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The HPLC method for the simultaneous determination of 2 PDE-5 (sildenafil and tadalafil) in herbal medicines was validated. Using acetonitrile and water as the mobile phase, Gemini C18 (250 x 4.6 nm, 5 µm) column, UV detection at 285 nm and flow at 1 ml/min, the PDE-5 were separated satisfactorily within 6 minutes,
Journal of military pharmaco-medicine 7-2013 SIMULTANEOUS DETERMINATION OF SILDENAFIL AND TADALAFIL IN HERBAL MEDICINES BY HPLC Nguyen Thi Hao*; Nguyen Van Bach*; Nguyen Van Thinh* Dao Van Don*; Pham Van Hinh*; Nguyen Thi Thanh Phuong** Summary The HPLC method for the simultaneous determination of PDE-5 (sildenafil and tadalafil) in herbal medicines was validated Using acetonitrile and water as the mobile phase, Gemini C18 (250 x 4.6 nm, µm) column, UV detection at 285 nm and flow at ml/min, the PDE-5 were separated satisfactorily within minutes The quantitation limits of sildenafil citrate and tadalafil were about 0.2 g/ml and 0.3 g/ml, respectively The calibration curve of each PDE-5 had a correlation coefficient close to The precisions were all less than 2% The recovery rates of extraction were 92 - 100% for all PDE-5 This method can be used to detemine sildenafil citrate and tadalafil simultaneously in herbal medicines * Key words: PDE-5; Sildenafil citrat; Tadalafil; HPLC; Herbal medicines INTRODUCTION Sildenafil citrate, 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3d]pyrimidin-5-yl)phenylsulfonyl]-4 methylpiperazine, is primarily indicated in the treatment of erectile dysfunction It is rapidly absorbed after oral administration, with absolute bioavailability of about 40% It is rapidly and extensively metabolized in the liver to the active N-desmethyl sildenafil metabolite Under steady state conditions, the plasma concentrations of N-desmethyl sildenafil were approximately 40% of those measured for sildenafil Tadalafil, (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-[3,4-(methylenedioxy) phenyl] pyrazino[1’,2’:1,6]pyrido[3,4-b]indole-1,4-dione [1] is a newly approved oral selective phosphodiesterase-5 (PDE5) inhibitor indicated for the treatment of erectile dysfunction [1, 2, 3, 4] The mechanism of action of tadalafil is similar to the other PDE5 inhibitors, sildenafil Through the inhibition on PDE5, tadalafil increases the concentrations of cyclic guanosine monophosphate (cGMP), producing smooth muscle relaxation and increased blood flow to the corpus cavernosum, thereby enhancing erectile response following appropriate sexual stimulation Because of its increasing popularity and potential side effects, the need for a procedure to detect both sildenafil and tadalafil in biological samples is becoming increasingly important The simultaneous determination of sildenafil and tadalafil is also necessary for pharmacokinetic and related studies The present work demonstrates that significant improvement in sensitivity was achieved by coupling HPLC with electrochemical detection The simultaneous detection and determination of both analytes was facilitated by the use of two internal standards namely, roxithromycin and clarithromycin, and the use of a one-step liquid–liquid extraction The developed method was validated for its application to a pharmacokinetic evaluation of sildenafil and N-desmethyl sildenafil in 25 healthy human volunteers MATERIALS AND METHODS Journal of military pharmaco-medicine 7-2013 Materials * Experimental subjects: - Blank sample: prepared by the extracts of herbals: 10 g of Epimedium grandiflorum; 10 g of Morinda officinalis; 10 g of Cortex eucommiae; 10 g of Semen cuscutae; 10 g of Cibotium barometz; 10 g of Radix dipsaci and 16 g of Herba cistanches - Test sample: prepare by blank sample added mg/g of SC and TA * Chemicals and reagents: - Standards: Sildenafil and tadalafil were purchased from Vietnam National Institute of Drug Control - Acetonitrile (Merck), water for HPLC * Analytical equipment: - HPLC system: HPLC Alliance Waters 2695D system, channel solvent, pump autosampler, column heating chamber, PDA detector, Gemini C18 column (250 x 4,6 mm, àm) Empower đ Software - Others: Sartorius analytical balance with precision of 0.1 mg, ultrasonic apparatus, glass flasks, precise pipettes Method * Sample preparation and chromatography: - Sample preparation: By the results reported [1, 2, 3], preparation procedures were conducted as follows: Accurately weight g of sample in 50 ml beaker Extract three times with 10 ml of acetonitrile Ultrasonic shaking for 15 minutes at 300C Filter the extract by filter paper permeated with MeOH into 50 ml flask Combine the extracts into the flask Shake and dilute to volume of flask The solution was filtered by 0,45 m membrane for HPLC injection - Chromatographic conditions: Gemini C18 (250 x 4,6 mm, µm) column, PDA detector, Analyses were run at a flowrate of ml/min, injection volume of 50 µl Investigation of mobile phase was carried out with MeOH/ H2O and ACN /H2O * Validation of the analytical procedures: The validations for the following analytical procedures are included: System suitability testing, selectivity - specificity, calibration curve and linearity, precision, accuracy, limit of detection, lower limit of quantification Journal of military pharmaco-medicine 7-2013 RESULTS AND DISCUSSION Chromatographic conditions Sample preparation an chromatographic conditions was performed according to 2.2.1 The HPLC method was achieved on a Gemini C18 (250 x 4,6 mm, µm) column, PDA detector; ACN:H2O (60:40, v/v) as mobile phase; flow rate of ml/min; injection volume of 50 µl Fig showed chromatogram of sildenafil and tadalafil t (phút) Fig 1: The HPLC chromatograms of sildenafil and tadalafil Validation of the analytical procedures * System suitability testing: System suitability testing was examined in six independent samples consisting of SC and TA at concentraction of 50 µg/ml Results was showned in table Table 1: Results of system suitability testing of SC and TA TA No SC tR (min) Spic (μV.s) tR (min) Spic (μV.s) 4.537 486233 5.421 1356987 4.540 479921 5.433 1375009 4.521 480234 5.445 1380855 4.539 485289 5.428 1386043 4.528 478981 5.439 1369309 4.532 489907 5.458 1368990 X 4.533 483427.5 5.44 1372866 SD 0.0074 4372.306 0.0130 10216.34 RSD (%) 0.16 0.90 0.24 0.74 As can be seen in table 1, the good system suitability was achieved under the chromatographic conditions The relative standard deviation (RSD) of retention time and peak area Journal of military pharmaco-medicine 7-2013 of TA and SC were below 1% showing that the HPLC method can be applied to analyze samples with reliable results * Selectivity - specificity: Analyze mixtured standard samples of PR, BE, TR and DA at concentration of µg/ml with test sample and blank samples Chromatograms were shown in fig TA SC a b c TA SC Fig 2: Chromatogram of SC and TA in mixed standard sample (a), blank sample (b) and test sample (c) TA also displayed appropriate chromatographic retention with its peak sufficiently separated from that of SC in the present method The peaks of the analytes in the test sample were symmetrical and completely separate from each other, showing that the HPLC method has good selectivity and specificity * Calibration curve and linearity: Prepare a series of standard solutions of PR, BE, TR and DA at concentrations ranging from - 100 µg/ml and subject to HPLC Results were shown in table and figure Table 2: Results of the linear correlation between concentration and peak area of SC and TA Journal of military pharmaco-medicine 7-2013 C (µg/ml) (TA) 1.04 2.08 5.2 10.4 20.8 52 104 Speak (TA) (μV.s) 9076 17759 46380 91145 186085 486095 934635 C (µg/ml) (SC) 1.02 2.04 5.1 10.2 20.4 51 102 Speak (SC) (μV.s) 28799 57968 105945 268540 546850 1358095 2670189 y(TA) = 9411x + 268 ; R = 0.9996 Calibration curve Equation y(SC) = 26808x - 525 ; R = 0.9998 S (µV.S) C (µg/ml) Figure 3: The calibration curve of SC and TA As can be seen from the table 2, peak area and concentration of PR, BE, TR and DA are correlated with R2 ≈ * Limit of detection (LOD) and lower limit of quantitation (LOQ): Based on the baseline noise level of blank and test samples, LOD values of TA SC were 0.1 µg/ml; 0.05 µg/ml, respectively LOQ of SC and TA were 0.3 µg/ml and 0.2 µg/ml, respectively * Precision: Weigh accurately g of test sample containing 0.2% of mixed standards Precision was measured using six determinations at concentration of 0.2% The results were showned in table Table 3: Results of precision of the HPLC method (n = 6) Content of TA added Content of TA found Content of SC added Content of SC found (%w/w) (%w/w) (%w/w) (%w/w) 0.21 0.196 0.19 0.180 0.21 0.198 0.19 0.183 0.21 0.201 0.19 0.188 STT Journal of military pharmaco-medicine 7-2013 0.21 0.199 0.19 0.184 0.21 0.200 0.19 0.187 0.21 0.194 0.19 0.179 X 0.198 0.184 SD 0.0026 0.0036 RSD(%) 1.31 1.96 The relative standard deviations were less than 3%, suggesting that the method have good repeatability * Accuracy: Weigh accurately g of the sample with six independent determinations Add 400 µl of mixture of standards at concentration of 1000 µg/ml Tables showed the results of accuracy of the method Table 4: Precision for the determination of SC and TA Sample no Amount added (µg) Amount found (µg) Recovery (%) 400 390 97.5 400 389 97.25 400 370 92.5 400 387 96.75 400 379 94.75 400 385 96.25 400 384 96.0 400 372 93.0 400 386 96.5 400 392 98.0 400 384 96.0 400 393 98.25 TA SC X = 95.83% SD = 1.90 RSD = 1.98% X = 96.29% SD = 1.89 RSD = 1.96% The mean recovery of TA was 95.83% (n = 6), and that of SC was 96.29% The high recoveries from sample preparation with RSD < indicated that the method offers good accuracy CONCLUSION Journal of military pharmaco-medicine 7-2013 A method which employed high-performance liquid chromatography coupled with PDA detector was developed for the simultaneous determination of sildenafil and taladafil The method was developed and validated for purposes of analysis in herbal medicines High precision and accuracy were demonstrated A limit of quantitation (LOQ) of 0.3 µg/ml was achieved for sildenafil whereas a LOQ of 0.3 µg/ml was obtained for tadalafil The mobile phase consisted of ACN:H2O (60:40, v/v) as mobile phase The column was a Gemini C18 (250 x 4.6 mm, µm) column The method is suitable in analysis of sildenafil and tadalafil in herbal medicines REFERENCES Phan Thanh Mau Study on determination of sidenafil spiked in functional foods (LC-MS HPLC) Pharmacy undergraduate dissertation Hanoi University of Pharmacy 2011 Hassan Y Aboul-Enein and Mohamed M Hefnawy RapiddDetermination of sildenafil citrate in pharmaceutical preparations using monolithic silica HPLC column Journal of Liquid Chromatography and Related Technologies 2003, Vol 26, No 17, pp.2897-2908 Samuel R.Gartz PhD E.D drugs and analogs: Characterization of phosphodiesterase-5 Inhibitors in suspect couterfeit and herbal products USP Annual Scientific Meeting 2007 ... g of test sample containing 0.2% of mixed standards Precision was measured using six determinations at concentration of 0.2% The results were showned in table Table 3: Results of precision of. .. suitability testing: System suitability testing was examined in six independent samples consisting of SC and TA at concentraction of 50 µg/ml Results was showned in table Table 1: Results of system...Journal of military pharmaco-medicine 7-2013 Materials * Experimental subjects: - Blank sample: prepared by the extracts of herbals: 10 g of Epimedium grandiflorum; 10 g of Morinda officinalis;