INTRODUCTION:

Amitriptyline (3-(10, 11-Dihydro-5H-dibenzo [a, b] cyclohepten-5-ylidene)-N, N-dimethyl-1-propanamine) is a tricyclic antidepressant ¹ commonly administered to patients having major depression. It acts primarily as a serotonin-norepinephrine reuptake inhibitor, with strong actions on the norepinephrine transporter, and moderate effects on the serotonin transporter.²

Propranolol (1-[(1methylethyl) amino]-3-(1-naphthalenyloxy)-2-propanol) is a beta-adrenoceptor blocking agent (antihypertensive) mainly used in the treatment of hypertension.³ It is a non-selective beta blocker, which blocks the action of epinephrine and norepinephrine on both β₁- and β₂-adrenergic receptors.⁴ It was the first successful beta blocker developed.⁵

Atenolol (4-[2-hydroxy-3-[(1-methylethyl) amino] propoxy]benzeneacetaminde) is also a beta-adrenoceptor blocking agent (antihypertensive)which is commonly administered to patients suffering from hypertension.⁶ It is a selective β₁ receptor antagonist, a drug belonging to the group of beta blockers, a class of drugs used primarily in cardiovascular diseases. It works by slowing down the heart and reducing its workload.⁷

The present work illustrates a fully validated method for the simultaneous determination of the above said drugs using High Performance Thin Layer Chromatography. HPTLC is a powerful chromatographic technique as it is highly reliable, reproducible, simple and can be carried out in a short duration of time. The technique is very cheap as it utilises only minimum amount of solvents with minimum sample clean up. It allows the application of dirty or turbid samples as well. Moreover, HPTLC permits the simultaneous determination of various components in any matrices like pharmaceutical dosage forms, blood, urine etc.^8-11 The method was developed and validated using pure standard reference materials of the corresponding drugs and studied the application of the method in formulations. No work has been reported so far on the simultaneous determination of the said drugs. In forensic context the developed method will be significant as the studied drugs are forensically encountered at times. The analytical method validation was carried as per the guidelines given International Conference on Harmonisation. (ICH)^{12, 13.}The Figure 1 given below gives the structures of the corresponding compounds being undergone study in the present work.

Amitriptyline Propranolol

Atenolol

Figure1. Structures of the studied compounds

EXPERIMENTAL:

Instrumentation:

A Camag ATS4 sampling device is used as the sample applicator, Camag TLC Scanner II controlled by win Cats 1.4.2 version software used for the scanning of the developed plates. Merck HPTLC plates coated with silicagel 60 F₂₅₄ (0.2 mm thickness) on aluminium sheets were used as the stationary phase. The chromatograms were developed in a twin-through chamber (Camag).

Solvents and chemicals:

Reference standards of AMTRL, ATL and PRPL were supplied by M/s Intas Pharmaceuticals Pvt Ltd, Ahmedabad, India. The mobile phase solvents, benzene, acetone, methanol and ammonia solution (25%) were procured from Merck India Ltd. All are of analytical grade.

Working Standard stock solutions:

Standard stock solutions of 1 mg/ml of ATL, AMTRL and PRPL were prepared by dissolving 10 mg reference standards of each of the drugs in 10 ml methanol. The standard mixture of all the three compounds was prepared by dissolving 10 mg each in 10 ml methanol.

Sample preparation:

Currently marketed tablets, Aten-25 (containing 25 mg ATL), Inderal- 40 (containing 40 mg PRPL), Tryptomer (containing 25 mg AMTRL) were taken for the quantitative studies. They were crushed to a fine powder and appropriate amounts of each one, corresponding to about 500 µg of each of the three were weighed in a 50 ml volumetric flask. After addition of 40 ml methanol and sonication (30–45 min), the samples made up to volume with methanol and filtered through a Whatman filter paper (No.1). The filtrate was used for the analysis. For recovery studies, standard additions of 10 mg, 20 mg, and 30 mg of the corresponding standards to the crushed tablet powder were done and prepared the analyte as described above.

Mobile phase:

Benzene: methanol: acetone: ammonia solution (25% NH3) (5:3:2:0.2; v: v) was mixed and centrifuged. Centrifugate was used as mobile phase.

Calibration procedure:

Aliquots of standard stock solution of ATL, AMTRL and PRPL were taken in eight different 10 ml standard volumetric flasks and diluted to the mark with the methanol, such that the final concentrations were in the range 1–1000 µg/ ml. Ten microlitres of each of these solutions were spotted on HPTLC plates as 6 mm bands. The plates were developed with the mobile phase up to 90 mm height in a saturated twin trough chamber. The plates were removed and then dried. Each band was scanned densitometrically at 254 nm, and the peak areas were recorded to plot the peak areas versus concentrations in µg/ ml. The linear regression coefficient and the range of linearity were determined from the calibration curve drawn.

Method validation studies:

Precision:

The precision of the method developed was tested by experimenting with the repeatability of the peak area and the Rf value of three different concentrations ie.2 µg/ spot, 4 µg/ spot, 6 µg/ spot with three replicates of each concentration. The intra and inter day precision studies of the same has been carried out and expressed in terms percent relative standard deviation.

Robustness:

The robustness of the method was examined by making slight variations in the mobile phase volume, composition, saturation time and activation period of the TLC plates. The variation in the peak area of the resulting chromatogram is observed for a single concentration, 4µg/spot and the % R.S.D is calculated to identify the robustness of the method.

Detection limits (LOD/LOQ):

The standard mixture of different lower concentrations coming down up to 0.01 µg/spot were prepared and HPTLC analysis was done as earlier to determine the LOD/LOQ values for the corresponding drugs. The blank methanol was also spotted and signal to noise ratio was determined. LOD was considered as 3:1 and LOQ was considered as 10:1 as per the signal to noise ratio.

Recovery:

The recovery studies were carried out by the standard addition technique. The pure standards of AMTR, PRPL and ATL were added to the samples of formulations in different proportions as 0 mg, 10 mg, 20mg and 30 mg and the HPTLC analysis was carried out with developed method.

Ruggedness:

The ruggedness of the particular method was studied using the analysis of a particular concentration, 10 µg/spot at different time intervals (0 hrs, 6 hrs, 24 hrs, 48 hrs, 72 hrs). The data were taken and % RSD was calculated to obtain the ruggedness of the method.

Specificity:

The specificity of the method was determined by comparing the analytical results given by the standard drugs and formulations. The Rf values and the spectra of the standard drug mixture and that of the formulations are compared to obtain the specificity. The peak purity of the three drugs was assessed by comparing the spectra at three different levels, at peak start, at peak apex and at peak end of the corresponding densitograms.

RESULTS AND DISCUSSION:

Development of mobile phase:

Different mobile phases were tried for the successful separation and determination of the compounds.

1. hexane: acetone: diphenylamine:: 6:4:0.1 ; v/v

2. benzene: acetone: methanol: ammonia::6:2:2:0.1 ; v/v

3. benzene: methanol: acetone: ammonia::5:3:2:0.2 ; v/v

The first two mobile phase compositions were not satisfactory as the former failed with tailing and the latter was unsuccessful with the resolution of PRPL and AMTR. But the third system was one which overcame the drawbacks of the former ones and it gave sharp and well defined spots at Rf values as given below:

ATL: 0.37, PRPL: 0.69, AMTR: 0.80

Figure 2. Thin layer chromatogram developed with pure standards and that extracted from the tablets using the mobile phase benzene: methanol: acetone: ammonia:: 5:3:2:0.2 ; v/v

The figure 2 given below is the TLC plate developed using the final mobile phase and viewed under UV light of 254nm wavelength which clearly depicts the separation of the components both in the standard and the tablet sample. The figure 3 shows the densitogram of the standard mixture containing the three compounds which is scanned using UV light of 254 nm wavelength.

Figure 3. Densitogram recorded at 254 nm for the standard mixture

Calibation curve:

The calibration curve (plot of peak area versus concentration of standards of AMTR, PRPL and ATL) was found be linear with linear regression equation as follows

y _ATL = 6.3283x - 420.03 with linear regression coefficient R² = 0.9862

y_PRPL= 1.1674x + 1765.7 with linear regression coefficient R² = 0.9881

y_AMTR= 16.461x + 2735.1 with linear regression coefficient R² = 0.9975

The range of linearity of the different standards was found to be 0.1µg/spot to 10µg/spot for PRPL and AMTR and for ATL it was found to be 0.5µg/spot to 10µg/spot with respect to the peak area.

Method validation studies:

Precision:

The precision of the method developed was examined over both intraday and inter day which was found to be highly precise. The mean peak area of the triplicate spots each of the three concentrations along with the standard deviation and the %R.S.D of the three compounds are given below in the tables (Table 1, 2&3). The lower values of %R.S.D (<1%) for the peak areas recorded for the three different concentrations shows that the method is highly precise.

Robustness:

The method was found to be robust as the slight variation in different parameters does not make significant deviation in the values of peak area for the particular concentration, 4 µg/spot. The lower values (given in the Table 4) for %R.S.D (<0.4%) makes it clear that the method is robust.

Table 1. Data for intra and inter day precision study of AMTR

Amount (µg/spot)	Mean area (AU)		Standard deviation		% R.S.D
Amount (µg/spot)	Intra day	Inter day	Intra day	Inter day	Intra day	Inter day
2	8158.3	8188.05	16.1220	25.95	0.1980	0.3169
4	11984.5	11909.2	62.86	43.13	0.5250	0.3620
6	14649.7	14549.1	102.53	39.74	0.6990	0.2730

Table 2. Data for intra and inter day precision study of PRPL

Amount (µg/spot)	Mean area (AU)		Standard deviation		% R.S.D
Amount (µg/spot)	Intra day	Inter day	Intra day	Inter day	Intra day	Inter day
2	3953.1	3876.1	2.687	35.497	0.0679	0.9157
4	6656.1	6728.0	10.112	29.98	0.1519	0.4456
6	9134.9	9062.2	35.072	81.88	0.3839	0.9035

Table 3. Data for intra and inter day precision study of ATL

Amount (µg/spot)	Mean area (AU)		Standard deviation		% R.S.D
Amount (µg/spot)	Intra day	Inter day	Intra day	Inter day	Intra day	Inter day
2	954.65	909.05	5.586	4.879	0.5851	0.5367
4	1858.95	1848.1	9.8287	11.031	0.5287	0.5908
6	3151.55	3106.5	11.9501	9.334	0.3791	0.3004

Table 4. Data for robustness of HPTLC method for AMTR, PRPL and ATL

Parameter	Mean peak area			Standard Deviation			% R.S.D
Parameter	AMTR	ATL	PRPL	AMTR	ATL	PRPL	AMTR	ATL	PRPL
Mobile phase volume (8 ml, 10 ml, 12 ml)	11909.2	1859.05	6660.15	43.1335	2.4748	4.7376	0.3620	0.1331	0.0711
Mobile phase composition	11947.2	1863.95	6675.15	16.2634	2.7577	2.3334	0.1360	0.1480	0.0350
Saturation time (20min, 25 min, 30 min)	11953.2	1859.65	6655.15	17.6776	0.6363	4.7376	0.1480	0.0342	0.0712
Activation time for TLC plates (3 min, 6 min, 8 min)	11952.7	1863.65	6673.15	9.8994	1.4849	5.1618	0.0830	0.0796	0.0774

Detection limits (LOD and LOQ):

The detection limits were determined with the signal to noise ratio method for the three compounds and given in the Table 5 below.

Table 5. The LOD and LOQ values for AMTR, PRPL and ATL

Compounds	AMTR	ATL	PRPL
LOD ng/µl	10	70	30
LOQ ng/ µl	34	234	100

The lower values of the detection limits of the corresponding drugs clearly indicate the better sensitivity of the developed method.

Recovery Studies:

The recovery studies data (given in Table 6, 7 & 8) shows that the recovery of all the three compounds was better as they have given recovery of more than 98%. Hence the adopted method is highly accurate.

Table 6. Data for recovery study of AMTR

Excess standard added to analyte %	Theoretical content µg/ml	Amount recovered µg/ml	Recovery %
0	524.6	511.53	97.51
40	724.2	709.7	97.99
80	921.34	908.79	98.64
120	1152.4	1139.9	98.92
%R.S.D			0.6477

Table 7. Data for recovery study of ATL

Excess standard added to analyte %	Theoretical content µg/ml	Amount recovered µg/ml	Recovery%
0	514.0	508.5	98.93
40	729.6	714.13	97.88
80	911.2	908.41	99.69
120	1170.4	1150.81	98.32
%R.S.D			0.7955

Table 8. Data for recovery study of PRPL

Excess standard added to analyte %	Theoretical content µg/ml	Amount recovered µg/ml	Recovery%
0	520.0	507.92	97.67
40	726.0	720.0	99.17
80	912.0	903.1	99.02
120	1120.0	1108.68	98.99
%R.S.D			0.7086

The amount of each of the drugs present in the formulations is determined using the HPTLC method and calculated using the recovery studies data which is given in the table 9.

Table 9. Amount of drugs present in the tablets determined using HPTLC.

Brand name	Amount of drug claimed (mg/tablet)	Amount determined (mg/tablet) by HPTLC	Amount (%) by HPTLC
Tryptomer (AMTR)	25	24.3775	97.51
Aten-25 (ATL)	25	24.7325	98.93
Inderal-40 (PRPL)	40	39.0680	97.67

The amount of the drugs present determined by the developed HPTLC method was found to be in close agreement with amount claimed by the manufacturers. The recovery studies also make it clear that the method is highly accurate.

Ruggedness:

The ruggedness of the method was examined with a particular concentration 10 µg/spot at different time intervals. The data (given in Table 10) shows that the method is rugged for the particular compound of interest. The lower value of the %R.S.D (<0.2%) clearly indicates that the particular compounds are stable over the period and can be examined with the developed method successfully.

Table 10. Data for ruggedness of HPTLC method for AMTR, PRPL and ATL

Time (hrs)	ATL (peak area)	PRPL (peak area)	AMTR (peak area)
0	5845.8	13626.8	19284.8
6	5828.9	13604.7	19265.6
24	5822.7	13597.3	19257.2
48	5819.6	13592.4	19255.2
72	5818.5	13590.5	19254.6
Mean	5827.1	13602.34	19263.48
%RSD	0.1923	0.1083	0.0659

Specificity:

The specificity of the method was examined by comparing the retention factor (Rf) values and the corresponding spectra given by the standards and the formulations. The Rf values of both the standards and formulations are matching exactly and the UV spectra of the standards and formulations are matching precisely which is clearly proved by the overlaid UV spectra of the standard and formulations which is depicted in the figure 5 given below.

Figure 5. Overlaid UVspectrum of the standards and the formulations.

CONCLUSIONS:

The discussed High Performance Thin Layer Chromatography method which is developed and validated is a highly sensitive and robust one for the simultaneous determination of the targeted compounds, Amitriptyline, atenolol and propranolol. The method is easy to carry out and it is precise and accurate. In addition, the method can be extended to the determination of the drugs from various biological matrices also if required.

ACKNOWLEDGEMENT:

The corresponding author is highly thankful to The Chief Forensic Scientist, Directorate of Forensic Science, Government of India, New Delhi for providing the fellowship to carry out the research work.

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Received on 03.03.2011 Modified on 13.04.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1059-1063