HPTLC Method Development and Validation for Nortriptyline Hydrochloride in Bulk and it’s Tablet

 

Arun Maruti Kashid*, Shivanjali Balasaheb Sable, Amey Chandrakant Khilare,

Aditya Mahesh Shinde

Department of Pharmaceutical Chemistry, Sinhgad Technical Education Society’s Sinhgad Institute of Pharmacy, Narhe, Pune, Maharashtra, India 411041.

Department of Pharmaceutical Quality Assurance, Sinhgad Technical Education Society’s Sinhgad Institute of Pharmacy, Narhe, Pune, Maharashtra, India 411041.

*Corresponding Author E-mail: arunkashid2006@gmail.com

 

ABSTRACT:

The simple, accurate, precise and sensitive High Performance Thin Layer Chromatography (HPTLC) method was developed and validated. The stationary phase used was precoated Silica gel F254 plates. The mobile phase used for method development contains methanol: ethyl acetate: ammonia (6: 4: 0.2 v/v). From the overlain spectra of standard and tablet the detection wavelength of 245 nm was selected. The validation parameters like linearity, precision, accuracy, limit of detection, limit of quantification and robustness were performed. For the linearity concentration range selected was 100-500 ng/band. The coefficient of regression (R2) was found to be 0.9900. The percent Relative Standard Deviation (%RSD) for intraday and interday precision study was found to be 0.3301-1.9046%. The % recovery was performed to study accuracy and the results were found to be 98.70-100%. The limit of detection and limit of quantitation was found to be 527.04 and 1597.45 ng/band. The robustness of the method was studied by making deliberate changes in the mobile phase composition and saturation time, the %RSD was found to be 0.9137 and 1.0869. The forced degradation study was performed in acidic, basic, oxidative and neutral conditions. The degradants were observed. The method was validated as per ICH guidelines and developed method can be used for routine analysis in quality control laboratories.

 

KEYWORDS: HPTLC, Nortriptyline hydrochloride, Validation, Forced degradation, ICH.

 

 

 

INTRODUCTION:

NORT is a type of antidepressant that belongs to the tricyclic class. It's derived from amitriptyline and acts similarly in the body. The IUPAC name of NORT is methyl (3- {tricyclo [9.4.0.0^ {3,8}] pentadeca-1(15),3,5,7,11,13-hexaen-2-ylidene} propyl) amine. The chemical name of NORT is C19H21N. NORT works by either stopping serotonin from being reabsorbed at the nerve endings in the brain or by affecting beta-adrenergic receptors. It's particularly good at preventing the reabsorption of noradrenaline, which might explain why symptoms improve more with NORT treatment. Unlike some other medications, tricyclic antidepressants like NORT don't affect dopamine reabsorption or block monoamine oxidase. Like other drugs in its class, NORT also affects several other receptors in the brain, including mACh receptors, histamine receptors, and 5-HT receptors. Figure 1. Depicts the chemical structure of NORT.

 

 

Figure 1. Chemical structure of NORT

 

High Performance Thin Layer Chromatography (HPTLC) is the most powerful advanced form of Thin Layer Chromatography (TLC) and consists of chromatographic layers of utmost separation efficiency and the application of sophisticated instrumentation for all steps in the procedure include accurate sample application, standardized reproducible chromatogram development and software-controlled evaluation. HPTLC is a concept that includes a widely standardized methodology based on scientific facts as well as the use of validated methods for qualitative and quantitative analysis. HPTLC meets all quality requirements for today’s analytical labs, to increase the resolution and to allow more accurate quantitative measurements. According to literature review, it was found that there are reported analytical methods for simultaneous estimation of NORT in bulk, pharmaceutical formulations along with combination of drugs, which include HPLC, RP-HPLC, HPTLC and UV-Spectroscopic method. The objective of this study to describe a simple, rapid, accurate, precise and robust HPTLC method for NORT in bulk and its tablet dosage form. The parameters used to validate the method were linearity, precision, accuracy, limit of detection, limit of quantification and robustness.

 

MATERIALS AND METHODS:

Materials:

Active pharmaceutical ingredient (API) working standard of NORT was purchased from Swapnaroop Pharmaceuticals Pvt. Ltd., Chhatrapati Sambhajinagar, Maharashtra, and tablet Sensival 25mg by Wallance Pharmaceuticals, was purchased from local pharmacy store. Methanol was used in mobile phase composition, and it is also used as a diluent, which was purchased from Hi Media Laboratories Pvt. Ltd. Mumbai. Other chemicals used were ethyl acetate, ammonia, toluene and ethanol of analytical grade was also purchased from Hi Media Laboratories Pvt. Ltd. Mumbai.

Instrumentation and Chromatographic Conditions:

The samples were applied in the form of bands of width 6mm with 100µl sample syringe on precoated silica gel 60F254 TLC plate (10cm × 10cm) with 250µm thickness, (E. MERCK, Darmstadt, Germany) using a Camag Linomat V sample applicator. The plates were washed prior to chromatography with methanol and then activated at 105şC for 15min The slit dimension was kept t 6mm x 0.45mm. The mobile phase consists of methanol: ethyl acetate: ammona (6: 4: 0.2 v/v). Linear ascending development was carried out in 10cm × 10cm twin trough glass chamber (Camag, Muttenz, Switzerland). The chamber saturation for mobile phase was done for 15 min, at temperature (25şC±5%), For UV- analysis Double beam UV spectrophotometer model V-730 manufactured by Jasco International Co. Ltd. Japan was used. FTIR Spectrophotometer of 4100 series manufactured by Jasco, Japan, was used for functional group detection. For accurate weighing of drug, analytical weighing balance model CB-50 was used, manufactured by Contech, Pune. UCB-40 Sonicator manufactured by Spectra Lab, Instruments, Mumbai was used for sonication purpose. Hot air oven 250 BSS manufactured by Pathak, Mumbai was used for drying purpose. Densitometric scanning was performed at 245nm using Camag TLC Scanner III controlled by WinCATs software version 1.4.3.6336.

 

Preparation of Standard Solution:

The 10mg NORT was accurately weighed and transfer into 10mL volumetric flask. The volume was makeup with methanol. The concentration of resultant solution was 1000µg/mL.

 

Preparation of Sample Solution:

Twenty tablets were accurately weighed, and average weight of the tablets was determined. The tablets were crushed using mortal and pastel and the powder was formed. An amount of powder equivalent to 2.4mg of NORT was then transferred in a 10mL volumetric flask. The 5mL of methanol was added into this volumetric flask and the solution was sonicated for 15 minutes. After sonication the volume was makeup with methanol.

 

Method Validation:

Linearity:

Linearity refers to the ability of an analytical method to produce test results that are directly proportional to the concentration of an analyte in a sample within a specified range. Standard solutions of NORT were applied on the TLC plate. Concentration range of 100-500ng/band was selected for linearity.

 

 

Accuracy:

The accuracy of the method was evaluated by employing the standard addition method, in accordance with the ICH guidelines. The known amount of standard solution was spiked against the tablet solution at different concentration levels of 80%, 100%, and 120%. The three replicates of each concentration were used for this study. For 100% standard concentration was assumed to be 300 ng/band. For 80% standard concentration was assumed to be 240ng/band. For 120% standard concentration was assumed to be 360ng/band. The concentration of sample solution was assumed to be 300ng/band. By calculating the recovery of NORT using this approach, the accuracy of the method was determined.

 

Precision:

Precision defines the consistency of measurements when the repeat analysis of the same sample under the same conditions is performed. It defines the closeness of measurements to each other. The three replicates of same concentration is used for interday as well as intraday precision.

 

Limit of Detection (LOD) and Limit of quantification (LOQ):

LOD is the lowest concentration in a sample that can be detected but not necessarily quantified under the stated experimental conditions and LOQ is the lowest concentration of an analyte in a sample that can be detected as well as quantified.

 

LOD = 3.3*(σ) / S

LOQ = 10*(σ) / S

Where, σ denotes standard deviation, S denotes slope.

 

Robustness:

Robustness of an analytical method refers to its ability to remain unaffected by small, deliberate variations in method parameters, providing an indication of its reliability during routine use. The change in mobile phase composition and saturation time were the parameters used to determine robustness of the method.

 

RESULTS AND DISCUSSION:

UV analysis:

The UV scanning was done from the wavelength range of 200-400 nm for determining the scanning wavelength for HPTLC analysis. The standard solution as well sample solution was used for UV analysis. From the overlain spectra of standard and sample solution, the 245 nm wavelength was selected. Figure 2 depicts the overlain UV spectra of NORT standard and Tablet.

 

 

Figure 2. depicts the overlain UV spectra of NORT standard and Tablet

 

Optimization of HPTLC Method:

The spotting was done on HPTLC plates with appropriate amounts of standard and sample solutions. The band application was done 8mm above the bottom and 15mm inward from the edges. Development was carried out in twin-trough chamber (10cm × 10cm). The optimised mobile phase was methanol: ethyl acetate: ammonia (6: 4: 0.2 v/v). The stationary phase used was precoated silica gel 60 F254 TLC plates. The band length was 6 mm. The saturation time for the mobile phase was 10min at room temperature (25±2°C). The distance covered by the solvent front was 8 cm, which took about 10min. Figure 3. depicts the densitogram of NORT.

 

 

Figure 3. Densitogram of NORT

 

Method Validation:

Linearity:

The linearity range of NORT was examined at concentrations of 100, 200, 300, 400, and 500 ng/band. The equation representing the linearity for NORT was determined as Y = 6.516x + 213.7. The coefficient of regression (R2) was found to be 0.9900. Figure 3. depicts the calibration curve of NORT. Figure 4. depicts the 3D densitogram of calibration curve. Table 1. depicts the linearity result.

 

 

Figure 3. Calibration curve of NORT

 

 

Figure 4. 3D densitogram of calibration curve

 

Table 1. Linearity result of NORT

Sr. No.

Application Vol. µl

Concentration (ng/band)

Peak Area

1

0.1

100

704.57

2

0.2

200

1604.50

3

0.3

300

2322.12

4

0.4

400

2893.54

5

0.5

500

3318.18

 

 

Calibration range

100-500

 

 

R2

0.9900

 

 

LOD/LOQ

527.05/1597.14

 

Accuracy:

The accuracy studies were performed at 80%, 100%, and 120% of the test concentration as per ICH guidelines. The % recovery of NORT at all three levels was found to be satisfactory. The % recovery for NORT at concentration levels of 80%, 100% and 120% was found to be 98.12-100.66. Table 2. depicts accuracy results.

 

Precision:

The %RSD for intraday and interday precision study was found to be 0.3301-1.9046%. The obtained results were within acceptable limits (Table 3).

Limit of detection and limit of quantitation:

The limits of detection (LOD) and quantification (LOQ) of NORT were found to be 527.04 ng/band and 1597.45 ng/band. Table 1. depicts the LOD and LOQ results.

 

Robustness:

The % RSD for change in mobile phase composition and saturation time was found to be 0.9137 and 1.0869. The method demonstrated consistent and reliable results across different conditions. Table 4. Robustness study: changes in mobile phase composition. Table 5. Robustness study: changes in saturation time.

 

Table 2. Accuracy result of NORT

Concentration (%)

Concentration of Tablet (ng/band)

Concentration of Standard (ng/band)

% Recovery

Average % Recovery

Run 1

Run 2

Run 3

80

300

240

97.57

98.08

98.71

98.12

100

300

300

99.08

99.61

98.99

99.22

120

300

360

101.14

100.02

100.83

100.66

 

Table 3. Precision result of NORT

Drug

Concentration (ng/band)

Precision Intraday (SD ± %RSD)

Precision Interday (SD ± %RSD)

NORT

300

(Morning)

26.75± 1.13

(Day 1)

35.94 ± 1.52

(Afternoon)

39.59 ± 1.68

(Day 2)

18.51 ± 0.79

(Evening)

7.86 ± 0.33

(Day 3)

44.77 ± 1.90

 

 

 

 

Table 4. Robustness study: changes in mobile phase composition

Mobile phase Composition (Methanol: Ethyl acetate: Ammonia 6: 4: 0.2 v/v)

NORT

6: 4: 0.2

2238.63

5.9: 4.1: 0.2

2198.09

6: 4.1: 0.1

2218.31

Mean

2218.34

SD

20.27

%RSD

0.9137

 

Table 5. Robustness study: changes in saturation time

Saturation time (min)

NORT

10

2382.05

15

2434.4

20

2408.2

Mean

2408.21

SD

26.17

%RSD

1.0869

CONCLUSION:

The newly developed HPTLC method was discovered to be fast, accurate, precise, and robust. The linearity, accuracy, precision, LOD, LOQ and robustness results were within the acceptable limits as per ICH guidelines. The HPTLC method presented in this study demonstrate its robustness and reliability in analyzing the target compounds. HPTLC offers several benefits over liquid chromatography methods, including the ability to analyze samples and standards concurrently on a single plate, minimal sample handling, short run times, reduced sample needs, and no sample pretreatment. As HPTLC continues to evolve with advancements in instrumentation and methodology, its role in analytical chemistry remains pivotal, providing efficient and cost-effective solutions for complex analytical challenges.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENT:

The authors are thankful to Swapnaroop Pharmaceuticals Pvt. Ltd., Chhatrapati Sambhajinagar for providing the API Sample of Nortriptyline hydrochloride. Authors are also thankful to Principal of Sinhgad Technical Education Society’s Sinhgad Institute of Pharmacy, Narhe, Pune, for providing necessary facilities.

 

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Received on 23.07.2024      Revised on 20.01.2025

Accepted on 14.05.2025      Published on 12.07.2025

Available online from July 21, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(3):170-174.

DOI: 10.52711/2231-5675.2025.00026

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