INTRODUCTION:

Tadalafil Hydrochloride (TDL) is selective phosphodiesterase type 5- inhibitor and approved by USFDA for the treatment of penile erectile dysfunction in 2003 and gets extended approval in 2009 for pulmonary arterial hypertension. The prescribed therapeutic dose of TDL is 2.5-20 mg daily under the brand name of Cialis.It is chemically (6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methylpyrazino [1',2':1,6] pyrido[3,4-b]indole-1,4-dione(fig.1).

TDL is white colored powder, insoluble in water and partly soluble on methanol^[1].Absorption of TDLis unaffected by the presence of high-fat food and drug is 94 % bound to plasma proteins. TDL getsmetabolized by CYP3A4 metabolizing enzyme by methylation and further gets conjugation to methyl catechol glucuronide conjugation. It is eliminated primarily in form of metabolites in faces (61%) and urine (36%)^[2]

Figure.1: Structure of Tadalafil Hydrochloride (TDL)

There are few analytical methods were reported for estimation of TDL in biological fluid such as plasma, serum and urine by LCMS/MS^[3]. Some analytical methods were reported for estimation of TDL in tablet dosage form by HPLC-UV method^[4]and stress degradation study by HPLC method^[5-7]. The validated electrophoretic method has been developed for the analysis of TDL in pharmaceutical preparations^[8].In addition one HPTLC method has been developed for estimation of TDL from tablet dosage form but the stability studies are not carried in their study^[9]. To our knowledge, no any literature reveals the presence of stability indicating high-performance thin layer chromatographic (HPTLC) determination of TDL in pharmaceutical dosage forms. The International Conference on Harmonization (ICH) guideline entitled ‘stability testing of new drug substances and products’ requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substance^[10-12].HPTLC has emerged as an important tool for the qualitative, semi-quantitative and quantitative analysis of phytochemical, herbal drugs and pharmaceutical formulations^[13,14,15]. The major advantage of HPTLC is that several samples can be analyzed simultaneously using a small quantity of mobile phase^[16,17]. The aim of the present study is to develop and validate stability indicating HPTLC method under ICH guidelines^[18,19,20]recommended test conditions for TDL and tablet dosage forms. More intensive force degradation studies in our laboratory were carried out on TDL. Accordingly, a stability-indicating method was developed, which could able to separate and quantify various degradation products of TDL.

MATERIALS AND METHODS:

Instrumentation:

HPTLC analysis was performed by using a CAMAG HPTLC system (Switzerland) comprising of CAMAGLinomat V semiautomatic sample applicator, CAMAG TLC Scanner III, CAMAG (Muttenz, Switzerland) flat bottom and twin-trough developing chamber (10 × 10cm), and UV cabinet. The CAMAG winCATS software was used for further data analysis.

Chromatographic conditions:

The samples were applied in the form of band with width 6mm on pre-coated silica gel aluminum plate 60F-254 with 250mm thickness, and distance of 10mm from bottom with Linomat III (Switzerland) sample applicator equipped with 100µL Hamilton syringe. The plates were prewashed by methanol and activated at 60˚C for 5 min prior to chromatography. The standard and sample solutions were applied with 100nL/s; at 5mm space between two bands under a stream of nitrogen. The elution solvent consists of Hexane: isopropyl alcohol: acetonitrile (5: 4: 1 v/v).Linear ascending development was carried out to distance of 8cm in 20/10cm twin trough TLC developing chamber CAMAG at room temperature and previously saturated with mobile phase for 30 min. TLC plates were dried in a current of air with the help of an air dryer. Densitometric scanning was performed on CAMAG TLC scanner III in the absorbance mode at 285nm. The source of radiation utilized was deuterium lamp. The slit dimensions were kept at 5*0.45mm micro and 5mm sec^-1scanning speed was employed.

Requirements:

The active pharmaceutical ingredient TDL was supplied by IPCA Laboratory Limited, Mumbai India. The solvents of hexane, isopropyl alcohol, acetonitrile used was of analytical grade (E-Merck Ltd.). All other chemicals and reagents used were of analytical grade and were purchased from Merck Chemicals, India.

Solution preparations:

Preparation of standard solution:

Stock solution of 1mg/ml was prepared by dissolving 10 mg of TDL in 10ml of acetonitrile. Intermediate stock solution of 100µg/ml was prepared by further dilution of standard solution of 1mg/ml. From the intermediate standard solutions 2.0, 3.6, 4.0, 4.8 and 6.0µl solutions were spotted on the activated HPTLC plate to obtain TDL concentrations of 200, 360, 400, 480 and 600ng spot^-1 respectively.

Calibration curve of TDL:

The intermediate stocks solution of 100µg/ml was prepared in acetonitrile. The different volumes of intermediate stock 2.0, 3.60, 4.0, 4.80 and 6.0µl were applied on plate to obtain a concentration of 200, 360, 400, 480 and 600ng spot^-1. The calibration curve was plotted using peak area against the concentration of drug and the linearity data was treated by the linear least square regression.

Precision:

Repeatability of sample application and measurement of peak areas were carried out using six replicates of the same spot. The intra-day and inter-day variation for the determination of TDL was carried out at a concentration of 200, 400 and 600ng spot^-1.

Accuracy:

Accuracy study was done at low quality control (LQC), medium quality control (MQC) and high-quality control (HQC) level concentration. i.e. at 200, 400 and 600ng spot^-1concentration level. It was measured in terms of recovery of drug at each concentration. The experiment was conducted in triplicate.

Repeatability of sample application:

The drug solution, TDL, 4µL (400ng/spot) was applied six times on a HPTLC plate; the plate was developed and dried. The spots were scanned and %RSD for measurement of peak areas was estimated.

Limit of detection and limit of quantification:

In order to estimate the limit of detection (LOD) and limit of quantitation (LOQ), blank acetonitrile was spotted six times. The signal to noise level was determined. LOD was considered as 3:1 and LOQ as 10:1.

Specificity:

The specificity of the method was ascertained by analyzing standard drug and sample. The spot for TDL in sample was confirmed by comparing the RF and spectra of the spot with those of standard. The peak purity of sample was assessed by comparing the spectra at peak start, peak apex and peak end positions of the spot.

Stock solution stability:

The stability studies were evaluated for two different concentration i.e. 200ng/spot and 400ng/spot, which were stored at refrigerated temperature (2-8°C) for a week. The stability wascalculated by comparing the results of six replicate injections of stored solutions with fresh samples.

Forced degradation of TDL:

All stress degradation studies were performed at initial concentration of 1000µg mL-1. For these studies, 10mg of TDL was accurately weighed and transferred to a 10 mL volumetric flask.

Acid and base induced degradation:

The 1000µg/ml of TDL was subjected to accelerated degradation under acidic and basic conditions by refluxing with 1 N HCl at 80°C for a period of 4hr and 0.1 N NaOH at 80°C for a period of 2 hr respectively. The accelerated degradation in acidic and basic media was performed in the dark in order to exclude the possible derivative effect of light on the drug.

The mixtures were allowed to cool and 0.6mL of these solutions were then transferred to 10mL volumetric flasks neutralized with 1.0mL of 1N NaOH for acid and neutralized with 1.0mL of 0.1N HCl for alkaline degradation and the volumes were made up with acetonitrile.

The resultant solutions were spotted in the form of bands by using Linomat IV, Switzerland applicator on pre-coated silica gel aluminum plate 60GF-254 to get 600ng/spot appropriately diluted and chromatograms were run after spotting. The hydrolyzed products were resolved from pure drug using HPTLC.

Preparation of hydrogen peroxide induced degradation product:

Accurately weighed 10mg of drug was dissolved in 10 ml of acetonitrile. Subsequently, 5ml of hydrogen peroxide 6.0% v/v was added and the solution was heated in boiling water bath for 1 h till the removal of excess hydrogen peroxide. The mixtures were allowed to cool and 0.6mL of this solution was then transferred to a 10mL volumetric flask and the volume was made up with acetonitrile. From this solution 2×50µL samples were plotted in the form of bands by using Linomat IV, applicator on pre-coated silica gel aluminum plate 60GF-254 to get 600ng per spot for TDL. The oxidized product was resolved from pure drug using HPTLC.

Preparation of Degradation under dry heat degradation product:

Dry heat studies were performed by keeping drug samples in oven (80˚ C) for 6 hrs. The samples were withdrawn, dissolved in acetonitrile, diluted suitably and appropriate volumes of resultant solution were applied on HPTLC plate and densitograms were developed.

Photo-degradation studies:

Photolytic studies were also carried out by exposure of drugs to UV light up to 200-watt hrs/sq. m. and subsequently cool fluorescent light to achieve an illumination of 1.2 million Lux.Hr.

Extraction and analysis of TDL in marketed formulation:

For determination of the content of TDL, marketed tablet dosage form under the brand name of Cialis (label claim: 20mg) was taken. 20 tablets were crushed and powder equivalent to 20mg of TDL was weighed. The extraction of drug was done by using acetonitrile as solvent. To ensure complete extraction of the drug it was sonicated for 30 min and volume made up to 20ml. The resulting solution was centrifuged at 3000rpm for 5 min and filtered. Laterthe solution was diluted to obtain final concentration of 100µg/ml. The 6µl of the resultant solution was spotted onto the plate followed by development and scanning. The analysis was repeated in triplicate. The possibility of excipients interference in the analysis was studied.

RESULTS:

Method development:

The HPTLC method was optimized with a view to develop a stability indicating assay method. Both drug and the degradation products were spotted on HPTLC plates and run in different solvent system. Initially Chloroform: toluene: Ethyl acetate (6:2:2 v/v/v), Toluene: t-ethyl butyl alcohol:Hexane (7:2:1 v/v/v) and Hexane:Isopropyl alcohol : acetonitrile (4:4:2 v/v/v) were tried. But the problems such as poor analyte retention and poor resolution were observed. The mobile phase consists of Hexane: Isopropyl alcohol: Acetonitrile (5:4:1v/v/v) was giving good resolution, sharp and symmetrical peak with RF value of 0.64 for TDL. Also, the spot for TDL was compact and not diffused (table1). The well-defined spots were obtained when the chamber was saturated with the mobile phase at room temperature. The representative densitogram is shown in figure.2

Method validation:

Calibration curve:

The linear regression data for calibration curves (figure.3) showed good linear relationship over the concentration range of 200-600ng spot^-1. The co-relation coefficient of the calibration equation was found to be r²= 0.995±0.0001. The polynomial regression data for the calibration curves (n=6) showed a good linear relationship over a concentration range of 200-600ng/spot.

LOD and LOQ:

The signal-to-noise ratios 3:1 and 10:1 were considered as LOD and LOQ respectively. The LOD of proposed method, with a signal-to-noise ratio of 3:1 was found to be 30ng per spot where as LOQ, with a signal to noise ratio of 10:1, was found to be 90ng per spot.

Figure.2: A typical HPTLC chromatogram of standard TDL

Table 1: Method development of TDL

Trial	Mobile phase composition	Mobile phase ratio (v/v/v)	Observation
1	Chloroform: toluene: Ethyl acetate	6:2:2	Broad peak shape
2	Toluene: t-ethyl butyl alcohol: Hexane	7:2:1	Poor retention of TDL
3	Hexane: Isopropyl alcohol: acetonitrile	4:4:2	Retention and peak shape improved but less resolution
4	Hexane: Isopropyl alcohol: Acetonitrile	5:4:1	Sharp peak shape with better resolution between analyte and degradation products

Figure.3: Calibration plot of TDL

Table 2: Intra- and inter-day precision of TDL at LQC, MQC and HQC level

Concentration of TDL(ng/spot)	Intraday precision (n=3)			Inter-day precision (n=3)
	Mean	SD	% RSD	Mean	SD	% RSD
200	1246.68	90.36	0.7	1229.4	85.50	0.069
400	2151.78	34.49	1.6	2187.70	54.33	0.024
600	3136.99	57.92	1.8	3146.54	84.01	0.026

SD = standard deviation; RSD = relative standard deviation

Table 3: Accuracy of TDL at LQC, MQC and HQC level

	Levels in ng/spot			Levels in ng/spot
	200	400	600	200	400	600
S. No	Area			Recovery
1	1124.03	2126.88	3145.75	182.35	397.32	615.73
2	1163.63	2141.45	3103.13	190.83	400.44	606.59
3	1259.23	2170.47	3108.91	211.33	406.66	607.83
4	1374.2	2101.43	3062.94	235.97	391.86	597.97
5	1292.08	2192.34	3176.21	218.37	411.35	622.26
6	1266.92	2178.13	3224.99	212.98	408.30	632.71
Mean	1246.68	2151.78	3136.98	208.64	402.66	613.85
%RSD	0.7	1.6	1.8
Accuracy	-	-	-	104.32	100.66	102.30

SD = standard deviation; RSD = relative standard deviation

Precision:

The repeatability of sample application and measurement of peak area were expressed in the terms of % RSD and the results are depicted in table 2, which revealed intra-day and inter-day variation of TDL at a concentration level of 200,400 and 600ng per spot.

Accuracy:

The proposed method when used for estimation of TDL at three concentration levels,the accuracy was found to be 100.0±5% in terms of mean recovery as listed in table 3. The data of summary of validation parameters are listed in table 3.

Repeatability of sample application:

400ng/ml sampleswere applied over the plate of six times. Areas for six injections were recorded and % CV was calculated. The % RSD was found to be 0.07.

SD = standard deviation;

RSD = relative standard deviation

Specificity:

The developed method was used for the estimation of TDL in bulk drug and pharmaceutical dosage form. The excipients did not interfere in the estimation. Interferences from the formulation and degradants were absent. These results indicated the specificity of the method. The method was quite selective for TDL. The baseline did not show any significant peak.

Stock solution stability:

Stability was calculated by comparing the results of six replicate injections of stored solutions with fresh samples. Results are tabulated in table 4.

Table 4: Solution Stability of TDL

Sr. No.	Fresh solution ng/spot		Stability solution
Sr. No.	200	400	200	400
1	1.7	3.9	1.94	4.1
2	1.8	3.93	1.73	4.11
3	2.05	3.99	2.06	4.17
4	2.2	3.84	2.2	3.98
5	2.1	4.03	2.21	4.2
6	2.7	4	2.2	4.14
Mean	1.98	3.94	2.05	4.11
%RSD	0.097	0.017	0.093	0.018
Accuracy	101.1	101.5	102.5	102.2

RSD = relative standard deviation

Forced degradation studies:

Acid and base induced degradation products:

The chromatogram of acid and base degradation of TDL (figure.4a and figure.4b) shows additional peaks at RF value 0.02, 0.10, 0.37 for acid and 0.65 for base. The concentration of drug was found to be changing from initial concentration indicating that TDL undergoes degradation in acid and base.

Hydrogen peroxide degradation products:

The samples degraded with hydrogen peroxide showed additional peak at Rf value of 0.27, 0.41 and 0.57. The spot of degraded product was well resolved from the drug spot (figure.5).

Photochemical degradation product:

The photo degraded sample showed no additional peak. The no significant degradation was observed in standard that were left in day light for 24 hr.

Dry heat degradation product:

The samples degraded under dry heat conditions showed no additional peak. This indicates that drug is susceptible to acid and base hydrolysis and oxidation. Lower RF value for acid and oxidation induced degradation products indicates that they were less polar and high RF value of base induced degradation products indicates that they were high polar than TDL.In forced degradation study maximum 16% degradation was observed by oxidation degradation. Acid and base hydrolysis shows 6-7% degradation. The results were tabulated in table 5.

Table 5: Forced degradation of TDL

Sr. No.	Sample name	% degradation found
1	Acid hydrolysis	6.6
2	Base hydrolysis	6.8
3	Oxidation	16

Assay of marketed formulation:

A single spot of TDL at RF 0.64 was observed in chromatogram of drug sample extracted from marketed tablets. There was no interference from excipients commonly present in conventional tablets. The drug content was found to be 94.78 %.

DISCUSSION:

A novel, precise and stability indicating high-performance thin layer chromatographic (HPTLC) method was developed for the estimation of TDL in bulk drug and in the marketed formulation. There is no any reported HPTLC methods for analysis of TDL. The developed method was found to be more accurate in terms of accuracy and precision. The developed method was sensitive and applied for the estimation of the marketed preparation of TDL which shows that the drug contents separated with no interfering peaks generated by the excipients in the marketed formulation. The method is versatile and can be applied for the forced degradation analysis of TDL. The developed method was found to obey all the validation parameters as per the ICH guidelines. This validated method can be used for quality control laboratories for the routine quantitative analysis of tablets consisting of TDL.

CONCLUSION:

The developed HPTLC technique is precise, specific, accurate and stability indicating. The method can be used to determine the purity of the drug available from various sources by detecting the related impurities. The statistical analysis proves that the method is reproducible and selective for the analysis of TDL as bulk drug and in pharmaceutical formulations. As the method could effectively separate the drugs from their degradation products it can be employed as a stability indicating one.

ACKNOWLEDGEMENT:

The authors wish to express their gratitude to the management of Prin. K. M. Kundanani College of Pharmacy, Mumbai for providing the research facilities, M/s IPCA laboratories, Mumbai for providing TDL as gift sample.

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Received on 22.08.2019 Modified on 06.10.2019

Research J. Pharm. and Tech 2020; 13(6):2608-2614.

DOI: 10.5958/0974-360X.2020.00464.3