentration on RTP lifetime of propranolol was studied and the luminescence kinetic parameters were calculated. It is found that the relation between I− concentration (x) and RTP lifetime (~) can be expressed as ~= 1.25e − 0.477x and the rate constants of phosphorescence emission kp was 0.800 per ms. The method was applied directly to determination of propranolol in urine and drug tablets with a satisfactory result. The recoveries were 96.6– 97.4% and the relative standard deviation was 2% for the 1.00 ×10 − 6 –4.00×10 − 6 mol l − 1 propranolol in spiked urine sample. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Propranolol; Non-protected room temperature phosphorescence; Iodide; Luminescence kinetic parameters; Urine; Pharmaceuticals
1. Introduction Propranolol, 1-(isopropylamino)-3-(1-naphthyloxy)-4-propanol hydrochloride, C16H21NO2 · HCl, is a kind of beta-blocking drug widely used as standard therapy in the treatment of high blood pressure, arrhythmias and angina pectoris. Besides, when administered chronically, it reduces the mortality due to hypertension and lengthen
* Corresponding author E-mail address: [email protected] (L.-D. Li). 1 Visiting scholar come from Jinggangshan Normal College.
survival in patients with coronary heart disease [1,2]. Different instrumental methods have been proposed in the literature for the determination of this important compound in numerous matrices, such as LC/MS [3], CE [4], HPLC [5], LC [6], indirect AAA [7,8] and SPME/LC/ESI-MS [9] etc. It is well known that luminescence analytical methods have high sensitivity and selectivity, there have been extensive studies on the fluorescence of propranolol and fluorimetric detection was widely used in HPLC, CE etc. methods for determination of propranolol. But till date, only a few investigations have been reported on phosphorescence properties of propranolol, es-
1386-1425/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 0 1 ) 0 0 7 2 9 - 6
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pecially at room temperature. Phosphorimetry is sensitive and more selective than fluorimetry for the analysis of many compounds, but the conventional low temperature phosphorimetry (LTP) is inconvenient and time-consuming. Recently, several methodologies have been proposed to obtain room temperature phosphorescence (RTP) signals of lumiphor solution. For a long time, a conventional point of view is that a protective medium, such as surfactant micelle, cyclodextrin, emulsion etc, is a necessary condition to induce RTP of a lumiphor in solution. Martinez et al. [10] reported the micelle stabilized room temperature phosphorimetry (MS-RTP) for the determination of propranolol, in which sodium dodecyl sulfate micelle was used as a protecting medium. More recently, the RTP emission phenomenon in a lumiphor solution without any protecting medium was discovered, which is a new kind of RTP phenomenon and was named non-protected fluid room temperature phosphorescence (NP-RTP) [11–22] or heavy atom induced room temperature phosphorescence (HAI-RTP) [23– 26]. NP-RTP has many advantages with respect to other RTP methods and allows one to develop very simple, fast, automatic analytical methods. After a careful review of the methods published in the literature for the analytical determination of propranolol, it is deduced that there are only a few RTP methods have been proposed for this compound and the NP-RTP method for determination of propranolol has not been reported. The aim of this work is the development of the first application of the NP-RTP for the determination of propranolol.
and emission slits were 15 and 20 nm, respectively, the gate time 2.0 ms and delay time 0.1 ms were kept constant.
2.2. Reagents The propranolol was purchased from Wu-Jin pharmaceutical factor (Jiangsu Province, China) and a 1.0× 10 − 3 mol l − 1 aqueous solution was prepared. KI and Na2SO3 (AR grade, Beijing Chemical Plant, China) were used as received. A 1.0 mol l − 1 solution of KI (or solid) and 0.1 mol l − 1 solution of Na2SO3 were used in the experiments. The Na2SO3 solution was prepared when used and kept in a tightly stoppered container. Propranolol drug tablets were purchased from Beijing Second Pharmaceutical Plant. All of the organic solvents used were AR grade, the water used was prepared by twice sub-boiling distillation.
2.3. General procedure A 0.1 ml aliquot of 1.0×10 − 3 mol l − 1 aqueous solution was added to a 10-ml quantitative test tube, a certain amount of KI (used as heavy atom perturber) solution (or solid) and Na2SO3 (used as chemical deoxygenator) solution were then added. The solution was filled to the mark with water. After the solution was shaken, the fluorescence spectra, phosphorescence spectra and lifetime were measured on the LS-50B apparatus with a 1-cm quartz cell.
3. Results and discussion 2. Experiment
2.1. Instrument
3.1. Fluorescence spectra of propranolol in the presence of KI and Na2SO3
All fluorescence and phosphorescence spectra were measured on a Perkin– Elmer LS-50B luminescence spectrometer, the RTP lifetime was made on the LS-50B apparatus with a pulsed xenon lamp and the Obey – Decay application program. Both excitation and emission slits were 3 nm for fluorescence measurements, but for all of the phosphorescence measurements, the excitation
Fig. 1 shows that fluorescence peak wavelengths of propranolol aqueous solution, uex/uem, are 228, 288/339, 353 nm. When Na2SO3 was added into the solution, the fluorescence peak of excitation spectrum at shorter wavelength (near 228 nm) was disappear immediately. If an external heavy atom perturber (HAP), KI, was also added into the luminescent system, the fluorescence in-
W.-Q. Long et al. / Spectrochimica Acta Part A 58 (2002) 2185–2191
Fig. 1. Fluorescence spectra of propranolol. (1) C = 1× 10 − 5 mol l − 1; (2) C=1 ×10 − 5 mol l − 1 + 0.01 mol l − 1 Na2SO3 (3) C= 1 × 10 − 5 mol l − 1 + 0.01 mol l − 1 Na2SO3 + 0.1 g/10 ml KI; left, excitation spectra; right, emission spectra.
tensity was decreased obviously, which means that KI is a strong quencher for fluorescence of propranolol and a RTP signal may be induced for the system.
3.2. NP-RTP spectra of propranolol The experiment results show that propranolol aqueous solution can emit a strong and stable RTP signal in the absence of a protective medium, only using KI as a HAP and Na2SO3 as a deoxygenator (Fig. 2). The excitation spectrum exhibits a single peak, whereas two peaks were found for emission spectrum, the phosphorescence peak wavelength maxima uex/uem =288/494, 522 nm.
Fig. 2. NP-RTP spectra of propranolol; C =1× 10 − 5 mol l − 1, 1 g/10 ml KI, 0.01 mol l − 1 Na2SO3.
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Fig. 3. Effect of pH of the system on RTP intensity. uex/uem = 288/522 nm, C = 1 ×10 − 5 mol l − 1, 2.5 g/10 ml KI, 0.01 mol l − 1 Na2SO3.
3.3. Optimum conditions for RTP emission 3.3.1. Effects of system pH The effect of system pH on the RTP intensity of propranolol is very significant (see Fig. 3) because propranolol molecular contained a hydroxyl and an amino group, pH of the system will affect not only the deoxygenation efficiency[27], but also the existing forms of the luminescent moiety, which have various luminescence quantum yield. The experiment result shows that when pH of the system was controlled in the range of neutral to week basic (pH 7–8),the phosphorescence intensity is the highest. 3.3.2. Effects of the concentration of Na2SO3 Various volumes of 0.1 mol l − 1 Na2SO3 solution were added to a solution with a fixed amount of propranolol (0.1 ml of 1× 10 − 3 mol l − 1) and KI (2.5 g). As shown in Fig. 4, when the concentration of sodium sulfite in the system was larger than 0.005 mol l − 1, the maximum and stable RTP signal was obtained. A 1.0 ml of 0.1 mol l − 1 Na2SO3 solution was selected for the rest of experimental work. In this case, the concentration of Na2SO3 in the system is 1.0× 10 − 2 mol l − 1 and the pH of the solution falls just in the optimum range of pH described above, a buffer solution is not needed.
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Fig. 4. Effect of the concentration of Na2SO3 on RTP intensity. uex/uem =288/522 nm, C= 1× 10 − 5 mol l − 1, 2.5 g/10 ml KI.
3.4. Effects of concentration of KI on fluorescence and phosphorescence spectra of propranolol 3.4.1. Effects of concentration of KI on fluorescence intensity As described above, when the heavy atom perturber, KI, was added into the luminescent system, the fluorescence was quenched regularly (Fig. 5). The effect of the concentration of KI on the intensity of fluorescence was studied. According to Stern –Volmer equation, I0/I = 1 +Ksvx, here x
Fig. 6. Dependence of I0/I on the concentration of KI for fluorescence. uex/uem =288/353 nm, C =1 ×10 − 5 and 0.01 mol l − 1 Na2SO3.
is the molar concentration of KI, a plot of I0/I against x exhibits a good linear relationship (Fig. 6). The quenching constant calculated for fluorescence is Ksv = 107.2 l mol − 1 (r=0.9992, n=12).
3.4.2. Effects of concentration of KI on NP-RTP intensity and determination of phosphorescence kinetic parameters for propranolol The experiment results show that RTP intensity of the system was increased with every increase of the concentration of KI in a certain range. At the same time, RTP lifetime was decreased. When the concentration of KI was 1.5 mol l − 1 (added 2.5 g KI into 10 ml solution), the RTP intensity of the system comes to the highest (Fig. 7). According to the definition of phosphorescence lifetime, we have: ~0 = ~=
Fig. 5. Effect of the amount of KI on fluorescence intensity of propranolol. uex/uem =288/353 nm, C=1× 10 − 5 and 0.01 mol l − 1 Na2SO3.
1 kp
1 1 = [kp + ki] [kp + ax]
(1) (2)
where kp is a rate constant for phosphorescent decay; ki is a rate constant for triplet state radiationless deactivation from T1 S0; a is a constant that expressing the effect level of the molar concentration of KI to rate constant of radiationless deactivation. From the above relations, we have following relation:
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where x is the concentration of KI (mol l − 1), k is the decreasing rate of lifetime. When ln ~ was plotted as a function of the concentration in KI, we have also got another linear relationship: ln ~= ln ~0 − kCKI
Fig. 7. Effect of the amount of KI on NP– RTP intensity of propranolol. uex/uem =288/522 nm, C=1× 10 − 5 and 0.01 mol l − 1 Na2SO3.
ax ~0 [kp +ax] = 1+ = kp ~ kp 1 1 = +ax ~ ~0
(3)
When 1/~ or ~0 /~ was plotted as a function of the concentration in KI, we have got a linear relationship. On the other hand, as KI can promote the rate of intersystem crossing and radiative processes of triplet state, the phosphorescence lifetime was shortened. Thus: ~ =~0e − kx
(4)
(5)
According to the RTP lifetime obtained experimentally at different concentration of KI, we calculated luminescence kinetic parameters of propranolol based on the above relationships. the RTP lifetimes ~0 obtained by extrapolating curves of 1/~ = 1/~0 + ax and ln ~0 = ln ~0 − kx (ref. Fig. 8) were 1.27 and 1.23 ms, respectively, which were in good agreement with each other. Take an average value ~0 = 1.25 ms, we obtained luminescence kinetic parameters of propranolol, kp = 1/~0 = 0.800 ms − 1, a= 0.560 ms − 1 l − 1mol − 1 and k= 0.477 ms − 1 l − 1 mol − 1, respectively. So the relation between KI concentration (x) and RTP lifetime (~) can be expressed as ~= 1.25e − 0.477x. 4. Analytical parameters According to the experimental procedure and using the optimum conditions described above, the concentration of propranolol solution in the range of 8× 10 − 8 –2× 10 − 5 mol l − 1 has a good linear relationship with RTP intensity r= 0.9994, n =7. The calculated detection limit by methodology of IUPAC [28] based on three times the standard deviation of background was 3×10 − 8 mol l − 1.
Fig. 8. The plot of 1/~ (left) or ln ~ (right) vs. x (concentration of KI) for phosphorescence. C =1 ×10 − 5 and 0.01 mol l − 1 Na2SO3.
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Table 1 Standard recovery of propranolol in urine samples Added (mmol l−1)
Found (mmol l−1)
Average (mmol l−1)
Recovery (%)
R.S.D. (%)
1.00 2.00 4.00
0.952 0.986 0.962 1.984 1.944 1.912 3.812 3.952 3.824
0.967 1.947 3.863
96.7 97.4 96.6
1.8 1.9 2.0
5. Analysis for propranolol in real sample
5.1. Determination of propranolol in urine sample In order to mimic a real sample situation and eliminate the matrix effect, the quantitative analysis of trace propranolol in spiked urine samples has been carried out by standard additions method. The preparation and pretreatment of spiked urine sample is as follows: 1 ml urine sample, a various amount of propranolol standard solution and 0.2 ml of 0.5 mol l − 1 NaOH solution was added in turn to a 10 ml quantitative test tube, shaken for 10 min, then 5 ml dichloromethane was added and shaken for 10 min again, then the mixture was centrifuged (3000 rpm) for 15 min. The upper aqueous layer was discarded and 3 ml of transparent solution of down layer was pipetted into another 10 ml quantitative test tube and evaporated to dryness at 50 9 1 °C to move dichloromethane. The dry residue was redissolved in 0.5 ml of water (or acetonitrile) and the recovery experiment of the propranolol standard in the spiked urine sample was performed based on the general procedure described above. At the same time, the analytical curves in the presence and absence of 5%(v/v) acetonitrile were made for the real sample. The regression equations of the analytical curves were Ip = 0.2633+3.544× 106 C (mol l − 1), r =0.9992, n= 7 for without acetonitrile system; and Ip = 0.08300+ 4.116×106 C (mol l − 1), r = 0.9998, n= 7 for the system in the presence of 5% acetonitrile, respectively. As shown in Table 1, the recovery and precision are good. The results show that a litter of acetonitrile (5%) is suitable for determination of propranolol (Fig. 9). The slope of the analytical curve in the presence of 5% (v/v) acetonitrile was higher than that of in absence of
acetonitrile, and the intercept was just contrast. We think, maybe it is relative with the weekly self-association property of propranolol in aqueous-electrolyte solution [29], because the concentration of KI in the luminescent system is high (2.5 g/10 ml is about 1.5 mol l − 1). The presence of acetonitrile is suitable for dissociation of associated molecule of propranolol.
5.2. Determination of propranolol in drug tablet Ten drug tablets were ground to powder in an agate mortar, a certain quantity of powder sample (66.5 mg) was dissolved in water and was filled to 100 ml with water. The mixture was filtered with a piece of dry filter-paper to remove insoluble excipient in the drug tablet. Finally, a certain quantity of filtrate was pipetted into a 10 ml quantitative test tube and the general procedure described above was applied to the solution. The results show that the content of propranolol in
Fig. 9. The NP-RTP analytical curves of propranolol for urine sample in the presence and absence of acetonitrile. uex/uem = 288/522 nm, 0.01 mol l − 1 Na2SO3, 2.5 g/10 ml KI.
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drug tablet was 10.8, 11.0, 11.0 and 10.8 mg per tablet, respectively, for four determinations, which are in good agreement with the results of UV method [30]. The relative standard deviation (R.S.D.) of 1.06%, indicates also good precision.
6. Conclusions The NP-RTP method developed in this paper for the determination of propranolol is high sensitive and is simple compared with other analytical methods proposed for the determination of this compound. As no protected media is needed, the luminescent system is completely transparent, no precipitation and no foam problems exist as in cyclodextrin-induced RTP and in MS-RTP methods. This simplification improves the analytical characteristics, which means that NP-RTP has good potential uses with other analytical techniques such as HPLC and FIA etc. On the other hand, this paper demonstrated that the lifetime method based on the heavy atom effect can be used for the determination of the luminescence kinetic parameters.
Acknowledgements This work was supported by the National Natural Science Foundation of China.
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