Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 67 No. 4 pp. 321ñ326, 2010
ISSN 0001-6837 Polish Pharmaceutical Society
ANALYSIS
PHOTOSTABILIZATION OF PAPAVERINE HYDROCHLORIDE SOLUTIONS KAROLINA PIOTROWSKA, TADEUSZ W. HERMANN* and ALICJA PAWELSKA Department of Physical Pharmacy and Pharmacokinetics, PoznaÒ University of Medical Sciences, 6 åwiÍcickiego St., 60-781 PoznaÒ, Poland Abstract: The stability of aqueous and non-aqueous papaverine hydrochloride solutions exposed to the UV radiation is poor. In order to enhance its photo-stability suitable light absorbers may be used. There were four photo-protectors considered in this work: 4-aminobenzoic acid, sodium benzoate, methyl 4-hydroxybenzoate and propyl 4-hydroxybenzoate, whose UV absorption spectra characteristics match to some extent with the UV spectrum of papaverine. Approximately 20 mg/mL papaverine chloroform solutions with the above non-toxic additives in the concentrations 0.01; 0.05; 0.10% were exposed to the UV light of 254 nm. High performance capillary electrophoresis was used to determine the papaverine hydrochloride concentration loss as a function of time exposition to the light. It was found that papaverine hydrochloride photolysis proceeds according to the first-order kinetics. Methyl 4-hydroxybenzoate was found to be the best UV radiation-protective agent, and at the concentration 0.10%, the reaction rate constant decreases from 0.143 h-1 to 0.028 h-1. Both 4-hydroxybenzoate esters develop a more efficient UV radiation-protective activity than sodium benzoate, because the latter additive molar extinction coefficient is less significant. However, in spite of a high value of 4-aminobenzoic acid molar absorptivity coefficient, it is an unsuitable photo-protector for papaverine hydrochloride solutions, because its UV absorption spectrum does not match with that of papaverine. Keywords: papaverine hydrochloride, photo-stabilization, HPCE quantification, photo-protectors, methyl 4hydroxybenzoate, propyl 4-hydroxybenzoate, sodium benzoate, 4-aminobenzoic acid
zoate, methyl and propyl 4-hydroxybenzoates, because they roughly meet the theoretical requirements (8).
It is widely recognized that papaverine salts injections are subjected to degradation via oxygen dissolved in water, and therefore should be sealed under a neutral gas (nitrogen, carbon dioxide) (1ñ3). However, it is not so well known that papaverine salts injection solutions should be also sheltered from degradation processes as a result of their exposition to the UV light (1ñ4). Papaverine and its salts are especially susceptible to the degradation when exposed to the UV light in nonaqueous solutions (5ñ7). In order to enhance the photostability of papaverine salts injection solutions of medical importance, it is possible to use additives of light absorbers. To be a suitable photoprotective agent for a drug, an additive must absorb the light radiation over the same wavelength range (8). The objective of the project was to select the most suitable photoprotective agent toward papaverine hydrochloride (PHCl) chloroform solutions, because they are more labile than its aqueous solutions and contain the same degradation products. There were four photoprotectors under hitherto investigation: 4-aminobenzoic acid, sodium ben-
EXPERIMENTAL Materials 4-Aminobenzoic acid, Applichem, Germany; sodium benzoate, POCH, Gliwice, Poland; methyl and propyl 4-hydroxybenzoate, Caelo, Germany; uranyl oxalate, Chemapol, Prague, Czech Republic. All other chemicals were either previously specified (9) or of the reagent grade. Apparatus High performance capillary electrophoresis (CE), model 3D 1 apparatus with diode array UV detector (Agilent Technologies, Walbronn, Germany) was used for quantification of PHCl. A 3D apparatus was equipped with chemstation used for instrument control, data acquisition and data analysis. The system was controlled by Windows NT software. A low pressure mercury lamp 254 nm
* Corresponding author: phone: +48 61 854 6432; fax: +48 61 854 6430, e-mail:
[email protected]
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Original Hanau TNN 15/32 (Heraeus, Germany) was used in photo-kinetic experiments. A spectrophotometer UV-vis Specord (Carl Zeiss, Jena, Germany) was applied for UV spectra recording of PHCl and the photoabsorbers. CE methodology The basic procedure was reported previously (9). However, the temperature of the capillary was maintained by a thermostatic system at 35OC. The samples were automatically injected using hydrodynamic injections at the anode for 5 s (50 mbar). The capillary between subsequent injections was washed with 0.1 M NaOH, water and BGE (background electrolyte) for 3 min each. All experiments were carried out at the voltage 30.0 kV resulting in the current of 49.0 ± 0.5 µA. Calibration curve for PHCl Stock solutions of PHCl and loratadine (IS) were prepared in methanol at concentrations 35.0 and 20.0 mg/mL, respectively. Then, standard solutions of PHCl each of 5.0; 10.0; 12.0; 17.5 and 20.0 mg/mL were prepared by dilution with methanol. To 16 mm culture tubes were added 30 µL each of a standard solution, 50 µL IS and 920 µL mixture of methanol and BGE pH = 2.5 (1;1, v/v), and vortexed. The solution samples were injected onto the capillary as mentioned above. The peak area ratio of an analyte to IS was plotted as a function of PHCl concentration. In order to calculate precision and accuracy, different concentrations of the compound considered were determined within-day (WDA) and between-days (BDA). Every single determination was done in triplicate. The selectivity of CE separation was checked for the analyte, its well known oxidation products: papaverinol, papaveraldine, socalled PHCl injection solutions final brown degradation product X, and IS (1, 2, 4, 9). Finally, LOD and LOQ values were calculated for the PHCl CE separation procedure worked out. UV spectra of the parent drug and its photoprotective additives The UV spectra in methanol of PHCl, 4aminobenzoic acid, sodium benzoate, as well as methyl and propyl benzoates each were recorded and molar absorptivity coefficients of the additives were calculated at their λmax values from their concentrations ranges: (2.92ñ4.39)◊10-5 M, (2.78ñ4.16) ◊ 10-5 M, (2.63ñ5.26) ◊ 10-5 M, and (2.21ñ4.45) ◊ 10-5 M, respectively, at the initial time and after 8 h elapsed from the start of exposition to 254 nm radiation, to control their stability.
Kinetics of PHCl photooxidation processes Two percent (w/v) PHCl chloroform solution, containing the above photoprotectors at their concentrations 0.01, 0.05 and 0.10% (w/v) were exposed to UV light 254 nm in 25 mL cylindrical silica cells from the distance of 5 cm at ambient temperature and in aerobic conditions. At suitable time interval 30 µL samples withdrawn were diluted with 50 µL of IS stock solution and 920 µL of the mixture of methanol and BGE pH = 2.5 (1:1, v/v). The concentration of PHCl was determined according to the CE methodology. Photooxidation reactions of PHCl followed the typical first-order equation provided elsewhere (9). Measurement of the quantum yield A uranyl oxalate actinometer was applied to measure the quantum yield of the above photolysis processes (9) in absence and presence of the photoprotectors at their different concentrations. The actinometer was titrated with manganate(VII) potassium prior and after irradiation. The results of titrations (∆c) allow us to calculate the intensity of light absorbed by the substrate (Ia). The specific quantum yield of the actinometer is recognized to be Φ = 0.602 (10). The quantum yields of PHCl photolysis processes were calculated from the equation (9): ∆c ϕ = ñññññ Ia ∑ t where t denotes the time of irradiation for different degrees of conversion which were extrapolated to the zero degree of conversion indicating the specific quantum yield (Φ) of a substrate. RESULTS Validation of HPCE methodology for PHCl quantification An electropherogram presented in Fig. 1 indicates three PHCl oxidation products: the brown compound X (A), papaverinol (C), papaveraldine (E), which are separated between each other, as well as from PHCl (B) and loratadine (D, IS). The migration time (tm, min) of the above compounds increase from 11.80 ± 0.09 (A) through 12.38 ± 0.12 (B), 12.86 ± 0.27 (C) and 13.19 ± 0.24 (D, IS) to 15.15 ± 0.34 (E). A calibration curve of PHCl was constructed for its area under the peak ratio to IS peak area (A/AIS) as a function of PHCl concentrations. Evaluation of linearity of the calibration curve by means of the least square regression analysis can be learnt from a pretty good correlation coefficient (r = 0.9997) and its equation:
Photostabilization of papaverine hydrochloride solutions
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Molar absorptivity coefficients of photoprotective additives The UV molar absorptivity coefficients (in l mol-1 cm-1 ± SD) of all methanol additives solutions examined at their λmax are presented as follows: 4aminobenzoic acid (17790 ± 30), sodium benzoate (9420 ± 70), methyl 4-hydoxybenzoate (16180 ± 140), and propyl 4-hydroxybenzoate (1400 ± 150). First-order kinetics of PHCl solution degradation in absence and presence of photoprotective additives Typical first-order plots have been generated for photooxidation reaction of degraded PHCl chloroform solutions (e.g. Fig. 2).
Figure 1. Electrophoregram of a mixture of papaverine hydrochloride (B), its oxidation products: papaverinol (C), papaveraldine (E) and the brown degradation product X mentioned (A) as well as loratadine, IS (D). Experimental conditions: fused-silica capillary, 50 µm i.d., length 72 cm; temperature, 35OC; running buffer, 50 mM Na2HPO4 and 100 mM H3PO4, pH 2.5; separation voltage 30 kV; injection at the anode for 5 s (50 mbar)
A/AIS = (0.04 ± 0.01) + (25.5 ± 0.3)◊c, where the first and the second parameter denote the intercept and the slope, respectively. Precision and accuracy data have been also calculated for the measurements of WDA and BDA for different concentrations of the analyte. The precisionsí correlation of variations range from 1.9% to 7.3% and their accuracies lay out between 0.25 and 5.25%. The lower limit of detection (LOD) was calculated from the equation: 3.3 ∑ S LOD = ñññññññ a and the lower limit of quantification from the other equation: 10 ∑ S LOQ = ñññññññ a where S and a denote the standard deviation of the calibration curve intercept and its slope, respectively (9). Therefore, LOD = 1.29 mM and LOQ = 3.9 mM as calculated from the above equations.
Figure 2. Typical first-order plots for photodegradation of papaverine hydrochloride in its 2.0% (w/v) chloroform solutions in absence (PHCl) and presence of methyl 4-hydroxybenzoate of different concentrations: 0.01%; 0.05%; 0.01%, when exposed to UV light of 254 nm at ambient temperature
Since the concentrations of the analyte as a function of time obey the normal distribution, the least squares regression calculations were used to obtain, firstly, the pseudo-first-order rate constants according to a semilogarithmic equations as the absolute value of their negative slopes, and secondly, half-lives times (t1/2) from the above rate constants from a known formula (9) (Tab. 1).
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Table 1. First-order kinetics of papaverine hydrochloride 2.0% (w/v) chloroform solutions photodegradation in presence and absence of photoprotective additives, on exposition to UV light of 254 nm at ambient temperature.
Additive
Concentrations (%)
First-order rate constants K ± SDa (h-1)
Half-life times, t1/2 ± ∆t1/2b (h)
0.01
0.115 ± 0.002
6.0 ± 0.10
4-Aminobenzoic acid
Sodium benzoate
Methyl 4hydroxybenzoate Propyl 4hydroxybenzoate No additive a
0.05
0.110 ± 0.003
6.3 ± 0.17
0.10
0.104 ± 0.004
6.7 ± 0.26
0.01
0.143 ± 0.003
4.8 ± 0.10
0.05
0.120 ± 0.0.003
5.8 ± 0.19
0.10
0.125 ± 0.003
5.5 ± 0.13
0.01
0.132 ± 0.004
5.3 ± 0.16
0.05
0.061 ± 0.004
11.4 ± 0.74
0.10
0.028 ± 0.002
24.8 ± 1.77
0.01
0.115 ± 0.002
6.0 ± 0.21
0.05
0.075 ± 0.003
9.2 ± 0.37
0.10
0.052 ± 0.002
13.3 ± 0.51
0.00
0.143 ± 0.005
4.8 ± 0.17
SD ∑ ln2
Standard deviation (SD). b ∆t1/2 = /ñññññññññ/ k2
Table 2. Apparent quantum yields (ϕ) of 2% (w/v) papaverine hydrochloride (PHCl) chloroform solutions photooxidation, exposed to UV254 light at ambient temperature and aerobic conditions, in absence and presence (0.1%) of methyl 4-hydroxybenzoate (bold digits).
Time of exposition (min)
Change of PHCl concentration, ∆c (mol l-1)
Degree of conversion (%)
Apparent quantum yield (ϕ)
30
0.0057
10.7
0.313
ñ
ñ
ñ
0.0088
16.4
0.243
0.0013
2.4
0.036
0.0135
25.2
0.185
0.0021
3.9
0.029
0.0187
35.0
0.171
ñ
ñ
ñ
0.0251
46.9
0.172
0.0052
9.8
0.035
60 120 180 240 300 360 420 480
ñ
ñ
ñ
0.0052
9.8
0.029
0.0310
57.9
0.142
0.0075
14.1
0.034
ñ
ñ
ñ
0.0087
16.3
0.034
ñ
ñ
ñ
0.0112
21.0
0.038
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Photostabilization of papaverine hydrochloride solutions
Table 3. Specific (extrapolated to zero radiation time) quantum yields (Φ) of photooxidation reactions of 2% (w/v) papaverine hydrochloride chloroform solutions, exposed to UV254 light at ambient temperature and aerobic conditions, in absence and presence of photoprotective additives at different concentrations
Photoprotector
Concentration (%)
Specific quantum yields (Φ)
Absence
0.00
0.301
0.05
0.154
0.10
0.151
4-Aminobenzoic acid
Sodium benzoate
Methyl 4-hydroxybenzoate
Propyl 4-hydroxybenzoate
Quantum yields measurement Intensity of light absorbed by the substrate (PHCl) [Ia = (6.09 ± 1.00)◊10-4 mol◊l-1◊min-1] was calculated from the equation given above, taking into consideration the results produced on irradiation the uranyl oxalate actinometer (9). Apparent quantum yields (ϕ) as a function of the substrate degree of conversion were calculated from the same equation when transformed for ϕ (e.g. Tab. 2), and next, they were extrapolated to zero substrate degree of conversion to obtain specific quantum yields (Φ) (Tab. 3). DISCUSSION The stability of unprotected PHCl aqueous and nonaqueous solutions, especially when exposed to UV radiation, is insufficient for pharmaceutical applications. In order to enhance its photostability, the addition of suitable light absorbers may be of importance. To be a suitable photoprotective agent for pharmaceutical formulations, the additive must be non-toxic and absorb the radiation over the same range as the drug itself. Moreover, the absorber should have a high molar absorptivity coefficient at the desired spectral range (8). There were four photoprotective additives under this investigation that absorb the radiation in the region close to PHCl: 4-aminobenzoic acid, sodium benzoate, and methyl as well as propyl 4hydroxybenzoates. The objective of the project was
0.01
0.188
0.05
0.177
0.10
0.174
0.01
0.243
0.05
0.104
0.10
0.034
0.01
0.127
0.05
0.076
0.10
0.053
to select the most suitable photoprotective agent for the PHCl solutions. Moreover, each absorber was investigated at three different concentration levels. Approximately 20 mg/mL PHCl chloroform solution with the addition of additives considered were exposed to UV25light. A HPCE method was employed to evaluate the PHCl photodegradation progress. The HPCE methodology for determination of PHCl and its degradation products (papaverinol, papaveraldine, and a brown compound X (1) identified now as 2,3,9,10-tetramethoxy-12-oxo-12H-indolo[2,1a]isoquinolinylium chloride (4) has been now improved. The time of separation has been shorten from 29 min to 15 min (9) (Fig. 1), because the temperature was increased from 25∞C to 35∞C and the voltage was enhanced from 20 kV to 30 kV. The precision and accuracy of the HPCE method are good enough e.g., the correlation coefficients are not greater than 7.3% It was found that the PHCl photodegradation obeys the first-order kinetics (Fig. 2). A decrease in the reaction rate constant and an increase in the half-life time with an increase in the absorbers concentration substantiated their photoprotective activity toward PHCl (Tab. 1). The most effective photodegradation inhibition, except sodium benzoate, was observed for the highest concentration (0.1%) of a protective agent (Tab. 1). Methyl 4-hydroxybenzoate was found to be the best photoprotective agent among the investigated absorbers, which at the concentration 0.1% decreases the reaction rate constant from
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0.143 h-1 to 0.028 h-1, resulting in the reaction halflife time increase from 4.8 h to 24.8 h (Tab. 1). Furthermore, the specific quantum yield of PHCl photodegradation is lowered from 0.301 (no additive) to 0.034 (Tab. 3). It should be also mentioned that UV spectra characteristics of the drug and the absorber are the most similar in this case (Fig. 3) if compared to those of the other absorbers under investigation.
benzoate ester, it is a less effective photoprotector (Tab. 1). Both 4-hydroxybenzoate esters develop more efficient photoprotective activity if compared to sodium benzoate, because this last possesses the lowest molar absorptivity coefficient. In spite of a high value of the molar absorptivity coefficient, 4aminobenzoic acid has appeared to be an unsuitable absorber for PHCl solutions as a result of significant differences in its UV spectrum if compared to PHCl (Fig. 4). It should be mentioned that the photodegradation processes of PHCl in non-aqueous solutions are accelerated if compared to its aqueous solutions, because the solubility of oxygen in the former solvents is much greater. However, the same degradation products of PHCl are observed in both media (5ñ7). REFERENCES
Figure 3. UV spectra of papaverine hydrochloride and methyl 4hydroxybenzoate in methanol, fresh and exposed to UV254 light for 8 hours
Fig. 4. UV spectra of papaverine hydrochloride and 4-aminobenzoic acid in methanol, fresh and exposed to UV254 light for 8 hours
Since propyl 4-hydroxybenzoateís molar absorptivity coefficient is lower than that of methyl
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