Pharmaceutical Development and Technology, 11:409–416, 2006 Copyright © Informa Healthcare ISSN: 1083-7450 print / 1097-9867 online DOI: 10.1080/10837450600770106
LPDT
Stability of Atenolol, Acebutolol and Propranolol in Acidic Environment Depending on its Diversified Polarity Jan Krzek and Anna Kwiecien Atenolol, Acebutolol, and Propranolol in Acidic Environment
Collegium Medicum, Department of Inorganic and Analytical Chemistry, Jagiellonian University, Kraków, Poland
Marek Zylewski Collegium Medicum, Department of Organic Chemistry, Jagiellonian University, Kraków, Poland
The main objective of this research was to investigate the relationship between the polarity of atenolol, acebutolol, and propranolol described by logP and kinetic and thermodynamic parameters characterizing their degradation process in acidic solution. Hydrolysis was carried out in hydrochloric acid at molal concentrations of 0.1 mol/L, 0.5 mol/L, and 1 mol/L for 2 hr at 40°C, 60°C, and 90°C. Chromatographic-densitometric method was used for the determination of drugs under investigation. The identification of degradation products was carried out by using 1H NMR. The degradation processes that occurred in drugs under investigation are described with kinetic parameters (k, t0.1, and t0.5) and energy of activation (Ea). It has been found that the stability of drugs increases toward lipophilic propranolol in the assumed experimental model. The rate constants k decrease, contrary to t0.1, t0.5, and Ea, which vary comparably to logP, thus increasing from the most hydrophilic atenolol, through acebutolol, of lower polarity, to the most lipophilic propranolol. This study demonstrated that the stability of chosen beta-adrenergic blocking agents increases with their lipophilicity. Keywords beta-blockers’ stability, kinetics of degradation process, TLC, drug analysis, atenolol, acebutolol, propranolol
INTRODUCTION Atenolol, acebutolol, and propranolol belong to betablockers that block activity of beta-adrenergic receptors and are of wide spectrum of pharmacological action; used
Received 8 October 2005, Accepted 3 April 2006. Address correspondence to Jan Krzek, Collegium Medicum, Department of Inorganic and Analytical Chemistry, Jagiellonian University, Kraków, Poland; E-mail:
[email protected]
in the treatment of ischaemic heart disease, coronary failure, or as illegal doping agents.[1–3] OH O
NH
CH3
(a)
CH3
(b)
Ar R
OH R
NH R
There are two characteristic groups, alkanolamine and aromatic, present in beta-adrenolytic drugs of formula (a and b) presented above deciding on their physicochemical and pharmacological properties. The alkanolamine chain of typical pKa from 9.2 to 9.6 is responsible for their alkaline properties, whereas the aromatic ring is responsible for their lipophilic character. The structural diversity within this group of drugs results in highly diversified physicochemical properties, such as fat and water solubility, which is strictly related to pharmacological action, metabolic processes, and their toxicity.[4–6] Hydrophobicity is most often expressed with the octanol-water partition coefficient denoted as logP; its value is determined by using classical and chromatographic methods as well as dedicated software.[7–13] When reviewing the available results of kinetic analysis of beta-adrenolytic drug decomposition processes, it is easy to find that they are highly diversified depending on drug structure and destructive factor.[14,15]
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H
OH
O
O
NH
CH3
(1)
CH3
H2N
For atenolol [Eq. (1)] the photodegradation process in the solutions of various acidity was analyzed, while considering the effect of γ-radiation at dosages of 40–60 kGy, UV, and visual radiation. It was also found that the decomposition of atenolol depends on the type of solvent in which various products are formed.[16, 17]
OH CH3
O
• Normocard tablets containing 50 mg of atenolol (Polfa,
Warszawa) • Acecor tablets containing 200 mg of acebutolol (Societa
Prodotti Antibiotici SPA) • Propranolol tablets containing 40 mg of propranolol
(Polfa, Warszawa)
NH C(CH3)2H
(2)
CH3 NH O
The degradation process of acebutolol [Eq. (2)] was analyzed in 0.1 mol/L HCl environment at variable temperatures, while establishing the direction of occurred changes.[18]
H
Preparations
Solutions for Stability Testing
H O
Tarnów, Poland), glacial acetic acid (Odczynniki Sp. z o.o., Lublin, Poland), and purified water according to the European Pharmacopoea. All reagents were of analytical purity.
OH
O
H N
CH3
(3)
CH3
Propranolol [Eq. (3)] is typically placed among stable drugs not susceptible to changes.[19–21] When considering the significance of the partition coefficient logP in the development of novel drugs, the aim of this article was to check whether there is a relationship between the polarity of atenolol, acebutolol, and propranolol and kinetic and thermodynamic parameters specific for these drugs in terms of stability and ability to changes that may be usable in receptor testing. In available literature there are no reports of complete assessment including stability and polarity of beta-adrenolytic drugs, thus justifying the research studies presented in this article.
MATERIALS AND METHODS Materials Methanol (Merck, Darmstadt, Germany), chloroform (Chempur, Poland), ammonia 25% (Zaklady Azotowe,
The test solutions were prepared from weighed amounts of 10 powdered tablets corresponding to the following content of active substances in the form of free base: 0.025 g of atenolol (Normocard), 0.015 g of acebutolol (Acecor), and 0.025 g of propranolol (Propranolol). Directly before the determination the weighed amounts of drugs were dissolved in 5 mL of hydrochloric acid at concentrations of 1.0 mol/L, 0.5 mol/L, and 0.1 mol/L heated in water bath up to the desired temperature. Incubation was carried out for 2 hr at temperatures of 90°C, 60°C, and 40°C, taking 500 μL of solution every half hour and dissolving it, after cooling, with methanol at 1:1 ratio. The usage of pharmaceutical preparations in this experiment together with active substance contain excipients was preceded by experiments that proved that placebo constituents do not influence the obtained results.
Chromatographic Conditions The analysis was carried out by using the validated chromatographic-densitometric method described earlier.[22] On the HPTLC F254 chromatographic plates 10 μL of relevant solutions were applied in 1-cm bands and then developed over the distance of 85 mm by using chloroformmethanol-ammonia (15:7:0.2, v/v/v) as the mobile phase. Chromatograms were dried at room temperature and scanned at wavelengths corresponding to the maximum absorbance of particular substances: λ = 270 nm for atenolol, λ = 240 nm for acebutolol, and λ = 289 nm for propranolol. Chromatograms were also checked visually under a UV lamp at two wavelength ranges, 254 nm and 366 nm. For quantitative determination purposes, the surface areas of appropriate peaks were recorded and the percentage concentrations of each constituent were computed by using the internal normalization method, according to the
Atenolol, Acebutolol, and Propranolol in Acidic Environment
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formula: % i = (x i / ∑ x) 100%, where % i-th is constituent concentration and xi is peak area for the determined constituent; and ∑ x is sum of peak surface areas in the chromatogram. Presented results are the mean value of the three measurements.
Identification of Degradation Products To identify the degradation products, UV spectra were recorded directly from chromatograms, and 1H NMR spectra were registered after isolating individual constituents from the gel layer. Gel layers were scraped from the plates where constituents were located. Gel was shaken in a 50-mL volumetric flask with 30 mL of methanol and 2 mL of water heating the suspension in water bath at 50°C for approximately 2 hr. The mixture was filtered through a G-4 filter paper. The solution was evaporated to obtain a dry matter under nitrogen at 37°C. Similarly, a blind test was prepared by using the gel from the pure plate developed as described above for extraction. The residue was dissolved in 0.7 mL of dimethyl sulfoxide and subjected to 1 H NMR.
Figure 1. Densitogram of atenolol (2) and its degradation product (1,3) after incubation for 60 mins in 0.1 mol/L hydrochloric acid at 90°C.
Kinetic Testing Based on the relationship log(c) = f(t) the order of degradation reaction was determined for drugs under investigation, and then the rate constants k and t0.1 and t0.5 as well as energy of activation Ea were calculated.
RESULTS The densitograms recorded for individual drugs under consideration have also shown additional peaks that indicate the degradation products. The number of spots in chromatograms, its locations, and surface areas, varied depending on the type of drug, acid concentration, temperature, and incubation time. Under specified test conditions, well-separated components were obtained of different retention coefficients: atenolol (Rf = 0.46) and two degradation products 1 (Rf = 0.12) and 2 (Rf = 0.74); acebutolol (Rf = 0.53) and degradation product 1 (Rf = 0.40); propranolol (Rf = 0.73) and degradation product 1 (Rf = 0.98). The additional peak areas originated from the degradation products and recorded in densitograms increased contrary to decreasing peak areas of constituents under investigation. Examples of densitograms are presented in Figures 1, 2, and 3.
Figure 2. Densitogram of acebutolol (2) and its degradation product (1) after incubation for 105 mins in 1 mol/L hydrochloric acid at 90°C.
Figure 3. Densitogram of propranolol (1) and its degradation product (2) registered after incubation for 150 mins in 0.3 mol/L hydrochloric acid at 90°C.
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Active component and degradation product concentration variations are listed in Tables 1, 2, and 3. The kinetic parameters and energy of activation that describe the degradation process of compounds under conTable 1 Results of atenolol quantitation after incubation in hydrochloric acid solutions at 90°C, 60°C, and 40°C Temperature/ hydrochloric acid concentration 90°C 1M 0.5 M 0.1 M
60°C 1M 0.5 M 0.1 M
40°C 1M 0.5 M 0.1 M
Time (hr) / concentration (%) atenolol 0h
0.5 h
1h
1.5 h
2h
78.16 85.06 100
3.58 12.92 62.19
0 2.52 46.34
0 0 32.98
0 0 24.55
0h
0.5 h
1h
1.5 h
2h
84.99 91.93 100
47.91 69.74 92.64
33.02 60.54 87.60
20.82 50.39 83.59
16.42 35.45 80.90
0h
1h
2h
3h
4h
90.36 100 100
84.53 91.53 100
79.98 87.14 100
74.63 81.94 97.96
66.70 76.24 95.00
Table 2 Results of acebutolol quantitation after incubation in hydrochloric acid solutions at 90°C, 60°C, and 40°C Temperature/ hydrochloric acid concentration
0h
0.5 h
1h
1.5 h
2h
90°C 1M 0.5 M 0.1 M
100 100 100
36.36 57.87 89.49
13.32 41.78 80.08
5.80 28.75 75.07
3.55 21.99 69.32
0h
0.5 h
1h
1.5 h
2h
100 100 100
92.25 96.73 100
84.65 91.97 100
77.13 85.87 97.79
72.43 85.04 97.02
0h
1h
2h
3h
4h
95.10 98.18 100
91.48 96.66 100
89.35 94.37 98.68
60°C 1M 0.5 M 0.1 M
40°C 1M 0.5 M 0.1 M
Time (hr) / concentration (%) acebutolol
100 100 100
100 100 100
Table 3 Results of propranolol quantitation after incubation in hydrochloric acid solutions at 90°C and 60°C Time (hr) / concentration (%) propranolol
Temperature/ hydrochloric acid concentration
0h
0.5 h
1h
1.5 h
2h
90°C 1M 0.5 M 0.1 M
100 100 100
63.90 78.23 93.50
56.80 66.94 88.59
54.10 60.77 84.79
52.29 57.99 83.06
0h
0.5 h
1h
1.5 h
2h
100 100 100
100 100 100
97.92 100 100
96.64 100 100
96.40 100 100
60°C 1M 0.5 M 0.1 M
sideration were determined and the compliance with kinetics of pseudo-first-order reaction was found (Figure 4), for which the rate constant k, t0.5, and t0.1 and energy of activation were determined and shown in Table 4. It has been found that within the investigated range of hydrochloric acid concentrations the reaction rate reaches the minimum value at 0.1 mol/L and increases with HCl concentrations for all drugs under consideration. Temperature has a similar effect causing the values of k to increase with increasing temperature. The rate constants decrease from atenolol through acebutolol to propranolol, contrary to increasing t0.1 and t0.5 as well as energy of activation (Table 4). For propranol, the energy of activation is approximately 50% higher than that of atenolol and approximately 20% higher than that of acebutolol. To identify products of degradation for individual spots in atenolol chromatograms the absorption spectra were recorded. It was found that the spectra are of similar shape, contrary to various Rf values. This finding suggests that both products of atenolol degradation and original substance have similar chemical structure (Figure 5). To identify those products, the 1H NMR analysis was carried out for products of Rf ≈ 0.12 and Rf ≈ 0.74 isolated from chromatograms and for atenolol as a reference substance. The interpretation of Rf ≈ 0.12 spot showed that aromatic and isopropylamine group signal pattern remains almost the same in comparison with reference atenolol spectrum, indicating that there are no changes in this moieties. Signal of amide protons vanishes showing hydrolysis of this group. However, the influence of appearing carboxyl group on aromatic system as a stronger electron acceptor is diminished because of indirect connection of this group to aromatic
Atenolol, Acebutolol, and Propranolol in Acidic Environment
413
Figure 4. The relationship between log c and t for atenolol, acebutolol, and propranolol corresponding to pseudo-first-order reaction.
Table 4 Kinetic and thermodynamic parameters describing degradation process of atenolol, acebutolol, and propranolol in acidic solutions at different temperatures k (min−1) HCl (mol/L) Atenolol 1 0.5 0.1 Acebutolol 1 0.5 0.1 Propranolol 1 0.5 0.1
t0.5 (hr)
313 K
333 K
363 K
313 K
333 K
0.00126 0.00113 0.00021
0.01370 0.00794 0.00177
0.10284 0.05865 0.01170
9.17 10.22 55.00
0.00047 0.00024 0.00005
0.00269 0.00135 0.00025
0.02783 0.01263 0.00305
-
0.00030 -
0.00540 0.00454 0.00155
t0.1 (hr) 363 K
313 K
333 K
363 K
Ea (kJ/mol)
0.84 1.45 6.53
0.11 0.20 0.99
1.39 1.55 8.36
0.13 0.22 0.99
0.02 0.03 0.15
67.58 67.04 63.40
24.57 48.13 231.00
4.29 8.56 46.20
0.42 0.91 3.79
3.73 7.31 35.10
0.65 1.30 7.02
0.06 0.14 0.58
78.37 74.95 83.65
-
385.00 -
2.14 2.54 7.45
-
5.85 -
0.33 0.39 1.13
96.33 -
moiety through metylene group. Significant changes are observed in aliphatic fragment of the spectrum compared with reference atenolol spectrum, most probably due to dehydration process generating double bond coupled with free electron pair of nitrogen atom. The direction of double bond formation is confirmed by rather small value of chemical shift of olefin protons, which should be greater for double bond coupled with free electron pair of oxygen atom. Thus, the identified product of acid hydrolysis of atenolol is 4-(3-isopropyloamino)-2-propenoxy) phenylacetic acid. Because of a very small amount of Rf ≈ 0.74 product, 1 H NMR analysis did not bring any reliable results for the
structure determination. When considering earlier findings related to the similarity of UV spectra as well as published reports on products of atenolol hydrolysis in acidic environment, chromatographic analysis was carried out to confirm the presence of p-hydroxyphenylacetic acid. Chromatographic separation was carried out under conditions established for the presented method. A spot of Rf ≈ 0.74 was found in chromatograms. Such spot originated from hydrolysis of the drug and corresponded to the appropriate spot from the reference substance. The recorded absorption spectra for both constituents within UV range from 200 to 400 nm, had similar shape
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Figure 5. Absorption spectra of atenolol (2) and its degradation products (1, 3) after incubation for 60 mins in 0.1s mol/L hydrochloric acid at 90°C.
and characteristic inflexion within the range of 200–225 nm, and had absorption maximum at wavelength approximately 276 nm. On the basis of the obtained results and the literature,[14] one can assume that the acid hydrolysis product is an amide of p-hydroxyphenylacetic acid. The comparative 1H NMR analysis, as described above, was carried out for acebutolol and product of its degradation (Rf = 0.40) which has similar to the original substance absorption spectrum in UV (Figure 6). Degradation product's spectrum shows lack of proton signal of the butyric acyl group, whereas signal of free amine group appears, indicating hydrolysis of amide bond. This change in the structure is also confirmed by strong upfield shift of aromatic protons signals indicating appearance of highly electron donor moiety connected to aromatic ring. Apart from this multiplicity and integral values of aromatic protons, signals remain unchanged, which shows that there are no changes in a way of substitution of the aromatic system. The aliphatic part of spectrum strictly corresponds to the acebutolol alkaline hydrolysis product showing that this fragment of a molecule undergoes the same change—dehydration with forming a double bond toward nitrogen atom. The identified product of hydrolysis is 3-acetylo-4-[3(1-isopropylamino)-2-propenoxy] aniline. The formation of a free group connected with aromatic ring suggests that the degradation process brings also another product: butyric acid. There were no spots corresponding to this acid in chromatograms, although there was an intensive smell of free butyric acid in hydrolyzates. For propranol that is less susceptible to degradation than other drugs, it was impossible to isolate a degradation product at a sufficient amount to perform NMR analysis; thus, no identification of this product was made. Absorption spectra registered for propranolol and its
Figure 6. Absorption spectra of acebutolol (2) and its degradation product (1) after incubation for 60 mins in 1 mol/Ls hydrochloric acid at 90°C.
Figure 7. Absorption spectra of propranolol (1) and its degradation product (2) after incubation for 150 mins in 0.3 mol/ L hydrochloric acid at 90°C.
degradation product are characterized by maxima of absorbance at 220 and 285 nm and have similar shape within 250–340 nm, which indicates that the structure of degradation product is similar to propranolol (Figure 7).
DISCUSSION An investigation of the stability of atenolol, acebutolol, and propranolol in hydrochloric acid solution has indicated that acid concentration, temperature, and incubation time have an influence on the degradation of drugs under consideration. The changes of degradation products' concentration are proportional to concentration variations of active substances of analyzed drugs and depend on the type of drug. Under established degradation conditions for individual drug, the fixed number of degradation products of similar Rf values regardless of the increasing concentration
Atenolol, Acebutolol, and Propranolol in Acidic Environment
of hydrochloric acid, temperature and incubation time are observed. The analyzed drugs have different logP values, ranging from –0.83 for atenolol, −0.40 for acebutolol, and 1.16 for propranolol.[23] Kinetic analysis has indicated that hydrolytic degradation of the selected drugs proceeds as a reaction of pseudofirst order. In relation to logP values, the computed rate constants k decrease from the greatest values for atenolol, smaller for acebutolol, and the smallest one for propranolol. A reverse relationship has been obtained for calculated values of t0.1 and t0.5, increasing for drug sequence mentioned above, thus enabling to conclude that propranolol shows the highest stability, acebutolol lower, and atenolol the lowest one. The changes of energy of activation with an increasing tendency from atenolol to propranolol confirm that the structure of propranolol remains unchanged for longer time than those of acebutolol and atenolol. The parameters k, t0.5, and t0.1 change for all examined drugs depending on the temperature, hydrochloric acid concentration, and incubation time, contrary to the energy of activation that depends on the type of drug under investigation. It was found that atenolol was more susceptible to degradation than acebutolol, while minor degradation of propranolol was observed. The identified products of reactions in acidic environment appear to be of similar structure to those described in the literature.[14, 15, 18] For atenolol, contrary to acebutolol, in addition to the product of dehydration, the presence of p-hydroxyphenylacetic amide formed in acid hydrolysis of ether bond was observed. Such different behavior of acebutolol is shown by lack of de-alkylation results probably from an effect of acetyl substitute in ortho position to the ether bond. As a general conclusion drawn from the presented analysis, one can state that hydrophilic atenolol is more susceptible to degradation than less hydrophilic acebutolol and the most lipophilic propranolol. The rate constants k describing degradation of compounds mentioned above decrease with increasing degree of lipophilic properties, whereas t0.1, t0.5, and Ea show an increasing tendency from atenolol to propranolol, as observed also for logP values.
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