Determination of Nisoldipine and Its Impurities in Pharmaceuticals
2004, 60, 223–227
D. Agbaba1,&, K. Vucicevic1, V. Marinkovic2 1 Faculty of Pharmacy, Institute of Pharmaceutical Chemistry and Drug Analysis, P.O. Box 146, 11000 Belgrade, Serbia and Montenegro; E-Mail:
[email protected] 2 A.D. Zdravlje, Pharmaceutical and Chemical Industry, 16000 Leskovac, Serbia and Montenegro
Received: 6 October 2003 / Revised: 2 March 2004 / Accepted: 22 March 2004 Online publication: 9 July 2004
Abstract A method has been established for separation of nisoldipine and impurities, for example reactants, products of side-reactions, and photodegradation products, by HPTLC on LiChrospher Si 60 F254s plates with detection at 280 nm. The mobile phase, cyclohexane–ethyl acetate–toluene, 7.5:7.5:10 (v/v), enables acceptable resolution of nisoldipine, in large excess, and possible impurities. Regression coefficients (r 0.997), recovery (98–108%), and determination limit (0.02–0.2%) were validated and found to be satisfactory. The method is convenient for quantitative analysis and purity control of nisoldipine in its raw material and dosage forms.
Keywords Thin-layer chromatography Nisoldipine and impurities Pharmaceutical preparations
Introduction Nisoldipine (3-isobutyl-5-methyl-1,4dihydro-2,6-dimethyl-4-(2-nitrophenyl) pyridine-3,5-dicarboxylate) is a calciumchannel blocker with vasodilator properties. It is used in several commercial preparations for treatment of hypertension [1]. Nisoldipine has been the subject of many analytical chemical investigations, including crystal structure elucidation [2, 3] and its determination as the main component in dosage formulations by UV [4], polarography [5], voltametry [6], HPLC [7–9], and TLC [10]. Because nisoldipine is light-sensitive, previously published papers deal mostly with
Original DOI: 10.1365/s10337-004-0335-4 0009-5893/04/08
stability studies, kinetics of degradation, and determination of nisoldipine and its photodegradation products by use of different analytical techniques, for example UV [11–13], polarography [14], HPLC [15–17], and GC [18, 19]. 1,4-Dihydropyridine derivatives with non-identical ester functions have been synthesized by Michael’s cyclocondensation reaction between methyl-3-amino crotonate (I) and 2-isopropyl-2-(2-nitrobenzilidene) acetoacetate (II) [20, 21]. Inappropriate synthetic conditions also result in side-reactions and formation of 1,4-dihydropyridines with identical ester functions, for example 1,4-dihydro2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (IV)
and 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid diisopropyl ester (V). On exposure to daylight degradation of nisoldipine can occur, forming the nitrosophenylpyridine analog (VI). Nifedipine, a product of a side-reaction during synthesis of nisoldipine, is even more sensitive to daylight than nisoldipine, and gives its nitrosophenylpyridine analog (VII). A schematic diagram of the synthesis of nisoldipine and potential degradation products is given in Fig. 1. Regulations on the purity profile of bulk drug substances require determination of levels of impurities such as reactants and by-products. The literature on nisoldipine contains no chromatographic or other analytical method for simultaneous identification and determination of reactants (impurities I and II), by-products (impurity V), or degradation products (impurity VI). In most pharmacopeias thin-layer chromatography and high-performance liquid chromatography are recommended as official techniques for purity testing; the former is usually used semi-quantitatively and the latter for quantitative analysis. Because instrumental planar chromatography is regarded as a simple, rapid, accurate, and precise technique we have developed a qualitative–quantitative method for simultaneous identification and determination of impurities arising from the synthesis of nisoldipine and possible degradation products arising as a result of the photolability of nisoldipine.
Chromatographia 2004, 60, August (No. 3/4) 2004 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
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bia and Montenegro). Nizoldin 5-mg and 10-mg tablets were obtained from Slaviamed (Belgrade, Serbia and Montenegro). All solvents were of analytical grade.
Solutions Standard Solutions
Stock solutions (0.5 mg mL)1) of nisoldipine standard substance and of impurities II, IV, and VI were prepared in methanol. Preparation of Standard Curve
Nisoldipine calibration solutions containing 0.05–0.5 mg mL)1 were prepared by diluting the stock solution. Calibration solutions containing impurities were prepared by diluting the stock solutions to furnish solutions containing 0.01– 0.1 mg mL)1 for II and IV, and 0.005– 0.05 mg mL)1 for VI. These solutions were applied to the HPTLC plates. Fig. 1. Synthesis and degradation of nisoldipine and its derivatives
Sample Preparation
Nisoldipine raw material (100 mg) was dissolved in methanol (10 mL) and this solution was applied to the plate for assay of impurities. For nisoldipine assay 0.5 mL of the sample solution was diluted to 200 mL and this solution was applied to the plate.
Chromatography
Fig. 2. Densitograms obtained from a test mixture of nisoldipine (III) and impurities I, II, IV, VI, and VII
Experimental Apparatus TLC plates (LiChrospher Si 60 F254s, 20 cm · 10 cm) were purchased from Merck (Darmstadt, Germany). A Nanomat III (Camag, Muttenz, Switzerland) was used as sample-application device. A TLC Scanner II with computer system and CATS software (version 3.18) was also obtained from Camag.
Reagents 1,4-Dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid methyl
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2-methylpropyl ester (nisoldipine, III),1,4dihydro-2,6-dimethyl-4-(2-nitrophenyl)3,5-pyridinecarboxylic acid dimethyl ester (nifedipine, IV), methyl 2-methylpropyl 2,6-dimethyl-4-(2-nitrosophenyl) pyridine-3,5-dicarboxylate (nitrosophenylpyridine analog nisoldipine, VI), and dimethyl-2,6-dimethyl-4-(2-nitrosophenyl)pyridine-3,5-dicarboxylate (nitrosophenylpyridine analog nifedipine, VII) were obtained from Promed (Praha, Czech Republic). Methyl-3-aminocrotonate (I) and 2-isopropyl-2-(2-nitrobenzylidene) acetoacetate (II), analytical grade, were obtained from Rosh Chemie (Germany). Different samples of nisoldipine raw material (A, B, C, and D) were synthesized by Zdravlje (Leskovac, Ser-
Standard and sample solutions (3 lL) were spotted on the plates. Ascending chromatography was performed with cyclohexane–ethyl acetate–toluene, 7.5 + 7.5 + 10 (v/v), as mobile phase in a twin-trough TLC chamber previously saturated for 15 min with mobile phase vapor. The development time was 20 min. The plate was dried in air and spots were detected under UV light at 254 nm. The chromatogram was scanned at 280 nm with a Camag II TLC Scanner in reflectance/absorbance mode. Each plate could accommodate eighteen spots. All procedures including preparation of standard and sample solutions and development of chromatograms were performed with protection from daylight.
Results and Discussion Optimization of conditions for simple, rapid, and reproducible analysis involves
Chromatographia 2004, 60, August (No. 3/4)
Original
selection of the appropriate stationary or mobile phases. To obtain satisfactory resolution and to avoid peak tailing, optimization was performed with different mobile phases. With cyclohexane– ethyl acetate, 5:5 (v/v), peaks of III and impurities I, IV, VI, and VII were not sufficiently well separated to enable quantitative analysis. On adding toluene to reduce the polarity of the mobile phase all the compounds were separated. The best resolution was obtained with cyclohexane–ethyl acetate–toluene, 7.5:7.5:10 (v/v). The effect of different stationary phases on the separation was investigated by use of conventional TLC, HPTLC, and LiChrospher Si 60 F254s plates. The best resolution, sharpest zones for all the substances, and best peak area/peak height response were obtained by use of LiChrospher Si 60 F254s plates. Calculated peak symmetry (A0.05) values for II, III, IV, VI, and VII were 1.1, 0.92, 0.9, 1.0, and 1.12, respectively, and resolution factors (RS) for IV and III, VII and VI, VI and II, and III and VI were 1.0, 1.54, 1.75, and 3.12, respectively. Scanned profiles of HPTLC chromatograms illustrating separation of nisoldipine from its possible impurities are shown in Fig. 2. These chromatograms, which include all reactants, sidereaction products, and products of degradation in UV light and daylight, show the method can be used to monitor the stability of nisoldipine and the process used for synthesis. The migration distances of nisoldipine and impurities I, II, IV, VI, and VII were 35.6, 57.8, 61.6, 30.7, 51.8, and 44.4 mm, respectively. The spectra of the substances were recorded and are shown in Fig. 3. All quantitative measurements were performed at the wavelength maximum 280 nm. The relationship between peak area and the amount of substance applied was evaluated by use of linear and second degree polynomial regression functions. Fitting with a second-degree polynomial was evaluated because a wider concentration range is required for quantification of an impurity in a purity method. Regression coefficients are summarized in Table 1. The effect of larger amounts of the drug on the peak shape and resolution of the impurities must be determined to avoid systematic errors. The accuracy of method was therefore proved by determination of impurities II and IV in the presence of nisoldipine. A solution of Original
Fig. 3. In situ UV spectra of nisoldipine (III) and impurities I, II, IV, VI, and VII Table 1. Statistical data for calibration curves—calibration function y = a + bx + cx2 Substance
n ng/spot
a
Nisoldipine Impurity VI Impurity IV Impurity II
5 5 5 5
11.6 )34.7 28.5 7.65
15–150 15–150 30–300 30–300
b ± ± ± ±
c
SD
r
14.03 3.76 ± 0.58 0.003 ± 0.005 3.0 0.999 14.67 13.16 ± 0.52 )0.039 ± 0.0034 11.07 0.999 19.6 3.3 ± 0.3 )0.002 ± 0.0007 14.78 0.999 5.84 6.58 ± 0.92 )0.002 ± 0.003 44.06 0.997
Fig. 4. Densitograms obtained from a reference sample of nisoldipine (a), 30 and 300 ng standards of impurities II and IV (c and e, respectively), and a sample of nisoldipine spiked with 0.1% and 1% of impurities II and IV (b and d, respectively)
nisoldipine (1 mg mL)1) containing no detectable amounts of impurities was spiked with impurities II and IV at concentrations of 0.001 and 0.01 mg mL)1, respectively (corresponding to 0.1% and 1%). Scanned profiles obtained from the spiked samples of nisoldipine are presented in Fig. 4. Calculated recoveries were plotted against expected values (the corresponding to standards without nisoldipine). Recoveries and relative standard deviations (RSD) for impurities I and IV were acceptable for a purity method (Table 2). Chromatographia 2004, 60, August (No. 3/4)
Table 2. Recovery rates for different levels of impurities Impurity
Amount added (ng)
IV
625 ng* 97.8 62.5 ng** 105.3 600 ng* 100.9 60 ng** 107.9
II
Recovery RSD (%) (%) 6.1 6.8 2.0 1.6
*Equivalent to an impurity level of 1% **Equivalent to an impurity level of 0.1%
Method accuracy was determined by recovery of nisoldipine from a laboratory-prepared mixture of excipients
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Table 3. Accuracy of the method Nisoldipine RSD Amount (ng) (%)
Impurity VI RSD Amount (ng) (%)
Impurity IV Amount (ng)
RSD (%)
Impurity II Amount (ng)
RSD (%)
150 105 75
300 150 60
625 312 63
2.0 1.2 3.4
604 302 60.5
1.3 3.5 1.8
2.5 2.6 3.1
1.7 0.9 2.2
Table 4. Assay of nisoldipine and its impurities Sample
Nisoldipine (% ± RSD)
Impurity IV (% ± RSD)
Impurity II (% ± RSD)
Impurity VI (% ± RSD)
Nizoldin 5 mg Nizoldin 10 mg A B C D
103.55 101.13 87.16 96.53 99.91 98.43
– – 1.05 0.18 0.25 0.17
– – 0.99 0.51 0.27 0.09
