The Development and Validation of Novel, Simple High-Performance Liquid Chromatographic Method with Refractive Index Detector for Quantification of Memantine Hydrochloride in Dissolution Samples.

The present study was aimed to develop an analytical method for quantification of memantine (MEM) hydrochloride in dissolution samples using high-performance liquid chromatography with refractive index (RI) detector. The chromatographic separation was achieved on C18 (250 × 4.5 mm, 5 µm) column using isocratic mobile phase comprises of buffer (pH 5.2):methanol (40:60 v/v) pumped at a flow rate of 1.0 mL/min. The column effluents were monitored using RI detector. The retention time of MEM was found to be ~6.5 ± 0.3 min. The developed chromatographic method was validated and found to be linear over the concentration range of 5.0-45.0 µg/mL for MEM. Mean recovery of MEM was found to be 99.2 ± 0.5% (w/w). The method was found to be simple, fast, precise and accurate, which can be utilized for the quantification of MEM in dissolution samples.

A novel nanoproliposomes of lercanidipine: Development, in vitro and preclinical studies to support its effectiveness in hypertension therapy.

AIM: The aim of the present study was to develop nanoproliposomes of lercanidipine, in order to overcome its poor biopharmaceutical properties and to improve its therapeutic efficacy in treating hypertension. MAIN METHODS: The nanoproliposomes were prepared using a modified thin-film hydration method, and the formula was optimized by varying the ratio of lipids and the types of cryoprotectants. This optimized formulation was characterized in terms of its particle size, solid-state, drug release, in-situ absorption, in-vivo pharmacokinetics, and in-vivo anti-hypertensive activity in DOCA-salt induced hypertensive rats. Finally, a PK-PD correlation was established in order to understand the clinical implications of the developed novel nanoproliposomes. KEY FINDINGS: The nanoproliposomes showed a particle size of 174.7nm and an entrapment efficiency of 85.4%. The in-vitro release displayed initial rapid release (19.33%) followed by a sustained release profile, releasing 88.37% of the encapsulated drug. The in-situ studies showed a significant increase in absorption rate across the rat intestinal membrane. The pharmacokinetics of this novel form indicated a 2.75-fold increase in the absolute bioavailability as compared to pure lercanidipine. In addition, the nanoproliposomes were found to be efficient in treating hypertension in DOCA-salt induced hypertensive rats. The PK-PD correlation demonstrated no time lag between effect and exposure, indicating that a direct PK-PD relationship can be expected in the clinic. SIGNIFICANCE: These findings suggest that nanoproliposomes are promising carriers in improving the oral bioavailability and bioactivity of lercanidipine, and can be an effective therapy in the management of hypertension.

Development and evaluation of sunscreen creams containing morin-encapsulated nanoparticles for enhanced UV radiation protection and antioxidant activity.

The objective of present work was to develop novel sunscreen creams containing polymeric nanoparticles (NPs) of morin. Polymeric NPs containing morin were prepared and optimized. The creams containing morin NPs were also prepared and evaluated. Optimized NPs exhibited particle size of 90.6 nm and zeta potential of -31 mV. The entrapment efficiency of morin, within the polymeric NPs, was found to be low (12.27%). Fourier transformed infrared spectroscopy and differential scanning calorimetry studies revealed no interaction between morin and excipients. Transmission electron microscopy and atomic force microscopy revealed that the NPs were spherical in shape with approximately 100 nm diameter. Optimized NPs showed excellent in vitro free radical scavenging activity. Skin permeation and deposition of morin from its NPs was higher than its plain form. Different sunscreen creams (SC1-SC8) were formulated by incorporating morin NPs along with nano zinc oxide and nano titanium dioxide. SC5 and SC8 creams showed excellent sun protection factor values (≈40). In vitro and in vivo skin permeation studies of sunscreen creams containing morin NPs indicated excellent deposition of morin within the skin. Morin NPs and optimized cream formulations (SC5 and SC8) did not exhibit cytotoxicity in Vero and HaCaT cells. Optimized sunscreen creams showed excellent dermal safety. SC5 and SC8 creams demonstrated exceptional in vivo antioxidant effect (estimation of catalase, superoxide dismutase, and glutathione) in UV radiation-exposed rats. The optimized sunscreen creams confirmed outstanding UV radiation protection as well as antioxidant properties.

Simultaneous improvement of solubility and permeability by fabricating binary glassy materials of Talinolol with Naringin: Solid state characterization, in-vivo in-situ evaluation.

The aim of the current study was to prepare binary amorphous forms of Talinolol (TLN) by using Naringin (NRG) as a stabilizing agent. The secondary objective of this study was to study the effect of P-gp inhibitor NRG on the P-gp probe drug TLN. The binary amorphous samples were prepared by quench cooling technique in the molar ratios TLN:NRG (1:1), TLN:NRG (1:2), TLN:NRG (2:1). The prepared samples were characterized by DSC, FTIR and XRD. Amorphicity of the prepared binary amorphous samples was confirmed by spotting diffuse halo in the diffractograms and further corroborated by detecting glass transition event (Tg) in the thermograms of the respective samples. The Tgs for all prepared systems were found above room temperature, the highest being 45.43 °C. The systems were found physically stable at 25 °C and 40 °C at dry conditions for 60 days. The temperature stability of prepared amorphous forms may be attributed to strong intermolecular hydrogen bond interaction between TLN and NRG, which was confirmed by Gordon-Taylor calculations and FTIR data. The solubility of TLN in amorphous form was increased by approximately 9-fold as compared to its crystalline counterpart. The in-vivo bioavailability study conducted on wistar rats demonstrated 5.4-fold increase in the AUC0-t value for TLN as compared to its crystalline counterpart. Further to learn the contribution of P-gp inhibition by NRG on the permeability of TLN, In-vitro single pass perfusion studies were conducted on the ileum of wistar rats. The permeability of TLN in rat ileum in the presence of NRG was significantly increased to 3.16×10(-5) cm/s as compare to control value 2.48×10(-5) cm/s. The current study demonstrated the ability of binary amorphous technology to simultaneously overcome both the BCS barriers i.e. solubility and permeability.

Aqueous penetration of orally and topically administered moxifloxacin.

AIM: To assess the intraocular penetration of 0.5% moxifloxacin hydrochloride into aqueous humour after oral and topical administration. METHODS: A prospective, interventional study of 42 patients scheduled to undergo cataract surgery was carried out. Out of the 42 subjects, 21 were randomly categorised into Group I and received one drop of 0.5% topical moxifloxacin four times, at 15 min intervals starting 75 min before the surgery. Another 21 subjects were categorised into Group II and all subjects in this group were administered a single tablet of 400 mg of moxifloxacin, 12 h before the surgery. Estimation of moxifloxacin in aqueous samples was carried out using high-performance liquid chromatography. Results were analysed using Student unpaired 't' test and analysis of variance. The value of p<0.05 was considered to be significant. RESULTS: Mean aqueous concentration of moxifloxacin attained in the oral group (n=21) was 0.504±0.30 µg/mL while that in the topical group (n=21) was 2.04±0.72 µg/mL, and this difference in levels was statistically significant (p<0.005). The levels attained by both the groups well exceeded the MIC90 (minimum inhibitory concentration of antibiotic required to inhibit growth of 90% of bacteria strains) levels for most of the organisms causing endophthalmitis. Penetration of moxifloxacin in aqueous in both the groups was not affected by gender, intraocular pressure or comorbidities significantly. However, aqueous levels were found to be higher among the younger subjects within the topical group. CONCLUSIONS: Moxifloxacin has an impressive spectrum of coverage and this pharmacokinetic study reinforces its potential as a prophylactic drug against intraocular infections, given the high aqueous levels post topical administration.

Fabrication, solid state characterization and bioavailability assessment of stable binary amorphous phases of Ritonavir with Quercetin.

In the current study, Quercetin (QRT) was characterized for thermodynamic and kinetic parameters and found as an excellent glass former. QRT was paired with Ritonavir (RTV) (BCS class-IV antiretroviral) to form stable amorphous form and pharmacologically relevant combination. Binary amorphous forms of RTV and QRT in molar ratios 1:1, 1:2 and 2:1 were prepared by solvent evaporation technique and characterized by XRPD, DSC and FTIR. The prepared binary phases were found to become amorphous after solvent evaporation which was confirmed by disappearance of crystalline peaks from X-ray diffractograms and detecting single Tg in DSC studies. The physical stability studies at 40 °C for 90 days found RTV:QRT 1:2 and RTV:QRT 2:1 phases stable, while trace crystallinity was detected for 1:1M ratio. The temperature stability of RTV:QRT 1:2 and RTV:QRT 2:1 amorphous forms can be attributed to phase solubility of both components where the drug in excess acts as a crystallization inhibitor. Except for RTV:QRT 1:2 ratio, there was no evidence of intermolecular interactions between two components. Almost 5 fold increase in the saturation solubility was achieved for RTV, compared to crystalline counterpart. While for QRT, the solubility advantage was not achieved. In vivo oral bioavailability study was conducted for 1:2 binary amorphous form by using pure RTV as a control. Cmax was improved by 1.26 fold and Tmax was decreased by 2h after comparing with control indicating improved absorption. However no significant enhancement of oral bioavailability (1.12 fold after comparing with control) was found for RTV.

Preparation and characterization of co-amorphous Ritonavir-Indomethacin systems by solvent evaporation technique: improved dissolution behavior and physical stability without evidence of intermolecular interactions.

The aim of this study was to stabilize the amorphous form of Ritonavir (RTV) a BCS class-II drug with known amorphous stabilizing small molecule Indomethacin (IND) by co-amorphous technology. The co-amorphous samples were prepared by solvent evaporation technique in the molar ratios RTV:IND (2:1), RTV:IND (1:1), RTV:IND (1:2) and their amorphous nature was confirmed by XRPD, DSC and FT-IR. Physical stability studies were carried out at temp 25°C and 40°C for maximum up to 90 days under dry conditions. Solubility and dissolution testing were carried out to investigate the dissolution advantage of prepared co-amorphous systems. The amorphous mixtures of all tested molar ratios were found to become amorphous after solvent evaporation. The same was confirmed by detecting halo pattern in diffractograms of co-amorphous mixtures. The Tg values of all three systems were found to be more than 40°C, the highest being 51.88°C for RTV:IND (2:1) system. Theoretical Tg values were calculated by Gordon-Taylor equation. Insignificant deviation of theoretical Tg values from that of practical one, corroborated by FT-IR studies showed no evidence of intermolecular interactions between RTV and IND. Almost 3-folds increase in the solubility for both amorphous RTV and IND was found as compared to their respective crystalline counterparts. The study demonstrated significant increase in the dissolution rate as well as increase in the total amount of drug dissolved for amorphous RTV, however it failed to demonstrate any significant improvement in the dissolution behavior of IND.

Development and Validation of Liquid Chromatographic Method for Estimation of Naringin in Nanoformulation.

A simple, precise, accurate, rapid, and sensitive reverse phase high performance liquid chromatography (RP-HPLC) method with UV detection has been developed and validated for quantification of naringin (NAR) in novel pharmaceutical formulation. NAR is a polyphenolic flavonoid present in most of the citrus plants having variety of pharmacological activities. Method optimization was carried out by considering the various parameters such as effect of pH and column. The analyte was separated by employing a C18 (250.0 × 4.6 mm, 5 µm) column at ambient temperature in isocratic conditions using phosphate buffer pH 3.5: acetonitrile (75 : 25% v/v) as mobile phase pumped at a flow rate of 1.0 mL/min. UV detection was carried out at 282 nm. The developed method was validated according to ICH guidelines Q2(R1). The method was found to be precise and accurate on statistical evaluation with a linearity range of 0.1 to 20.0 µg/mL for NAR. The intra- and interday precision studies showed good reproducibility with coefficients of variation (CV) less than 1.0%. The mean recovery of NAR was found to be 99.33 ± 0.16%. The proposed method was found to be highly accurate, sensitive, and robust. The proposed liquid chromatographic method was successfully employed for the routine analysis of said compound in developed novel nanopharmaceuticals. The presence of excipients did not show any interference on the determination of NAR, indicating method specificity.

Osmotically controlled pulsatile release capsule of montelukast sodium for chronotherapy: statistical optimization, in vitro and in vivo evaluation.

The purpose of present study was to design, optimize and evaluate osmotically controlled pulsatile release capsule (PRC) of montelukast sodium (MKS) for the prevention of episodic attack of asthma in early morning and associated allergic rhinitis. Assembly of the capsular systems consisted of push, active and plug tablet arranged from bottom to top in hard gelatin capsule. The capsule system was coated with a semi-permeable membrane of cellulose acetate and drilled towards plug side in cap. A three-factor, three-level central composite design (CCD) with α = 1 was introduced to execute the experiments and quadratic polynomial model was generated to predict and assess the independent variables with respect to the dependent variables. The composition of optimal formulation was determined as weight of push tablet 138 mg (coded value: +0.59), plug tablet 60 mg (coded value: +0.49) and coating weight gain of 8.4 mg (coded value: -0.82). The results showed that the optimal formulation of PRCs had lag time of 4.5 h, release at 6 and 12 h are 61.95% and 96.29%, respectively. The X-ray radiographic imaging study was carried out to monitor the in vivo behavior of developed barium sulfate-loaded PRCs in rabbits under fasting conditions. In vivo pharmacokinetic study revealed Tmax of 2 h for marketed tablets; however 7 h for PRCs with initial lag time of 4 h. Thus designed capsular system may be helpful for patients with episodic attack of asthma in early morning and associated allergic rhinitis.

Informed consent: Issues and challenges.

Informed consent is an ethical and legal requirement for research involving human participants. It is the process where a participant is informed about all aspects of the trial, which are important for the participant to make a decision and after studying all aspects of the trial the participant voluntarily confirms his or her willingness to participate in a particular clinical trial and significance of the research for advancement of medical knowledge and social welfare. The concept of informed consent is embedded in the principles of Nuremberg Code, The Declaration of Helsinki and The Belmont Report. Informed consent is an inevitable requirement prior to every research involving human being as subjects for study. Obtaining consent involves informing the subject about his or her rights, the purpose of the study, procedures to be undertaken, potential risks and benefits of participation, expected duration of study, extent of confidentiality of personal identification and demographic data, so that the participation of subjects in the study is entirely voluntary. This article provides an overview of issues in informed consent: The obligations of investigator, sponsor and Institutional Review Board to protect rights and welfare of human research subjects. It discusses about the basic elements of informed consent and the process to be followed while obtaining informed consent. Some of the circumstances under which informed consent can be waived and ethical challenges faced by physicians in obtaining informed consent from subjects are also highlighted in this article.

Development and validation of RP-HPLC method with ultraviolet detection for estimation of montelukast in rabbit plasma: Application to preclinical pharmacokinetics.

OBJECTIVE: To develop a liquid-liquid extraction based reverse phase liquid chromatography method for estimation of montelukast in rabbit plasma. METHODS: Chromatographic separation was carried out using Phenomenex Luna C18 column (250 mm × 4.6 mm × 5 µm) with mobile phase composed of ammonium acetate buffer (20 Mm), pH 5.5 and acetonitrile in 20:80, v/v ratio. The analyte was monitored with UV detector at 345 nm. The developed method was validated with respect to linearity, accuracy, precision, specificity and stability. RESULTS: The peak area ratio of montelukast (MKS) to that of internal standard was used for the quantification of samples. Calibration curves were linear in the concentration range of 20-2000 ng mL(-1). The LOD and LLOQ of present method were found out to be 10 ng mL(-1) and 20 ng mL(-1) respectively. The intra-day and inter-day %CV values for MKS were below 6.06% and 8.43%. Intra-day and inter-day accuracies were within 95.81% and 110.90%, respectively. Extraction recoveries of drug from rabbit plasma were >66.47%. CONCLUSION: A simple, alternative, reproducible and sensitive HPLC-UV method was developed for MKS that can be used in preclinical pharmacokinetics.

High-performance liquid chromatographic assay for ziprasidone in plasma samples: application to pharmacokinetic studies in rats.

Ziprasidone (ZRS) is among the various antipsychotic drugs indicated for treating schizophrenia. The determination of the pharmacokinetic behavior of this drug is of utmost importance in evaluating its bioavailability. The objectives of the present study are: (1) to develop and validate a sensitive, specific, accurate and precise reverse-phase high performance liquid chromatographic method for quantification of ZRS in the plasma of rats; and (2) to apply the developed method to study the pharmacokinetic profile of ZRS in rats after oral administration. The method uses a C18 (250.0 × 4.6 mm, 5 µm) column and ultraviolet detector with wavelength set at 210.0 nm. The mobile phase is acetonitrile-phosphate buffer (pH 3.6) 28:72% v/v at a flow rate of 1.0 mL/min. The internal standard (IS) is escitalopram. The extraction procedure for ZRS and IS from the biological matrix (plasma) employs liquid-liquid extraction technique using a mixture of methyl tert-butyl ether-dichloromethane (70:30% v/v). The results show good accuracy and precision over a linearity range of 20.0-3,000.0 ng/mL with r(2) ≥ 0.9986. The mean recoveries of ZRS and IS are 79.32 ± 1.16 and 84.10 ± 3.2%, respectively. The method has been successfully utilized to study the pharmacokinetic profile of ZRS in rats after oral administration.

Novel liquid chromatographic method for simultaneous estimation of pioglitazone and glimepiride in rat plasma by solid phase extraction: application to preclinical pharmacokinetic studies.

The need for a reliable bioanalytical method is of primary importance during preclinical studies. The aim of the present study was simultaneous determination of pioglitazone (CAS 111025-46-8) (PIO) and glimepiride (CAS 93479-97-1) (GLM) in plasma of rats. A high-performance liquid chromatographic method has been developed and validated using C18 column and UV detector. A mobile phase composed of acetonitrile and ammonium acetate buffer pH 4.5 in the ratio of 55:45%. The plasma samples clean-up was carried out using solid phase cartridges. The method was in the linear range of 50-8000 ng/mL for PIO and 50-2000 ng/mL for GLM. The coefficient of regression was found to be > or = 0.99. Precision and accuracy were within the acceptable limits, as indicated by relative standard deviation varying from 1.5 to 6.1% for PIO and 3.1 to 7.0% for GLM whereas the accuracy ranged from 97.0 to 106.4% for PIO and 96.5 to 106.4% for GLM. The mean extraction recovery was found to be 90.2 +/- 4.5, 76.8 +/- 2.8 and 85.2 +/- 5.2% for PIO, GLM and internal standard, respectively. Moreover, PIO and GLM were stable in plasma, up to 30 days of storage at -70 degrees C and after being subjected to bench top, auto-sampler, and three freeze-thaw cycles. The developed method was applied for preclinical pharmacokinetic studies.

Validated HPLC method for quantitative determination of talinolol in rat plasma and application to a preclinical pharmacokinetic study.

BACKGROUND: A simple HPLC-UV method with a high reproducibility and sensitivity for the determination of talinolol in rat plasma was developed in this study. METHOD: After liquid-liquid extraction, the compounds were separated on a Vydac(®) C18 monomeric column (250 × 4.6 mm inner diameter × 5-µm particle size) using a mobile phase composed of acetonitrile and potassium dihydrogen phosphate buffer (34:66 v/v), delivered isocratically at a flow rate of 1.0 ml min(-1). Escitalopram was used as an internal standard. The chromatographic peak-area ratio, based on UV absorbency at 245 nm, was used for quantitative analysis. RESULTS: Calibration standards with concentrations over the range of 10-1000 ng ml(-1) were validated for routine sample analysis to support pharmacokinetic studies with talinolol in rats. The intra- and inter-day precision studies showed good reproducibility with coefficients of variation of less than 11.49%. The developed method is simpler and more sensitive than previously reported methods. DISCUSSION: The analytical sensitivity and accuracy of this assay were adequate for characterization of talinolol in rat plasma and the assay has been applied successfully to the in vivo kinetic study of talinolol in rats. After talinolol (10 mg kg(-1)) was given orally, the maximum concentration and the AUC(0-∞) were 341.8 ± 99.4 ng ml(-1) and 976.26 ± 173.37 ng h ml(-1), respectively. The oral bioavailability was approximately 52.14 ± 9.26%. CONCLUSION: The advantages of our method are a small sample volume (200 µl), short analysis time (13.5 min) and a simple sample extraction and clean-up compared with multiple extraction and washing steps and a longer analysis time in previously published methods.

Enhanced oral absorption of saquinavir with Methyl-Beta-Cyclodextrin-Preparation and in vitro and in vivo evaluation.

Saquinavir (SQV) is a weak base compound, whose solubility is strongly influenced by pH variations. Thus, in the present work, we thought it worthy of interest to investigate in-depth the combined effect of pH control and cyclodextrin (CyD) complexation on SQV solubilization. Phase-solubility studies were performed by adding excess drug to buffered (pH from 1.1 to 7.4) aqueous solutions containing increasing concentrations of Methyl-Beta-CyD (M-ß-CyD) in order to evaluate the role of the unionized species of SQV in improving solubility by CyD complexation and to be able to select the most suitable conditions for optimizing drug solubilization. Our study reveals that the integrated approach of pH adjustment and CyD complexation can be successfully used for improving the CyD solubilizing power towards an ionizable drug such as SQV, thus allowing a smaller quantity of CyD to solubilize a given amount of drug, offering clear economic and technologic advantages as well. When biopharmaceutics of the optimized cyclodextrin-based formulation of SQV was studied in Wistar rats after intravenous and oral administrations, we found that inclusion of SQV into M-ß-CyD could dramatically improve its oral bioavailability and decrease the variation of its oral pharmacokinetics. Compared to the control, the presence of M-ß-CyD significantly increased the area under the plasma concentration-time curve (439.7±161.35 to 2312.03±159.53, p<0.01) and the peak plasma concentration (117.24±35.77 to 1347.88±276.76, p<0.01) of orally administered SQV. The modulating effect of M-ß-CyD on the bidirectional transport of SQV was also investigated using a modified Ussing chamber system. The results demonstrated that the enhancing effect of M-ß-CyD on the oral bioavailability of SQV is due not only to its solubilizing effect on SQV but also, at least in part, to the inhibitory effect of M-ß-CyD on the P-glycoprotein (P-gp) mediated efflux of SQV in the gastrointestinal tract. The present results suggest that M-ß-CyD is particularly useful in designing oral preparations of SQV with an enhanced bioavailability and a reduced variability in absorption.

Preparation, in vitro characterization, pharmacokinetic, and pharmacodynamic evaluation of chitosan-based plumbagin microspheres in mice bearing B16F1 melanoma.

The present study was aimed to evaluate the anti-tumor efficacy and systemic toxicity of chitosan-based plumbagin microspheres in comparison to free plumbagin. The optimized formulation had a mean particle size of 106.35 mum with an encapsulation efficiency of 80.12%. Pharmacokinetic studies showed a 22.2-fold increase in elimination half-life (t(1/2)) of plumbagin from chitosan microspheres as compared to free plumbagin. Administration of plumbagin microspheres resulted in a significant tumor growth inhibition and reduced systemic toxicity. These results suggest that chitosan-based microspheres could be a promising strategy for the systemic delivery of anti-cancer agents like plumbagin.

High-performance liquid chromatography and pharmacokinetics of aceclofenac in rats.

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for quantification of aceclofenac in rat plasma. Ibuprofen was used as an internal standard (IS). The present method used protein precipitation for extraction of aceclofenac from rat plasma. Separation was carried out on reversed-phase C(18) column (250 mm x 4.6 mm, 5 micro) and the column effluent was monitored by UV detector at 282 nm. The mobile phase used was methanol-triethylamine (pH 7.0; 0.3% v/v in Milli-Q water) (60:40%, v/v) at a flow rate of 1.0 mL min(-1). This method was linear over the range of 50.0-3500.0 ng mL(-1) with regression coefficient greater than 0.99. The mean recovery of aceclofenac and IS were 84.62+/-3.23 and 89.19+/-1.57%, respectively and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for pharmacokinetic study of aceclofenac in rats.

Bioequivalence study of sildenafil citrate tablets in healthy human volunteers.

Newly developed sildenafil citrate (SC), a selective inhibitor of cyclic guanosine monophosphate (c-GMP) specific phosphodiesterase type 5 (PDE 5) in the corpus cavernosum is used for the oral treatment of erectile dysfunction. A convenient, sensitive and simple method for the determination of sildenafil in human plasma is presented. The analytical technique was based on reversed-phase high-performance liquid chromatography coupled with UV detector set at 295 nm. Rofecoxib was used as internal standard (I.S). Liquid-liquid extraction using diethyl ether was performed to recover sildenafil and rofecoxib. The retention time of I.S and sildenafil were 5.5 minutes and 7.2 minutes respectively. The method was validated over a linear range of 10 to 1000 ng/ml from plasma. Separate stability study showed that sildenafil is stable under conditions of analysis. The extraction efficiency from plasma varied from 79.69% to 81.13 %. The minimum quantifiable concentration was set at 10 ng/ml. (%o CV<12.5%). The method was used for Bioequivalence Study of Two Brands of Sildenafil citrate 50 mg tablets in healthy human volunteers. All pharmacokinetic parameter were calculated along with statistical evaluation.