Stability Indicating HPTLC Method Development and Validation for Estimation of Nortriptyline and Pregabalin in Tablet Dosage Form

Ghogare Jyoti D.*, Panchal Pranita P., Rathod Sayali P., Jadhao U. T.

Department of Pharmaceutical Quality Assurance, SVP College of Pharmacy (B. Pharmacy),

Hatta Tq. Basmat, Dist - Hingoli

*Corresponding Author E-mail: jyotighogare8275@gmail.com

ABSTRACT:

Chromatography is non-destructive procedure for resolving a multi-component mixture of solids, gases, Liquids. HPTLC is use of validated methods for qualitative and quantitative analysis. HPTLC is playing an important role in analytical world and a complementary method for HPLC. The analytical method was evaluated by using parameters such as Linearity, Precision, Accuracy, Limit of detection and Limit of quantification, Specificity, Robustness. In this method 100ng µL^-1 and 750ng µL^-1 volume of standard stock solutions of Nortriptyline and Pregabalin were taken, respectively. The mobile phase contains Toluene: Ethyl acetate: Methanol (6: 2: 1, v/v/v). Standard stock solutions were applied by over spotting on HPTLC plate with the help of CAMAG 100µl sample syringe, Linomat 5 sample applicator. The development chamber was saturated for 15 min. The plate was scanned at 210nm. The retention factors of PREGA and NORT were found to be PREGA: 0.48±0.03, NORT: 0.70±0.07. The % drug content (mean±S.D.) were found to be 99.32±1.39 for NORT and 99.75±1.15 for PREGA. The results of stress degradation studies revealed that NORT was prone to hydrolysis, oxidative, thermal and photolytic degradation whereas PREGA was found susceptible to hydrolysis, oxidative, thermal degradation but stable under photolytic stress conditions.

KEYWORDS: HPTLC, Nortriptyline, Pregabalin, Method development. ICH guidelines.

INTRODUCTION:

Nortriptyline (NORT) is chemically 3-(5,6-dihydrodibenzo[2,1-b:2',1'-][7]annulen-11-ylidene)-N-methylpropan-1-amine.² Nortriptyline inhibits the reuptake of the neurotransmitter serotonin at the neuronal membrane or acts at beta-adrenergic receptors. Pregabalin (PREGA) is chemically (3S)-3-(amino methyl)-5-methylhexanoic acid.¹ Pregabalin binds to an auxiliary subunit of voltage-gated calcium channels in central nervous system tissue with high affinity to the alpha2-delta site.

This combination is used for nerve damage pain, seizures, depression, anxiety disorder in adults and other conditions. Literature survey revealed that HPLC method had been reported for estimation of NORT and PREGA either as single drug or in combination with other drugs. To best of our knowledge, no reports were found for Development and Validation on HPTLC determination of NORT and PREGA in combined tablet dosage form.^9-17 HPTLC is widely standardized method used for validation method for qualitative and quantitative analysis.^6-8 HPTLC is sophisticated instrument, controlled by an integrated software ensure the reliability, reproducibility of generated data and highest possible degree the usefulness. HPTLC technique for simultaneous determination of NORT and PREGA as bulk and in tablet dosage form.^3-5 This present study was developed and validated as per ICH guidelines.¹⁸

Fig 1 Structure of NORT

Fig: 2 Structure of PREGA.

EXPERIMENTAL WORK:

Material and Chemical reagent:

Working standard of Nortriptyline was obtained gift sample from Alkem Laboratories Ltd, Mumbai, Maharashtra. Pregabalin was obtained gift sample from Cipla Pvt. Ltd, Mumbai. All chemicals were used as analytical reagent (AR) grade.

Instrumentation:

A HPTLC system (camag, Switzerland) equipped with sample applicator (Linomat V) And Hamilton syringe (100uL capacity), camag TLC Scanner-3 operated through WinCAT software 1.4.2 version, Camag twin trough chamber (20×10cm) were used for the TLC study.

Selection of Mobile Phase and Chromatographic Conditions:

Chromatographic separation studies were carried out on the mixed standard solution of NORT and PREGA. Different mobile phases containing various ratios of Toluene, Methanol, Ethyl acetate, and Chloroform, Glacial acetic acid, benzene, n-Hexane were examined. Finally, the mobile phase containing Toluene: Ethyl acetate: Methanol (6: 2: 1, v/v/v) was selected as optimal for obtaining well defined and resolved peaks. Other chromatographic conditions like chamber saturation time, run length, sample application rate and volume, sample application positions, distance between tracks, detection wavelength, were optimized to give reproducible R_f values, better resolution, and symmetrical peak shape for the two drugs.

Selection of Analytical Wavelength:

Densitometric scanning of the developed plates was carried out on CAMAG TLC scanner 3 in the reflectance-absorbance mode operated by WINCATS software version 1.4.2. The slit dimension was kept at 6 mm ´ 0.45 mm and 20 mm/spot scanning speed was employed. Source of radiation utilized was deuterium lamp emitting a continuous UV spectrum over the range of 200- 400 nm and the spectra were overlain. It was observed that both drugs showed considerable absorbance at 210 nm.

Preparation of Standard Stock Solution:

Standard stock solution of NORT was prepared by dissolving 10 mg of drug in 10 mL methanol to get working standard stock solution of 1000 ng µL^-1 from which 1 mL of the solution was diluted further with methanol to achieve final concentration 100 ng µL^-1. Standard stock solution of PREGA was prepared by dissolving 75 mg of drug in 10 mL methanol to get working standard stock solution of 7500 ng µL^-1 from which 1 mL of the solution was diluted further with methanol to obtain final concentration 750 ng µL^-1.

Analysis of formulation:

For the assay of marketed formulation, twenty tablets each containing 10 mg of NORT and 75 mg PREGA were weighed and finely powdered. Powder equivalent to 10 mg of NORT and 75 mg of PREGA were taken in 10 mL volumetric flask having 7 mL methanol. The contents were sonicated for 15 min and filtered and volume was made with solvent to obtain solution having concentration 1000 ng μL^-1 for NORT and 7500 ng μL^-1 for PREGA.  From this solution, one mililitre of the solution was diluted further using methanol to get the concentration of 100 ng μL^-1 for NORT and 7500 ng μL^-1 for PREGA.

Fig. 3: In situ overlain spectrum of NORT and PREGA measured from 200 to 400 nm.

Fig 4: Representative densitogram of marketed formulation containing 10 mg of NORT and 75 mg of PREGA.

Two μL of this solution was applied on the plate. After chromatographic development peak areas of the bands were measured at 210nm and the amount of each drug present in each sample was estimated from the respective calibration curve. Procedure was repeated six times for the analysis of homogenous sample. The % drug content (mean±S.D.) was calculated.

Method Validation:

According to ICH guidelines

Linearity:

The standard stock solutions of NORT (100ng µL^-1) and PREGA (750ng/µl) were applied by over spotting on HPTLC plate in range of 1, 2, 3, 4, 5 and 6µl with the help of CAMAG 100µl sample syringe, using Linomat 5 sample applicator to obtain final concentration 100-600 ng band^-1 for NORT and 750-4500ng band^-1 for PREGA. The plate was developed and scanned under above established chromatographic conditions. Each standard in six replicates was analyzed and peak areas were recorded. Calibration curves of NORT and PREGA were plotted separately of peak area Vs respective concentration. Linear response was observed in the concentration range 100-600ng band^-1 for NORT and 750-4500ng band^-1 for PREGA.

Precision:

The repeatability study was performed to check precision under same operating conditions over short interval of time. In Intra-day precision the following three standard solutions used viz. 300ng band^-1, 400ng band^-1, 500 ng band^-1 for NORT and 2250ng band^-1, 3000 ng band^-1, 3750ng band^-1 for PREGA and these solutions were spotted in three replicates and standard deviation of areas were noted. Inter-day precision was studied by analyzing three different concentrations of standard drug solutions within the linearity range on three different days for period of one week and %R.S.D (Percent Relative Standard Deviation) was determined.

Accuracy:

To check the accuracy of the method, recovery studies were carried out by spotting a mixture of standard drug solution to pre-analyzed sample solution at three different levels 50, 100 and 150%. Basic concentration of sample chosen was 200ng band^-1 for NORT and 1500 ng band^-1 for PREGA. The areas were noted after development of plate. The drug concentrations of NORT and PREGA were calculated by using regression equations.

_{Limit of detection (LOD) and Limit of quantitation (LOQ):}

LOD and LOQ were calculated as 3.3 σ/S and 10 σ/S, respectively. Where, σ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot.

_Specificity:

The specificity of the method was ascertained by analyzing standard drug and sample. The spots for both drugs in sample were confirmed by comparing the Rf values of both drugs and spectra of the spots in sample with that of standard drug spots.

Robustness:

The robustness of the method was studied, during method development, by small but deliberate variations in mobile phase composition (±2% methanol), wavelength (±1nm) One factor at a time was changed at a concentration level of 600ng band^-1 for NORT and 4500ng band^-1 for PREGA respectively, to study the effect on the peak area of the drugs.

Stress degradation studies of bulk drug:

Forced degradation studies were carried out to provide evidence on how stability of drug varies under the influence of variety of environmental conditions like hydrolysis, oxidation, temperature, etc. and to establish specific storage conditions, shelf-life and retest period. In order to achieve degradation within the limit recommended by ICH, stress degradation was performed under different stress conditions.

Acid treatment:

1 mL working standard solution of NORT (1000ng µL^-1) was mixed with 1mL of 0.1 N HCl (methanolic) and 8 mL of methanol. The mixture was refluxed at 45°C for 4 hours. 4µL of the resulting solution was applied to HPTLC. 1mL working standard solution of PREGA (7500ng µL^-1) was mixed with 1mL of 0.1 N HCl (methanolic) and 8 mL of methanol. The mixture was refluxed at 60°C for 3 h. 4µL of the resulting solution was applied to HPTLC.

Alkali treatment:

1 mL working standard solution of NORT (1000ng µL^-1) was mixed with 1mL of 0.1 N NaOH (methanolic) and 8 mL of methanol. The mixture was refluxed at 45°C for 6 hours. 4µL of the resulting solution was applied to HPTLC. 1mL working standard solution of PREGA (7500ng µL^-1) was mixed with 1mL of 0.1 N NaOH (methanolic) and 8 mL of methanol. The mixture was refluxed at 60°C for 3 h. 4µL of the resulting solution was applied to HPTLC.

Oxidation:

1 mL working standard solution of NORT (1000ng µL^-1) was mixed with 1 mL of 3 % solution of H₂O₂ and 8 mL of methanol. The mixture was refluxed at 45°C for 3 hours. 4µL of the resulting solution was applied to HPTLC. 1 mL working standard solution of PREGA (7500 ng µL^-1) was mixed was mixed with 1 mL of 3 % solution of H₂O₂ and 8mL of methanol. The mixture was refluxed at 60°C for 3 hours. 4µL of the resulting solution was applied to HPTLC.

Neutral hydrolysis:

1mL working standard solution of NORT (1000 ng µL^-1) was mixed with 1mL of water and 8 mL of methanol. The mixture was refluxed at 45°C for 5 hours. 4µL of the resulting solution was applied to HPTLC. 1mL working standard solution of PREGA (7500ng µL^-1) was mixed was mixed with 1 mL of water and 8mL of methanol. The mixture was refluxed at 60°C for 3 h. 4 µL of the resulting solution was applied to HPTLC.

Degradation under dry heat:

Dry heat study was performed by keeping NORT in oven at 60ºC for 10 hours and PREGA at 80ºC for 2 hours. A sample was withdrawn at appropriate times, weighed and dissolved in methanol to get solution of 100 ng µL^-1 for NORT and 750ng µL^-1 for PREGA.  4µL of the resulting solution was applied to HPTLC.

Photo degradation:

Photolytic studies were carried out by exposure of NORT and PREGA to UV light up to 200-watt h square meter^-1 for 1 and 7 d, respectively. Sample was appropriately weighed and dissolved separately in methanol to obtain concentration of 100 ng µL^-1 and 750 ng µL^-1 for NORT and PREGA, respectively.

RESULT AND DISCUSSION:

Method validation:

Linearity:

The linearity of proposed method was found in range of concentration 100-600 ng band^-1 for NORT and 750-4500 ng band^-1 for PREGA. Calibration curves of NORT and PREGA were plotted separately of peak area Vs respective concentration. Calibration curve shown in fig 3 and 4. Linear response was observed in the concentration range100-600 ng band^-1 for NORT and 750-4500 ng band^-1 for PREGA. Results are shown in table 1 and 2.

Fig 5 : Calibration curve for NORT.

Fig 6: Calibration curve for PREGA.

Table 1: Linearity results for NORT

Conc. Spotted (ng band^-1)	Average Area	S.D.*
100	1208	43.56
200	1982	39.68
300	2697	34.96
400	3435	89.34
500	4126	103.84
600	4770	58.64

*n=6

Table 2: Linearity results for PREGA

Conc. Spotted (ng band^-1)	Average Area	S.D.*
750	2865	60.27
1500	4057	48.98
2250	5169	24.77
3000	5945	90.46
3750	6839	60.47
4500	7784	57.90

*n=6

Precision:

The % RSD was found to be in the range of 0.85-1.15 for NORT and 0.39-0.99 for PREGA. The results obtained for intraday variations for NORT and PREGA are shown in Table 3. The % RSD was found to be in the range of 1.04-1.57 for NORT and 0.40-1.06 for PREGA. The results obtained for Inter day variations for NORT and PREGA are shown in table 4.

Table 3: Intra-day precision of NORT and PREGA

Drug	Amount spotted (ng band^-1)	Average Area	% Assay	S.D.	% R.S.D.*
NORT	300	2670	99.57	0.8742	0.8780
	400	3384	99.67	1.1477	1.1514
	500	4079	99.22	0.8511	0.8577
PREGA	2250	4944	99.44	0.6577	0.6615
	3000	5922	99.97	0.9951	0.9954
	3750	6894	100.16	0.3967	0.3961

                                 *n = 3

Table 4: Inter-day precision of NORT and PREGA

Drug	Amount spotted (ng band^-1)	Average Area	% Assay	S.D.	% R.S.D.*
NORT	300	2670	99.58	1.5650	1.5715
	400	3365	99.03	1.4558	1.4700
	500	4074	99.07	1.0319	1.0415
PREGA	2250	4954	99.79	0.9708	0.9728
	3000	5922	99.96	1.0666	1.0670
	3750	6885	99.97	0.4073	0.4074

*n = 3

Accuracy:

The drug concentrations of NORT and PREGA were calculated by using regression equations. The % mean recovery was found to be 99-101 for NORT and 99-100 for PREGA. The results obtained are

shown in table 5.

Table 5: Recovery Studies of NORT and PREGA

Drug	Sample conc. (ng band^-1)	Added conc. (ng band^-1)	Total conc. found (ng band^-1)	% Recovery	S.D.	% R.S.D.^*
NORT	200	100	298.72	99.57	1.604	1.61
	200	200	400.96	100.23	1.443	1.44
	200	300	499.92	99.98	0.76	0.76
PREGA	1500	750	2240.91	99.59	0.667	0.66
	1500	1500	3002.85	100.09	0.887	0.88
	1500	2250	3720.66	99.21	0.756	0.76

^*Average of three determinations.

LOD and LOQ:

The LOD of NORT and PREGA were found 15.48ng band^-1 and 136.97ng band^-1, respectively. The LOQ of NORT and PREGA were 46.92 ng band^-1 and 415.06ng band^-1, respectively.

Robustness:

The robustness of the method was calculated. %RSD by Variation in mobile phase composition (± 2% methanol) was found to be 1.52% and 0.640% for NORT and PREGA, respectively. %RSD by variation in wavelength (± 1nm) was found to be 1.09% and 0.429% for NORT and PREGA, respectively. Results are shown in table 6.

Table 6: Robustness Data in Terms of % RSD of Retention Time

Sr. No.	Parameters	Variation	S. D.		% R.S.D*
			NORT	PREGA	NORT	PREGA
1.	Mobile phase composition	+2% Methanol	65.50	32.59	1.377	0.421
		-2% Methanol	78.58	65.31	1.679	0.859
		± 2 % Methanol	72.04	48.95	1.52	0.640
2.	Wavelength	+1 nm	59.55	37.89	1.271	0.490
		-1 nm	42.71	28.47	0.911	0.369
		± 1 nm	51.13	33.18	1.09	0.429

*Average of three determinations.

Analysis of formulation:

The % drug content (mean±S.D.) was found to be 99.32±1.39 for NORT and 99.75±1.15 for PREGA. The results obtained are shown in table 7 and 8.

Table 7: Analysis of formulation

Amount taken (ng band^-1)		Amount found (ng band^-1)		% Drug content
NORT	PREGA	NORT	PREGA	NORT	PREGA
200	1500	199.94	1492.21	99.97	99.48
200	1500	197.28	1512.46	98.64	100.83
200	1500	198.12	1479.75	99.06	98.65
200	1500	201.06	1498.44	100.53	99.89
200	1500	195.88	1475.85	100.74	98.39
200	1500	194.05	1519.47	97.02	101.29

Table 8: Statistical data for analysis of formulation

Drug	Mean*	S.D.*	%RSD
NORT	99.32	1.39	1.403
PREGA	99.75	1.15	1.159

* Average of six determinations.

Stress degradation studies of bulk drug:

Acid treatment:

NORT was found to be labile to acid hydrolysis in 0.1N HCl at 45°C. About 12.32 % degradation was observed forming degradation products showing Rf 0.51.  After refluxing at 60°C for 30 min, PREGA was degraded by 16.86% with forming the degradation products at Rf values 0.25 and 0.56. The representative densitogram obtained after acid treatment for NORT and PREGA are shown in fig. 7, 8 and table 9.

Fig 7: Representative densitogram of NORT after acid hydrolysis with degradation product (D1, Rf = 0.51).

Fig 8: Representative densitogram of PREGA after acid hydrolysis with degradation products (D2, Rf = 0.25) and (D3, Rf = 0.56).

Fig 9: Representative densitogram after alkali treatment for NORT with degradation product D4 (Rf = 0.78).

Fig 10: Representative densitogram after alkali treatment for PREGA with degradation product D5 (Rf = 0.18).

Alkali treatment:

15.61 % degradation of the NORT was observed in 0.1N NaOH at 45°C. The degradation peak appeared at Rf 0.78, Whereas 16.32 % degradation was seen in PREGA in alkaline condition. One peak was generated at Rf 0.18. The representative densitogram after alkali treatment for both drugs are shown in fig. 9, 10 and table 9.

Oxidation:

16.59 % degradation was observed for NORT with degradation peak at Rf 0.83 and 15.47 % degradation was observed for PREGA with degradation peaks at Rf values 0.23 and 0.26. The representative densitogram after oxidative degradation for both drugs are shown in fig. 11, 12 and table 9.

Fig 11: Densitogram of NORT after treatment with 3 % H₂O₂ with degradation peak D6 (Rf = 0.83).

Fig 12: Densitogram of PREGA after treatment with 3 % H₂O₂ with degradation products D7 (Rf = 0.23) and D8 (Rf = 0.26).Neutral hydrolysis:

In neutral condition, NORT was found to be degraded with 88.48 % recovery. Additional peak of degradation was shown by drug at Rf 0.60 in fig 13. On the other hand, 7.16 % degradation of PREGA was seen after neutral hydrolysis. Reduction in the peak area of drug was observed without appearance of any degradation peak.

Degradation under dry heat:

Thermo labile property of NORT was clearly observed when it was exposed to dry heat at 60ºC for 10 hours. Profound degradation (14.70 %) was seen with a single degradation peak at Rf 0.86. shown in fig 14. On the other hand, 8.10 % degradation was shown by PREGA without any product of degradation.

Photo degradation:

There was no change in the peak area of PREGA indicating that it is stable under photolytic degradation. However mild degradation was observed for NORT without any peak of degradation.

Fig 13: Densitogram of NORT after neutral hydrolysis with degradation product D9 (Rf = 0.60).

Fig 14: Densitogram obtained after dry heat degradation for NORT with degradation peak D10 (Rf 0.86).

Table 9: Stress degradation summary for NORT and PREGA

Mode of degradation		% Degradation		% Recovery
NORT	PREGA	NORT	PREGA	NORT	PREGA
Acid (0.1 N HCl) Reflux at 45°C for 4 hrs.	Acid (0.1 N HCl) Reflux at 60° C for 3 hrs.	12.32	16.86	87.68	83.14
Alkali (0.1N NaOH) Reflux at 45°C for 6 hrs.	Alkali (0.1 N NaOH) Reflux at 60° C for 3 hrs.	15.61	16.32	84.39	83.68
Oxidation (3 % H2O2) Reflux at 45°C for 3 hrs.	Oxidation (3 % H2O2) Reflux at 60° C for 3 hrs.	16.59	15.47	83.41	84.53
Neutral (H2O) Reflux at 45°C for 5 hrs.	Neutral (H2O) Reflux at 60° C for 3 hrs.	11.51	7.16	88.48	92.83
Photolysis (UV light for 1 day)	Photolysis (UV light for 7 day)	6.19	Stable	93.81	99.89
Dry heat at 60º C for 10 hrs.	Dry heat at 80º C for 2 hrs.	14.70	8.10	85.30	91.90

CONCLUSION:

Over the past decades, HPTLC has been successfully used in the analysis of pharmaceuticals, plant constituents and bio-macromolecules. Literature survey revealed that no stability indicating HPTLC method has been reported so far for the simultaneous estimation of NORT and PREGA in pharmaceutical tablet dosage form.

Therefore, stability indicating HPTLC-Densitometric method for simultaneous estimation of NORT and PREGA as bulk drugs and in pharmaceutical tablet dosage form has been developed and validated as per ICH Guidelines.

The standard deviation, % RSD calculated for the method are low, indicating a high degree of precision of the method. The results of the recovery studies performed show a high degree of accuracy of the proposed method. The results of the stress studies indicated the specificity of the method. The method gives well-resolved peaks of NORT and PREGA even after exposure to different stress conditions when analyzed individually. The method can be used to determine the purity of the drugs available from various sources by detecting the related impurities.

As the method is stability indicating one it may be extended to study the degradation kinetics of NORT and PREGA in combination. Hence, it can be concluded that the developed TLC-densitometry method is accurate, precise, and selective and can be employed successfully in the estimation of NORT and PREGA in bulk and in pharmaceutical formulation.

ACKNOWLEDGEMENT:

Authors are grateful to Alkem Laboratories Ltd, Mumbai for providing Nortriptyline as a gift sample and also grateful to Cipla Pvt. Ltd, Mumbai for providing Pregabalin as a gift sample. The authors are also thanks to the Director, School of pharmacy, S. R. T. M. University, Nanded, for providing research facilities.

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Received on 28.10.2022       Modified on 16.11.2022

Accepted on 30.11.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(1):21-29.

DOI: 10.52711/2231-5675.2023.00004