determination of tadalafil in pure powder and tablet dosage

DRUG FORMULATIONS AND CLINICAL METHODS

Determination of Tadalafil in Pure Powder and Tablet DosageForm by High-Performance Liquid Chromatography

TUSHAR G. BAROT and POPATBHAI K. PATEL

M.G. Sciences Institute, Department of Chemistry, Ahmedabad Gujarat, India

A simple and accurate method to determinetadalafil (TAD) in pure powder and tablet dosageform was developed and validated using HPLC.The separation was achieved on an Xterra RP18column (150 ´ 4.6 mm id, 3.5 mm) in the isocraticmode using buffer–acetonitrile (70 + 30, v/v),adjusted to pH 7.00 ± 0.05 with triethylamine as themobile phase at a flow rate of 1.0 mL/min. Thephotodiode array detector was set at 225 nm.Quantification was achieved over theconcentration range of 50.7–152.10 mg/mL withmean recovery of 100.26 ± 0.75%. The method wasvalidated and found to be simple, accurate,precise, and specific. The method wassuccessfully applied for the determination of TADin pure powder and tablet dosage form withoutinterference from common excipients ordegradation products.

Tadalafil (TAD) has the chemical name(6R-12aR)- 6- (1,3-benzodioxol-5-yl)- 2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino [1¢, 2¢:1,6] pyrido

[3,4-b]indole-1,4-dione (Figure 1). Erectile dysfunction (ED) is defined as the consistent or recurrent inability of a man to attainand/or maintain penile erection sufficient for sexual activity (1). ED has significant effects on mood state, interpersonalrelationships, and quality of life (2–5). One explanation for thisis that ED can result in a sexual aspiration/achievement gap(i.e., personal distress) that can negatively impact sexualsatisfaction and lead to reduced overall life satisfaction (6).

Psychological factors that correlate to ED include anxiety,depression, and anger (1–4, 7). Despite strong associationsbetween ED and psychological and interpersonal factors,these factors historically have been inadequately addressed intreatment outcome research until recently (8–10). TAD is an oralphosphodiesterase-5 inhibitor for the treatment of ED (11, 12).It has a half-life of 17.5 h (13), is well tolerated, and is efficacious in the majority of men for up to 36 h after dosing (14). TAD isavailable as bulk material and tablet dosage form (15).Reviews of its preparation (16–18), pharmacology (19–21), drug interaction (22–24), chiral synthesis (25), structure activity

relationship (26), clinical data (27–29), metabolism (30–33),pharmacokinetic and clinical experience (34–36), and clinicalevaluation (37) have been published. A literature survey revealed that analytical methods for determination of TAD in human urine based on HPLC (38) and HPLC/MS/MS (39, 40) have beenreported. This paper describes a precise and accurate HPLCmethod for determination of TAD in pure powder and tabletdosage form (41).

Experimental

Apparatus

A Waters (Milford, MA) Alliance HPLC 2996 instrument equipped with photodiode array (PDA) detector 2996, anautoinjector with a 200 mL loop, and an Xterra RP18 column(150 ´ 4.6 mm id, 3.5 mm particle size) was used. A Sartorius CP224S (Gottingen, Germany) analytical balance and anultrasonic cleaner (Frontline FS 4, Mumbai, India) werealso used.

Samples and Reagents

TAD pure powder and TAD tablets (TAD 1, 20 mg/tablet;TAD 2, 20 mg/tablet) from Zydus Cadila Ltd (Ahmedabad,India) were purchased in a local market. HPLC gradeacetonitrile was purchased from S.D. Fine Chem Ltd(Ahmedabad, India). The water for HPLC was prepared bytriple glass distillation and filtered through a nylon 0.45 mm,47 mm membrane filter (Gelman Laboratory, Mumbai, India). Potassium dihydrogen orthophosphate, orthophosphoric acid, and triethylamine were procured from S.D. Fine Chem Ltdand were of analytical grade.

HPLC Conditions

The Xterra RP18 column was used in the isocratic mode,with buffer–acetonitrile (70 + 30, v/v), adjusted to pH7.00 ± 0.05 with triethylamine as the mobile phase at a flowrate of 1.0 mL/min. The mobile phase was filtered through anylon 0.45 mm, 47 mm membrane filter and degassed beforeuse. The elution was monitored at 225 nm, and the injectionvolume was 10 mL.

Preparation of Buffer

Dissolve 6.8 g potassium dihydrogen orthophosphate in1000 mL water. Sonicate to dissolve, and then adjust the pHto 7.0 ± 0.1 with triethylamine.

516 BAROT & PATEL: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010

Received April 18, 2008. Accepted by SW December 3, 2008.Corresponding author’s e-mail: [email protected]

Preparation of Diluent

Prepare a mixture of buffer–acetonitrile (50 + 50).

Preparation of TAD Standard Solutions

Transfer accurately weighed TAD (25 mg) to a 25 mLvolumetric flask and dilute to the mark with diluent. Dilute5 mL of this solution to 50 mL with diluent (100 ppm TAD).

Preparation of Sample Solutions

Weigh and powder 20 tablets. Accurately weigh powderequivalent to 100 mg TAD, transfer it to a 100 mL volumetricflask, and add diluent (50 mL). Sonicate the solution for15 min with intermittent shaking. Then cool the mixture to

room temperature and dilute to volume with diluent.Centrifuge the resulting solution at 3000 rpm for 10 min, thenfurther dilute 5 mL of this solution to 50 mL in a volumetricflask.

Method Validation

(a) Interference from excipients.—Specificity wasdemonstrated by injecting blank, placebo, sample, andstandard.

(b) Interference from degradation products.—Specificitywas further demonstrated by carrying out forced degradationof the sample with 5 M HCl, 1 M NaOH, 30% H2O2, heatingin a water bath at 80°C for 15 min, and keeping under UV light at 254 nm for 24 h. Prepare samples as per the test preparationand inject into the HPLC system. In each case, the purityfactor for the TAD peak should be more than 980.

(1) Acid degradation.—Accurately weighed intact tabletsequivalent to 100 mg TAD were transferred to a 100 mLvolumetric flask, 50 mL diluent was added, and the solutionwas sonicated for 15 min. A 5 mL volume of 5 M HCl wasadded, and the solution was heated in a water bath at 80°C for15 min. The solution was then cooled and neutralized with5 mL 5 M NaOH, the volume was made up with diluent, andthe solution was mixed well and centrifuged at 3000 rpm for10 min. A 5 mL aliquot of this solution was further diluted to50 mL with diluent, followed by injection into the HPLCsystem.

(2) Alkali degradation.—The method was the same as in(1) except for adding 1 mL 1 M NaOH instead of 5 mL 5 MHCl, and neutralizing with 1 mL 1 M HCl instead of 5 mL 5 M NaOH.

BAROT & PATEL: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010 517

Figure 1. Structure of TAD.

Figure 2. Chromatogram of a sample.


Table 1. Analysis results for degradation of TAD tablets

Condition and time Assay, % Purity angle Purity threshold

Sample 90.56 0.041 0.242

Acid degradation (5 mL sample, 5 M HCl, heat in water bath at 80°C for 15 min) 88.30 0.041 0.248

Alkali degradation (1 mL sample, 1 M NaOH, kept at room temperature for 15 min) 89.81 0.045 0.253

Peroxide degradation (1 mL sample 30% H2O2, heat in a water bath at 80°C for 15 min) 66.55 0.050 0.238

UV degradation (sample, 24 h at 254 nm) 91.65 0.044 0.257

Thermal degradation (heat in water bath at 80°C for 15 min) 90.42 0.048 0.255

Figure 3. Chromatogram of the blank—diluent.

Figure 4. Chromatogram of the placebo.

(3) Peroxide degradation.—The method was the same asin (1), except 1 mL 30% H2O2 was added in place of 5 mL 5 M HCl, and no neutralization was required. Heat in a water bathat 80°C for 15 min.

(4) UV degradation.—Accurately weighed intact tabletsequivalent to 100 mg tadalafil (previously kept under UV light at 254 nm for 24 h) were transferred to a 100 mL volumetricflask. The remainder of the procedure was the same as in (3),except the solution was not heated at 80°C for 15 min.

(5) Thermal degradation.—The method was the same asin (1) without addition of HCl or neutralization.

(c) Calibration curve linearity.—The calibrationcurves were plotted over the concentration range of50.7–152.10 mg/mL. Accurately measured working standardsolutions of TAD (2.5, 4.0, 5.0, 6.0, and 7.5 mL) weretransferred to a series of 50 mL volumetric flasks and dilutedto the mark with diluent. A 10 mL volume of each solution was injected under the operating HPLC conditions as describedabove. Calibration curves were constructed by plotting peakareas versus concentrations of TAD, and the regressionequations were calculated. Each value was the average ofthree determinations.

(d) Accuracy (recovery).—The accuracy of the methodwas determined by calculating recoveries of TAD by thestandard addition method. Known amounts of standardsolution of TAD (0.5, 1.0, and 1.5 mg/mL) were added to aprequantified sample solution of tablet dosage form. Theamount of TAD was determined by applying these values tothe regression equation of the calibration curve.

(e) Method precision (repeatability).—The precision waschecked by repeatedly injecting (n = 6) standard solution ofTAD (1.5 mg/mL) for the HPLC method.

(f) LOD and LOQ.—The LOD and LOQ of the drug werecalculated using the following equations as per InternationalConference on Harmonization (ICH) guidelines (42):

LOD S= ´33. /s

LOQ S= ´10 s /

where s = the SD of the response and S = the slope of theregression line.

(g) Robustness.—Three sample solutions of same lot ofTAD were prepared and analyzed using the followingchromatographic conditions:

(1) Change in flow rate by ±0.2 mL/min;(2) Change in organic solvent composition of the mobile

phase by ±5.0%;(3) Change in the pH of the buffer solution by ± 0.2 unit; (4) Change in the temperature of the column by ± 0.2 unit.(h) Stability of the analytical solutions.—This was

evaluated by injecting freshly prepared TAD standard andsample solutions, and repeating the injections after 24 h.

Analysis of TAD Tablet Dosage Form

Tablets containing TAD were purchased from a localmarket. The responses of the tablet dosage form weremeasured at 225 nm for quantification of TAD by HPLC. The


Figure 5. Linearity of response. y = area counts andx = concentration.

Table 2. Regression analysis of the calibration curvesfor TAD

Parameter HPLC method

Linearity range, mg/mL 50.70 to 152.10

r 0.9999

Table 3. Summary of validation parameters for TAD

Parameter HPLC method

LOD, mg/mL 0.0509

LOQ, mg/mL 0.0509

Accuracy, % 98.43–100.65

RSD, % 0.24

Table 4. HPLC system suitability parameters for TAD

Parameter TAD ± RSD, %

Retention time, min 11.7 ± 0.01

Tailing factor 0.96 ± 0.02

Asymmetry 0.98 ± 0.02


Figure 6. System precision study chromatogram.

Figure 7. System suitability study chromatogram.

Table 5. Results of robustness study

ParameterMean assay of TAD, %

Intermediate precision 92.50

Change in flow rate by –0.2 mL (0.8 mL/min) 92.78

Change in flow rate by +0.2 mL (1.2 mL/min) 92.37

+5% organic solvent concentration in mobile phase 93.45

–5% organic solvent concentration in mobile phase 93.01

Change in pH of buffer solution by –0.2 unit (6.8 units) 92.75

Change in pH of buffer solution by +0.2 unit (7.2 units) 92.32

Change in temperature of column oven by +0.2 degrees (42°C) 92.43

Change in temperature of column oven by –0.2 degrees (38°C) 92.26

amount of TAD present in sample solutions was determinedby fitting the responses into the regression equation of TAD.

Results and Discussion

HPLC Method

To optimize the HPLC parameters, several mobile phasecompositions were tried. A satisfactory separation and goodpeak symmetry for TAD was obtained with the mobile phaseconsisting of buffer–acetonitrile (70 + 30, v/v), adjusted to pH 7.0 ± 0.05 with triethylamine (Figure 2). Quantification wasachieved with PDA detection at 225 nm based on peak area.

Validation of the Proposed Method

Specificity.—There was no peak at the retention time ofTAD, indicating that there was no interference from the blankand excipients. Figure 3 shows a chromatogram of theblank-diluent. Figure 4 shows a chromatogram of theplacebo. Figure 2 shows a chromatogram of a sample. In allcases for acid, alkali, peroxide, UV, and thermal degradation,the purity factor was less than the purity angle for the TADpeak (Table 1).

Linearity.—Linear correlation was obtained between peakarea and concentration of TAD in the range of50.7–152.10 mg/mL. The linearity of the calibration curveswas validated by the r value of 0.9999. Data for the regressionanalysis of the calibration curves are shown in Table 2 and thecalibration curve in Figure 5.

Accuracy (recovery), precision, and system suitabilitystudies.—The recovery study was carried out by the standardaddition method. The mean recovery obtained was100.08 ± 0.75% (Table 3), which is satisfactory. The RSD forTAD was found to be 0.24% (Table 3), indicating that theproposed method is repeatable. The system suitability test

parameters are listed in Table 4. Figure 6 shows the systemprecision study chromatogram, and Figure 7 shows thechromatogram obtained in the system suitability study.

LOD and LOQ.—The LOD and LOQ of TAD were foundto be 0.0509 and 0.0509 mg/mL, respectively (Table 3).

Robustness.—Table 5 shows that no change was found inresults after changing chromatographic conditions.

Stability of the analytical solution.—Table 6 shows theanalytical solution stability data for peak response over 24 h.

Analysis of TAD Tablet Dosage Form

The proposed method was successfully applied todetermine TAD in a tablet dosage form. The results obtainedfor TAD were comparable to the corresponding label claim(Table 7).

Conclusions

The results of the analysis of a tablet dosage form by theproposed method are reproducible, reliable, and in goodagreement with the label claim of the drug. The method wasvalidated and found to be simple, sensitive, specific, accurate,and precise. Neither the excipients present in the tabletdosage form nor the degradation products interfered withdetermination of TAD. Hence, the method could be usedsuccessfully for the routine determination of TAD in a tabletdosage form.

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Table 6. Results of solution stability study

Time, h

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Standard Sample Standard (NMTa ± 3.0%) Sample (NMT ± 3.0%)

Initial 5148408 5588198 0.0 0.0

24 5263973 5598441 2.2 0.2

a NMT = Not more than.

Table 7. Analysis results for TAD tablets by theproposed HPLC method

Formulation TAD, % ± SDa

A (TAD 20 mg, 1) 91.8 ± 1.36

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determination of tadalafil in pure powder and tablet dosage

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