Technique for Quantitative Estimation of the Pharmaceutical Compounds - Essay Example

?Literature-based study on chromatographic and/or spectroscopic-based analysis of a case-study counterfeit drug: Viagra Pharmaceutical counterfeitingis becoming a major issue in the world now. Counterfeiting affects the consumers, drug manufacturers, health care providers and the governments. These counterfeit drugs may or may not contain the active ingredients, harmful substances and create a potential threat to the patients. The pharmaceutical manufacturers Association (phRMA) has stated that counterfeiting is becoming a major health threat in the developing countries because of the weak regulatory systems and the poor intellectual property acts in these countries. phRMA has requested China, Mexico, Indonesia, Lebanon and many countries in the Central America to have a close watch on the drugs used in their countries. They have also warned those countries that the illegal drugs were found to get originated from Czech Republic, Sweden and Switzerland. (Oslen et al., 2002).The Drug manufacturer take many necessary steps to prevent counterfeiting of the drugs but their efforts are getting veined off because of the different methods that they adopt in trade and their distribution systems are very complex for tracing off. The illegal drug manufacturers are very smart enough to steal the name, basic formula, holograms and are able to prepare the counterfeit drug very smartly as the real drug. This has become a head ache for all the drug manufacturers. The only possibility for them is to develop analytical methods that can easily identify the authentic and fake products through simple testing methods. Sildenafil Citrate is a drug that is used to treat the pulmonary arterial hypertension and erectile dysfunction. Its Brand Name is Viagra, developed and marketed by Pfizer.the chemical composition of Sildenafil citrate is 1-( [3- ( 6, 7- dihydromethyl -7- oxo -3- propyl -1H – pyrazolo [4,3 - d] pyrimidin – 5 – yl ) - 4- ethoxy – phenyl ] sulfonyl ])- 4 – methyl piperazine. Viagra is used as a novel oral medicine for penile erectile dysfunction, the inability to keep hard erect penis for satisfactory sexual intercourse. Sildenafil citrate inhibits the cyclic guanosine monophosphate- specific phosphodiesterase present at the penis and making the cGMP to get accumulated at the corpus cavemonsum. This pill when taken inside, starts its effect after the twenty minutes of inhalation and lasts up to three hals lifes ( 18 hours ). Viagra came in to the market by 1998 after the US Food and Drug Administration approved it. Viagra was recommended for Erectile dysfunction as the over the counter drug. By the year 2002, Viagra sales accounted for about 92% of the global market for the pill to treat erectile dysfunction. The safety of this medicine was established in many pre- and post- approval studies. Phosphodiesterase family type five classes.(PDE). The PDE - 5 class receptors are found in corpus cavernoscum, platelets, skeletal muscle, vascular and visceral muscles. Of these at penis there are larger amount of PDE-5 than any other region. PDE-5 breaks down cGMP which causes the contraction of the penile arteries and smooth muscles. As the chemical structure of sildenafil is similar to the cGMP, the binding of sildenafil inhibits the cGMP from binding to the PDE-5 through the competitive inhibition mechanism. (McCullough, 2002). Amphetamine is a phenlyethylamine class drug that acts as a psycho stimulant drug. This drug is generally used as a performance enhancer. Amphetamine is found to increase the energy level, wakefulness and they are able to make the body work even at appetite and fatigue conditions. Because of these characteristics Amphetamine is chosen as a counterifeit drug for Viagra by the illegal drug manufacturers. In the counterfeit Viagra’s, Sildenafil was present at lower concentration and the other drugs such as amphetamine, methamphetamine and ring substituted analogs. A rapid screening technique is essential for the quick identification of the counterfeit drug from the real ones. Various methods were used for the analysis of sildenafil citrate in the pills. Some of them include spectroscopic method, chromatographic methods, resonance ray light scattering and ion-selective electrode. Recently the Raman spectroscopy and Fourier Transform Infrared Spectrodscopy (FTIR) are also used. FTIR is the quantitative method that is used for the determination and structural analysis of Sildenafil citrate. FTIR is a rapid screening method used for analyzing the powders and tablets and giving the indications about their batches in both mixed state and homogeneity states. A more rapid, sensitive, robust and accurate precise method is required for the analysis of sildenafil citrate. (Amin, 2009). Near Infra-Red spectroscopy is one among them. In NIR both the qualitative and the quantitative analysis are possible. NIR can analyze the physical and chemical nature of the sample constituents and it can also be performed rapidly. NIR can detect the solvents, active ingredients potency, identification of the tablet and the level of capsule tampering occurred in the tablets. First A standard spectrum was established using the original sample of Sildenafil citrate. These were done based on the age of the tablet. The tablet was powdered and dropped into the glass vial and the sample was scanned with the 16 nm light. Then the spectral comparisons were done for the sample with the standard ones. The graphs will help us to identify the fake ones from the real ones. This is a very rapid technique with sampling time of 1 min/sample and a very sensitive technique. (Oslen et al., 2002). Another technique used for the analysis of the samples was the High Temperature Liquid Chromatography using the thermal gradient. In this technique, the sildenafil citrate (Pfizer Viagra) and the fake sample were analyses using HTLC. Both were supposed to have 100 mg active ingredient. In this technique, the pills were powdered and extracted with 50:50 methanol: water sample by sonicating it for 20 minutes. The samples were then centrifuged at 300 rpm for 15 minutes and the supernatant was collected. HTLC containing selerity blaze C8 column, using 35:65 acetonitrile : water with 0.1% TFA as mobile phase and temperature ranging from 40?C to 100?C can be used. The flow rate must be adjusted to 1 ml/min and 5 µl of sample is used for the analysis. The two samples were analyzed 220 nm. (Tseng and Lin, 2002). The concentration of Sildenafil citrate in both the tablets can be calculated from the area of the peak obtained. By comparing the areas of the two samples we can calculate the concentration of the active ingredient. The main advantage of this technique is that the active ingredient does not get degraded. (Tseng and Lin 2002). High performance Liquid Chromatography can be used to detect the presence and concentration of Sildenafil citrate in the tablets. This is a very rapid and very sensitive technique. A monolithic silica column chromatography with reverse phase HPLC technique is performed with UV detection. With monolithic Silica column as the stationary phase and the isocratic solution of 60:40 acetonitrile: water solvent as mobile phase, the analysis was carried out at 292 nm with a flow rate of 2ml/min. (Aboul-Enein and Hefnawy, 2003). The tablets were powdered and extracted with the mobile phase. The sample was then sonicated for 15 min and centrifuged at 3000 rpm for 10 minutes. 20 µl of the sample was injected into the column. This type of HPLC is more specific and more sensitive to the sildenafil citrate in the solution. This is a very rapid technique. (Aboul-Enein and Hefnawy, 2003). A rapid analysis method was determined by Daraghmeh et al. (2001) using HPLC. They used µBondapak C 18 column with ammonium acetate and acetonitrile in the ratio of 1:1 at a pH of 7. The samples were analyzed at 240 nm. The flow rate was 1 ml / min. (Daraghmeh et al., 2001).The main advantage of this technique was its stability and the resolution property among the sildenafil citrate related compounds. Even when slight changes in the mobile phase ratio, detection wave length, sonication, flow rate and filtration time did not affect the HPLC. This method can also be used for assay and for determining the impurities that are present in the tablets at the solid form. It is very specific, accurate, robust, precise and has very good reproducibility (Daraghmeh et al., 2001). UV Spectroscopic method can also be used to determine the sildenafil citrate concentration in the counterfeit drugs. The spectroscopic absorbance of the sample can be found at 292 nm. The absorbance values obtained in this experiment can be compared with the results of the FIA and concentration of the sample can be found. Higher mean and lower standard deviation percentage (RSD %) and better clearance values (CL) can be found using spectro photometric methods. (Mahmoudian, 2005). Thin Layer Chromatography method can also be used to determine the presence of sildenafil citrate in the given tablet. The sample is dissolved in the solvents and applied to the silica gel 60 F254plates. The lower layer containing chloroform/Ammonia solution/ methanol in the ratio of 15:3:2. (Mahmoudian, 2005). The developing solvent was found to contain chloroform/diethyl amine/ methanol in the ratio of 15:3:2. (Mahmoudian, 2005). The component was allowed to separate and then the spots were analyzed in the UV radiation at 254 nm. The identified spots were sprayed with the Dragendorff’s reagent. The concentration of the dark blue spots in the plate indicates the presence of sildenafil. This method can be used to find out the concentration of sildenafil up to 45 milli grams per tablet. (Mahmoudian, 2005). Flow injection analysis can also be used to determine the concentration of sildenafil citrate concentration in the given pill. The counterfeit pill will contain less amount of this active ingredient than the real one. This variation in the concentration of sildenafil citrate can be found by FIA. The solvent that is used to dissolve the activ3e ingredient is 0.2M phosphate buffer at a pH of 8 containing 10% Methanol. (Altiokka et al., 2001). The flow rate for the solvent was adjusted to 1 ml/min and the sample was viewed under 292 nm. (Altiokka et al., 2001).The limit of detection and the limit of quantification was found for the given sample. These results were then compared with the results obtained from the UV- Spectrophotometry. It was found that the results were in good agreement between FIA and UV – Spectrophotometry, (Altiokka et al., 2001). The determination of the toxicologically active ingredient in the drugs can be done very conveniently using the mass spectrophotometric approach. Elecrto spray ionization method mass spectrometry was used in positive as well as in negative ion mode for the quadrupole- quadrupole- time of flight instrument. In this method the dissolved samples were injected using the flow injection method in mass spectrophotometer. Two results are obtained from this experiment. Mass spectrum and Tandem Mass spectrum. (Pavlic et al., 2010).These data were then fed into the mass spectral library. The active ingredient of the sample, their concentration can be easily found by this experiment. The ionization polarities are the most important things for the determination of the samples. These samples can also be sent for further verification to the GC/MS. Thus by using these techniques, the presence of the active ingredient sildenafil citrate in the pill can be easily identified. (Pavlic et al., 2010). Ion associate extraction spectroscopy is another newer technique for quantitative estimation of the pharmaceutical compounds. The formation of the ion- sensitive complexes between the organic compounds and the dyes is used for the detection of the compounds in the given sample. The intensity of color will give us the idea about the concentration of the sample. For the estimation of sildenafil citrate, Congo Red, Sudan II, Gentian Violet are used as chromogenic reagents. (Amin et al., 2009). The Sildenafil citrate is dissolved in methanol and used for the analysis. The absorptions were done using the UV spectrophotometer at a wavelength of 400nm/min. for this experiments, the pH range of 2-12 can be used. Similarly acetate, borate, phosphate and universal buffer solutions can be used for the estimation of Sildenafil citrate. (Amin et al., 2009). Apart from the above mentioned techniques Capillary Electrophoresis, Gas Chromatography – Fourier transformed infrared spectroscopy (GC-FTIR), Nuclear magnetic resonance spectroscopy (both qualitative and quantitative), Quantitative TLC and Solid phase- micro extraction- gas chromatography, CE-MS and LC-MS are the other techniques that can be used for the analysis of sildenafil citrate.(United Nations, 2006). References: Aboul-Enein, HY and Hefnawy, MM, 2003, “Rapid Determination of Sildenafil Citrate in Pharmaceutical Preparations Using Monolithic Silica HPLC Column”, Journal Of Liquid Chromatography & Related Technologies, Vol. 26, No. 17, pp. 2897–2908. Altiokka, G., Atkosar, Z., Sener, E., and Tuncel, M. 2001, “FIA of sildenafil citrate using UV-detection”, Journal of Pharmaceutical and Biomedical Analysis, Vol.25, No.2 339-342. Amin, AS., Moustafa, ME.,El-Dosoky, R., 2009, “Colorimetric determination of sildenafil citrate (viagra) through ion-associate complex formation”, Journal of AOAC International, Vol.92, No.1, 125- 130 Daraghmeh, N., Al-Omari, M., Badwan, AA and Jaber,AM., 2001, “Determination of sildenafil citrate and related substances in the commercial products and tablet dosage form using HPLC”, Journal of Pharmaceutical and Biomedical Analysis, Vol.25, 483-92 Mahmoudian, M 2005, “Sildenafil Determination in Various Matrices: A Review”, Iranian Journal Of Pharmacology & Therapeutics, vol.4, no.2, 72-75. McCullough, AR., 2002,” Four-Year Review of Sildenafil Citrate”, Reviews in Urology, Vol.4, S26-S38 Oslen, BA., Borer, MW., Perry, FM. and Forbes, RA, 2002, “Screening for Counterfeit Drugs Using Near-Infrared Spectroscopy”, Pharmaceutical technology, Vol. 26, No.6 , 62-71. Pavlic M., Schubert M., Libiseller, K., and Oberacher, H., 2010, “Comprehensive identification of active compounds in tablets by flow-injection data-dependent tandem mass spectrometry combined with library search”, Forensic Science International, Vol.197, 40-7 Tseng, MC, and Lin, JH, 2002, “Determination of sildenafil citrate adulterated in a dietary supplement capsule by LC/MS/MS”, Journal of Food and Drug Analysis .Vol. 10, 112-119. United Nations, 2006, “Recommended methods for the identification and analysis of amphetamine, methamphetamine and their ring-substituted analogues in seized materials”, United Nations office on Drugs and Crime, United Nations Publications. Read More