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2003 The Japan Society for Analytical Chemistry Separation and Determination of Synthetic Impurities of
Sildenafil (Viagra) by Reversed-Phase High-Performance
Liquid Chromatography
Velupula NAGARAJU,*† Dasari SREENATH,** Jammula TIRUMALA RAO,**
and Ramisetti NAGESWARA RAO***
*Chemistry Section, A. P. State Forensic Science Laboratory, Red Hills, Hyderabad-500 004, India **Department of Chemistry, Dr. Hari Singh Gour University, Sagar-470 003, India ***Analytical Chemistry Division, Indian Institute of Chemical Technology, A simple and rapid high-performance liquid chromatographic method for the separation and determination of process-related impurities of sildenafil was developed. The separation was achieved on a reversed-phase C18 column usingacetonitrile–0.05 M potassium dihydrogen orthophosphate (70:30 v/v) as a mobile solvent at a flow rate of 1.0 ml/min andUV detection at 230 nm. The method was used not only for quality assurance, but also for monitoring the chemicalreactions during the synthesis of sildenafil. It was found to be specific, precise and reliable for the determination of allprocess-related impurities of sildenafil in bulk drugs and formulations.
(Received January 6, 2003; Accepted April 9, 2003)
reaction products, unreacted raw materials, and intermediates, Introduction
since they may possess unwanted toxicological effects due towhich the benefit from administration of SLD may be Sildenafil,1-{[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H- outweighed. Therefore, a close monitoring of related pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]}-4- substances is of great importance for controlling the quality of methylpiperazine (SLD), popularly known as Viagra, is a novel SLD in the final products. A thorough literature search has oral agent for the treatment of penile erectile dysfuntion, which indicated that the voltammetric behavior of SLD using square- consists of an inability to achieve or maintain a hard, erect penis wave and adsorptive stripping techniques in pharmaceutical sufficient for sexual intercourse.1,2 It is an active inhibitor of the type V-cyclic guanosine monophosphate (cGMP)-specific method using UV detection for the determination of SLD in phosphodiesterase on penile erectile activity, and causes cGMP pharmaceutical preparations was reported.11 methods were found not to be suitable for differentiating the involving pharmacokinetic, efficacy and safety evaluations have related substances from SLD due to a lack of selectivity. Thus, revealed that SLD is an effective and well-tolerated drug in the HPLC is the technique of choice for the separation and treatment of male erectile dysfuntion.7 Its synthesis8 involves determination of related impurities in pharmaceutical several steps, in which 4-amino 1-methyl-3-propylpyrazole-5- preparations. A few methods based on HPLC were reported for carboxamide (AMP) is initially obtained from 1-methyl-4-nitro- the determination of sildenafil citrate in biological and 3-propylpyrazole-5-carboxamide (MNC) by reduction with pharmaceutical products. A reversed-phase HPLC method SnCl2 under refluxed conditions. It is then condensed with 2- using acetonitrile–phosphate buffer–water (28:4:68 v/v/v) with ethoxybenzoyl chloride and oxidized with H2O2 to yield 5-(2- detection at 230 nm was utilized for the simultaneous ethoxyphenyl)-1-methyl-3-propyl-6,7-dihydro-1H-pyrazolo- determination of sildenafil and its metabolite (UK-103, 320) [4,3-d]pyrimidin-7-one (EMP), which is then sulfonated with using the automated sequential trace enrichment of dialysates.12 chlorosulfonic acid to give 5-[5-(chlorosulfonyl)-2- This method was found to be selective and precise, but not ethoxyphenyl]-1-methyl-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]- stable, thus indicating that most of the impurities have short pyrimidon-7-one (CMP). In the last step, CMP is reacted with retention times close to that of the solvent front, and not methyl piperazine to produce sildenafil (SLD), which is resolved from each other. Segall et al. have proposed a generally converted into citrate and used for treatment without reversed-phase HPLC method using 70 mM potassium further purification.9 Since it is intended for oral consumption, phosphate monobasic buffer of pH 3.0 containing 100 mM its purity and safety can be thoroughly ensured before using it in triethylamine:acetonitrile (7:3 v/v) as the mobile phase at 225 different formulations. Its quality depends not only on the nm for the separation and determination of the degradation adopted procedures, but also on the synthetic precursors, side products of sildenafil citrate formed due to oxidation.13 Dineshet al. developed another reversed-phase HPLC method for the † To whom correspondence should be addressed.
determination of SLD in pure and pharmaceutical forms using a Effect of the concentration of acetonitrile on the retention of chromatographic and integrated data were recorded using an Reactions involved in the synthesis of sildenafil.
HP-Vectra (Hewlett Packard, Waldron, Germany) computersystem.
Lichrospher C18 column with water–acetonitrile as the mobile The mobile phase was acetonitrile–0.05 M potassium phase and UV detection at 245 nm.14 A reversed-phase HPLC dihydrogenphosphate (70:30 v/v); before delivering into the method for the determination of its related substances in system it was filtered through 0.45 µm PTFE filter and degassed commercial formulations and tablets was reported.15 However, using a vacuum. The analysis was carried out under isocratic it did not attempt to separate 1-methyl-4-nitro-3- conditions using a flow rate of 1.0 ml/min at room temperature propylpyrazole-5-carboxylic acid (MNP), which is most likely (28˚C). Chromatograms were recorded at 230 nm using an to be present as an impurity in the final products of SLD. In this paper we describe a simple and rapid reversed-phase HPLCmethod for the separation and determination of small amounts of all probable impurities of SLD, not only for quality Samples (5 mg) were dissolved in the mobile phase (10 ml) assurance, but also to monitor the procedures followed during and a 20 µl volume of each sample was injected and chromatographed under the above conditions. Syntheticmixtures containing CMP, MNP, AMP, MNC, SLD, EMP andbulk drugs were analyzed under identical conditions. The Experimental
amounts of impurities were calculated from their respectivepeak areas.
Analytical reagent-grade potassium dihydrogen orthophosphate (E. Merck, Mumbai, India) and HPLC-grade Results and Discussion
acetonitrile obtained from Qualigens, Mumbai, India was used.
Glass-distilled and deionized water (Nanopure, Barnsted, USA) Figure 1 shows the chemical reactions generally followed in the was used throughout the study. Sildenafil and its synthetic synthesis of SLD in a bulk drug manufacturing unit. It can be impurities were synthesized by D. S. in his laboratory seen from Fig. 1 that there are as many as six compounds, department of chemistry (Dr. Hari Singh Gour University, which include the starting materials and intermediates, that could be present as potential impurities in SLD. The presentstudy was aimed at developing a chromatographic system capable of eluting and resolving SLD and its impurities An HPLC system was composed of two LC-10 AT VP originating from synthesis. In a preliminary experiment, all of pumps, an SPD-M 10A VP diode array detector, an SIL-10AD these impurities and SLD were subjected to separation by VP auto injector, a DGU 12 A degasser and an SCL-10 VP system controller (all from Shimadzu, Kyoto, Japan). A acetonitrile–water as an eluent. Two compounds viz., EMP and reversed-phase C18 (YMC, Kyoto, Japan) column (25 cm × 4.6 SLD were retained on the column when the concentration of mm i.d., particle size 5 µm) was used for separation. The acetonitrile was kept below 40%. However, upon increasing its Retention and response data for SLD and potential CMP, 5-[5-(chlorosulfonyl)-2-ethoxyphenyl]-1-methyl-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-7-one; MNP, 1-methyl-4-nitro-3-propylpyrazole-5-carboxylic acid; AMP, 4-amino-1-methyl-3-propylpyrazole-5-carboxamide; MNC, 1-methyl-4-nitro-3-propylpyrazole-5-carboxamide; SLD, sildenafil; EMP, 5-(2-ethoxyphenyl)-1-methyl-3-propyl-6,7-dihydro-1H-pyrazolo-[4,3-d]pyrimidin-7-one.
a. RRT: relative retention time.
b. RRF: relative response factor.
n = 6.
Recovery data for standard mixtures containing CMP, Typical chromatogram of a synthetic mixture containing (1) CMP (5 µg), (2) MNP (5 µg), (3) AMP (5 µg), (4) MNC (5 µg), (5)SLD (5 µg) and (6) EMP (5 µg).
concentration, these two compounds were eluted, but the separation of the other compounds was found to be effected.
This would most probably be due to the adsorption of these twocompounds by exposed silinols on C column. This behavior of SLD and EMP is reasonable becauseboth compounds have basic functional groups with pKa valuesof 8.7 and 8.0 with a weak acidic moiety on the parent determined on 10 replicate injections of SLD solution, and compound. In another attempt, the water was replaced by 0.05 reported as the relative standard deviation (RSD), 0.89%. The M potassium dihydrogenphosphate, and the effect of the signal-to-noise ratio was determined to be 4.0 for the detection concentration of the organic modifier viz., acetonitrile upon of impurities as low as 0.02 × 10–9 g. It can be seen from Table separation was studied (Fig. 2). When the concentration of 2 that the measured amounts agree well with the actual values; acetonitrile was at 70%, all of the impurities and SLD were the mean recovery of SLD from authentic samples was found to eluted and separated from one another. The typical be 99.75 ± 0.25%. The UV detector was set at 230 nm for both chromatogram of a synthetic mixture containing SLD and its detection and quantification. The wavelength was selected impurities is shown in Fig. 3. The peaks were identified by based on observations that the resolution between the injecting and comparing with the retention times of the chromatographic peaks of SLD and its synthetic impurities were individual compounds. It can be seen from Fig. 3 that SLD was well separated from all of the impurities examined in the present A bulk drug of SLD was spiked with low-level impurities and study, and that these impurities were also well separated from chromatographed. The chromatogram is shown in Fig. 4. The each other. The specificity of the method was checked by high-level (75% – 120%) linearity of SLD as well as the low- subjecting the bulk drug under UV light at 254 nm and to stress level (0.05% – 0.5%) linearity data of impurities were conditions, like 0.1 M HCl, 0.1 M NaOH and 3% H2O2 determined, and are recorded in Table 3. Good linearity was solutions at 60˚C and for 24 h. All of the degraded products found between the mass and the integral response for each of were found to be well-separated from SLD, and did not interfere the compounds under examination. Table 3 gives the linearity with any of the process impurities, indicating that the method is equation, mass range and correlation coefficients for all of the quite specific. The chromatographic data, including the compounds. At 0.001 A.U.F.S., the limit of detection (LOD) of retention times (tR), retention factors (k), number of theoretical SLD was 6.00 × 10–9 g; the impurities are recorded in Table 3.
plates (N), tailing factors (Tf), relative response factors (RRF) The robustness of the method was evaluated by a deliberate and wavelength of absorption maxima (λmax), are given in Table slight variation of the parameters, such as the mobile-phase composition, strength of potassium dihydrogenphosphate, A synthetic mixture containing small quantities of impurities temperature, flow rate and wavelength of absorption for viz., MNP, MNC, AMP, CMP, EMP and SLD, was prepared detection. No significant change was observed in the and chromatographed to check that these quantities were chromatographic results of SLD and its impurities, even after accurately reflected in their peak areas. All of the estimations changing the experimental parameters.
were carried out thrice and the percentage of error was The quality of SLD in bulk drugs was thoroughly checked. A calculated (Table 2). The precision of the method was typical chromatogram (T0h) of a bulk drug of SLD is shown in Linear-regression analysis data of SLD and its related impurities HPLC determination of potential process impurities in Fig. 5. The amounts of various impurities were determined, andthe purity of SLD was calculated. The results are recorded inTable 4. To determine the stability of SLD in the mobile phase,the drug was stored in the mobile phase for 24 h andchromatographed on the following day. The chromatogram isshown in Fig. 5. It can be seen from the chromatograms of Fig.
5, T0h and T24h that no significant change was observed. Fromthese results, it is clear that the method is precise and accuratefor the separation and determination of small quantities of some Typical chromatogram of (5) SLD (20 µg) spiked with low- of the process impurities that are generally present in SLD.
level impurities containing (1) CMP (0.5 µg), (2) MNP (0.5 µg), (3)AMP (0.5 µg), (4) MNC (0.5 µg) and (6) EMP (0.5 µg).
Conclusion
A robust and sensitive HPLC procedure has been developed forthe rapid determination of SLD and its synthetic impurities viz.,MNP, MNC, AMP, CMP and EMP. The developed HPLCmethod is suitable not only for the separation and determinationof process impurities, but also for monitoring the syntheticprocess of SLD. The method is thus suitable for the processdevelopment and quality assurance of SLD and relatedproducts.
Acknowledgements
One of the authors (V.N.) wishes to thank Dr. K. P. C. Gandhi,Inspector General of Police (PSS) and Director, A. P. StateForensic Science Laboratory, Hyderabad and also Dr. K. V.
Raghavan, Director, Indian Institute of Chemical Technology,Hyderabad, for their encouragement and support.
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