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Stability Indicating Method Development and Validation for the Determination of Armodafinil in Pharmaceutical Tablet Dosage Form by RP-HPLC

Nalini Kanta Sahoo1*, Ramya G2, Harini D3, Tarun Kumar Padhiary3

1SRM Modinagar College of Pharmacy, SRMIST (Deemed to be University), Delhi-NCR Campus, Uttar Pradesh, India

2Yalamarty Pharmacy College, Visakhapatnam, Andhra Pradesh, India

3Marri Laxman Reddy Institute of Pharmacy, Telangana, India

*Corresponding Author:
Nalini Kanta Sahoo, Principal
SRM Modinagar College of Pharmacy
SRMIST (Deemed to be University)
Delhi-NCR Campus, Uttar Pradesh, India.
Tel: +91 9550741536
E-mail: [email protected]

Received Date: 02/06/2021; Accepted Date: 26/08/2021; Published Date: 02/09/2021

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Abstract

Therapeutic drug monitoring in terms of quality is very much important in pharmaceutical industries. Some of the barriers are like finding out the purity and maintaining uniformity content in pharmaceutical dosage form. To overcome such problems, the present research deals with the development of a stability indicating reverse phase HPLC with PDA detector method for the determination of Armodafinil Agilent XDB-C18, 150 × 4.6 mm, 5 μm or Equivalent column. The flow rate was kept at 1.0 ml/min and the injection volume 10 mL and the run time is 8 min and drug Rt is 3.354. The separation was performed at 30°C. Eluents were monitored by PDA detector set at 223 nm. The developed method was statistically validated and results for the linearity is 0.999 and for System suitability, theoretical plates are 2500 and its tailing factor is 1.64, Precision is 0.1, LOQ is 1.00 μg/ml, LOD is 0.33 μg/ml, accuracy is 100.19, Robustness (flow rate, mobile phase) is complied.

Keywords

RP-HPLC, Armodafinil, Forced degradation.

Introduction

Armodafinil [1-3] is the Enantiomer pure compound of the euro-genic modafinil (Provigil). It consists of only the (R) (−) enantiomer of the racemic modafinil. Armodafinil is currently FDA-approved to treat excessive daytime sleepiness associated with obstructive sleep apnea, narcolepsy and shift work disorder. It is commonly used off-label to treat attention deficit hyperactivity disorder, chronic fatigue syndrome and major depressive disorder. It has been shown to improve vigilance in air traffic controllers. Literature review [4-7] reveals very less works done, it became very interesting to pursue the work and to implement the therapeutic drug monitoring in terms of stability.

Experimental Procedure

Drug Profile

IUPAC name : (–)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide

Molecular formula : C15H15NO2S

Molecular weight : 273.35

PKa value : 8.839

Melting Point : 156-158°C

HPLC Instrumentation and Conditions

Instrumentation and analytical conditions: The analysis of the drug was carried out on a HPLC system equipped with a reverse phase HPLC with PDA detector method for the determination of Armodafinil Agilent XDB-C18, 50 × 4.6 mm, 5 μm or Equivalent column. A mobile phase consisting of Phosphate Buffer: Acetonitrile (65:35 v/v) was employed in this study. The flow rate was kept at 1.0 ml/min and the injection volume 10 mL and the run time is 8 min. The separation was performed at 30°C. Eluents were monitored by PDA detector detector (Waters 2695 Separation Module Equipped with 2996 PDA) set at 223nm.

Chemicals and Reagents

All the chemicals used were of analytical grade and procured from Qualigens India Ltd., Rankem Chemicals Ltd. The chemicals used for the study were, Potassium di-hydrogen phosphate purchased from Merck, Methanol purchased from Rankem, Water and Acetonitrile from Rankem and other chemicals are Ortho Phosphoric acid, Hydrochloric Acid, Sodium Hydrogen Peroxide and Sodium Hydroxide.

Preparation of phosphate buffer

Accurately weighed 2.72 gm of potassium dihydrogen phosphate in 1000 ml of Volumetric flask add about 900 ml of milli-Q water and sonicate and make up to the final volume with milli-Q water, add 1 ml of Triethylamine and then PH 5.6 is adjusted with dilute orthophosphoric acid solution.

Preparation of mobile phase

Mix 600 ml of phosphate buffer pH 5.6 and 400 ml of Acetonitrile (HPLC grade) in a ratio of (65:35% v/v) degassed in ultrasonic water bath for 5 minutes. Filtered through 0.45 μ filter under vacuum filtration

Diluent

Prepare filter and degass the mixture of HPLC grade water & methanol in a Ratio of (20:80% v/v).

Preparation of standard solution of Armodafinil

Accurately Weighed and transferred 10 mg of Armadofinil working Standards into a 10 ml clean dry volumetric flask, add 7 ml of methanol, sonicated for 5 minutes and make up to the final volume with methanol (standard stock 1000 μg/ml). From the filtered solution 0.5 ml was pipette out into a 10 ml volumetric flask made up with diluents.

Assay of Armodafinil 1 N Armod 20 Tablets

Preparation of sample solution of Armodafinil

Twenty tablets were weighed and crushed into powder, in order to calculate the average weight of each tablet. From that powder weight equivalent to 50 mg of Armodafinil was transferred into a 100 ml volumetric flask, 70 ml of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution 0.2 ml was pipetted out into a 10 ml volumetric flask and made up to 10 ml with diluent. The peak areas were measured at 223 nm and concentrations in the samples were determined by interpolation from calibration plot previously obtained.

Estimation of Armodafinil in tablet dosage form

Assay was performed by using the regression equation (y = 100769x + 27300, R2=0.9991) obtained from the standard curve of Temozolomide API.

Forced Degradation Studies

The drug was subjected to stress conditions in various ways to observe the rate and extent of degradation that is likely to occur in the course of storage and/or after administration to body. The various degradation pathways studied are Acid degradation, alkaline degradation, Oxidative degradation, Thermal degradation, Photo degradation.

Method Validation

Linearity and Calibration Curve

The linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analytes in samples within a given range or proportional by means of well-defined mathematical transformations. Linearity may be demonstrated directly on the test substance (by dilution of a standard stock solution) and/or by using separate weight of synthetic mixtures of the test product components, using the proposed procedure [8-11].

Accuracy

Accuracy was best determined by the standard addition method. Previously analyzed samples of Armodafinil API were added with standard drug solutions and are analyzed by the proposed method. Recovery (%) and RSD (%) were calculated for each concentration.

Precision

Precision was determined as both repeatability and intermediate precision, in accordance with ICH guidelines. Repeatability of sample injection was determined as intra-variation and intermediate variation was determined by measurement of inter day variation. For these determinations, three concentrations of the solutions of Armodafinil API were used.

Robustness

The concept of robustness of an analytical procedure has been defined by the ICH as “a measure of its capacity to remain unaffected by small but deliberate variations in method parameters”. The robustness of a method is the ability to remain unaffected by small changes in parameters such as pH of the mobile phase, temperature, % organic solvent strength and buffer concentration etc. To determine the robustness of the method experimental conditions are purposely altered and chromatographic characters are evaluated. Influence of small changes in chromatographic conditions such as change in flow rate (± 0.1 ml/min), temperature (± 2°C), wavelength of detection (± 2 nm) and water content in mobile phase (± 2%) were studied to determine the robustness of the method.

Limit of Detection (LOD)

The limit of detection (LOD) of an analytical method may be defined as the concentration, which gives rise to an instrument signal that is significantly different from the blank. For spectroscopic techniques or other methods that rely upon a calibration curve for quantitative measurements, the IUPAC approach employs the standard deviation of the intercept (Sa), which may be related to LOD and the slope of the calibration curve, b, by

Limit of Quantitation (LOQ)

The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represent the concentration of analyte that would yield a signal-to-noise ratio of 10.

LOQ = 10 Sa/b

Where, Sa is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the drugs and b is slope of the corresponding calibration curve.

Specificity

The specificity of the method was determined by exposing the drug sample to acidic (0.1 N HCl), basic (0.1N NaOH) and oxidizing (3% H2O2) stress conditions. The resulting solutions were then analyzed and the analyte peak was evaluated both for peak purity and for resolution from the nearest eluting peak.

Stability

Stability of pharmaceutical product may be defined as, the capacity of a particular formulation in a specific container or closure system, to remain within its physical, chemical, microbiological, therapeutic and toxicological specifications.

Stability of Armodafinil API was determined after storage of the drug solution for 24 hours at room temperature (25 ± 2°C).

Results and Discussion

Optimization of Chromatographic Conditions

The chromatographic conditions were optimized by different means i.e. using different column, different mobile phase, different flow rate, different detection wavelength and different diluents for sample preparation etc. and finally the prescribed method is accepted. Chromatographic conditions were listed in Tables 1-3 and Figures 1-3.

Trial Column Flow rate (ml/min) Temp Mobile phase Wave length Observation Remarks
1 Agilent XDB, C18, 50×4.6 mm, 5 µm or Equivalent 1.0 ml/min 30°C Phosphate Buffer: Methanol (70:30) 223 nm Broad peak was observed. Method rejected
2 Agilent XDB, C18, 150×4.6 mm, 5 µm or Equivalent 1.0 ml/min 30°C Phosphate Buffer: Acetonitrile (90:10) 223 nm No peak was observed Method rejected
3 Agilent XDB, 150×4.6 mm, 5 µm or Equivalent 1.0 ml/min 30°C Phosphate Buffer: Acetonitrile (80:20) 223 nm No peak was observed Method rejected
4 Agilent XDB, C18, 150×4.6 mm, 5 µm or Equivalent 1.0 ml/min 30°C Phosphate Buffer: Acetonitrile (75:25) 223 nm Poor plate count Method rejected
5 Agilent XDB, C18, 250×4.6 mm, 5 µm or Equivalent 1.0 ml/min 30°C Phosphate Buffer: Acetonitrile (70:30) 223 nm Peak with more tailing is observed. Method rejected
6 Agilent XDB, C18, 150×4.6 mm, 5 µm or Equivalent 1.0 ml/min 30°C Phosphate Buffer: Acetonitrile (65:35) 223 nm A sharp peak with good plate count is observed. Method accepted

Table 1. Analytical method development trials.

Column Agilent XDB C18, 150 × 4.6 mm, 5 m
Detector wavelength 223 nm
Column temperature 30°C
Injection volume 10 mL
Run time 8 min
Diluent Water: Methanol (20:80)
Mobile phase Buffer: Acetonitrile (65:35)
Drug RT 3.354
Elution technique Isocratic

Table 2. Optimized chromatographic conditions.

  Retention Times Peak Area Tailing Factor Theoretical Plates
1 3.301 1822152 1.65 2447
2 3.307 1825225 1.68 2408
3 3.31 1810134 1.67 2482
4 3.325 1810210 1.63 2483
5 3.326 1810976 1.61 2363
6 3.35 1815203 1.61 2456
Mean -- 1815650 -- --
SD -- 6573 -- --
% RSD -- 0.3 -- --

Table 3. System suitability parameters.

chemistry-structure-armodafinil

Figure 1: Chemical structure of Armodafinil.

chemistry-calibration-curve

Figure 2: Calibration curve of Armodafinil API.

chemistry-optimized-trial

Figure 3: Chromatograph of optimized trial.

Accuracy - Recover Study

The recovery of the method, determined by adding a previously analyzed test solution with additional drug standard solution, was 100.19%. The values of recovery (%) and RSD (%) listed in Tables 4 and 5 indicate the method is accurate-chromatogram was shown in Figures 4 and 5.

Linearity Level (%) Concentration (ppm) Area
20 12.5 524067
50 25 1056889
70 37.5 1616960
100 50 2120262
120 62.5 2647995
150 75 3210254
-- Linearity concentration 12.5-75PPM
-- Slope 42691
-- Intercept 4295
-- Correlation coefficient 0.999

Table 4. Calibration of the HPLC method for armodafinil.

Accuracy Area % Recovery Mean Recovery
S1: 50% 919713 101.11 Mean=100.72%
S2: 50% 913107 100.38 S.D. = 0.365
S3: 50% 915759 100.67 % RSD = 0.36
S4: 100% 1810501 99.52 Mean = 99.88%
S5: 100% 1824997 100.31 S.D. = 0.404
S6: 100% 1815611 99.80 % RSD = 0.40
S7: 150% 2725468 99.87 Mean =99.98%
S8: 150% 2735077 100.23 S.D. = 0.2119
S9: 150% 2734699 99.84 % RSD = 0.21

Table 5. Results of accuracy.

chemistry-suitability-standard

Figure 4: Chromatogram of System suitability standard.

chemistry-chromatogram-accuracy

Figure 5: Chromatogram of accuracy.

Precision: System Precision and Method Precision

This method was carried out and the high values of mean assay and low values of standard deviation and % RSD (RSD NMT 2.0%) within a day and day to day variations for armodafinil revealed that the proposed method is precise and the final result obtained % RSD is 0.1. Results obtained are shown in Figures 6 and 7 and Tables 6 and 7.

chemistry-system-precision

Figure 6: Chromatogram of System precision.

chemistry-method-precision

Figure 7: Chromatogram of Method precision.

System Suitability Areas
1 1822152
2 1825225
3 1810134
4 1810210
5 1810976
6 1815203
AVG 1815650
SD 6573.01
% RSD 0.36

Table 6. Results of system precision.

Sample No Sample Areas % Assay
1. 1815404 99.79
2. 1811942 99.60
3 1819899 100.03
4 1816055 99.82
5 1815040 99.77
6 1816870 99.87
AVG 1815868 99.81
Standard Deviation 2592.30 0.1425
Relation standard
Deviation (% RSD)
0.10 --

Table 7. Results of method precision.

Robustness

Influence of small changes in chromatographic conditions such as change in flow rate (± 0.1 ml/min), Temperature (± 2°C), Wavelength of detection (± 2 nm) & buffer in mobile phase (± 2%) studied to determine the robustness of the method are also in favor of (Table 4, % RSD < 2%) the developed RP-HPLC method for the analysis of Armodafinil API. Results obtained are shown in Figure 10 and Table 8.

Inj. Sample Change parameter modification Peak area 1 Peak area 2 mean % RSD
Armodafinil Flow rate 0.8 ml/min 2018950 2008764 2008857 0.1
1 .1 ml/min 1620562 1615142 1617852 0.23
Mobile phase 60:40 1805335 1811345 1808340 0.23
70:30 1816046 1815471 1816258 0.1
temperature 25°C 1803514 1807444 1805479 0.2
35°C 1799955 1798639 1799297 0.1

Table 8. Results of robustness.

Linearity

In the linearity, correlation coefficient obtained is 0.999. Chromatograph of calibration curve is shown in Figure 2 and observe Table 4.

LOD & LOQ

The Minimum concentration level at which the analyte can be reliable detected (LOD) & quantified (LOQ) were found to be 0.33 μg/ml and 1.006 μg/ml respectively. Chromatograms obtained are shown in Figure 8.

chemistry-method-precision

Figure 8: Chromatogram of LOD and LOQ.precision.

Specificity: No peak was found at the retention time of armodafinil peak. Observe the peaks in Figure 9.0, 9.1, 9.2.

chemistry-drug-peak

Figure 9: Chromatography of Mobile phase; Chromatography of placebo; Chromatographyof drug peak.

chemistry-chromatograms-robustness

Figure 10: Chromatograms of robustness.

System Suitability: Theoretical plates are about 2500 and tailing factor obtained was 1.64. Observe the Table 3 and chromatograms obtained are shown in Figure 4.

Summary: Method validation parameters are shown in Table 9.

S. No Parameters Limit Observation
1 System suitability Theoretical plates should not less than 2000
Tailing factor should not more than 2.0
Theoretical plates
2500
Tailing factor 1.64
2 Precision RSD NMT 2.0% 0.1
3 Linearity Correlation coefficient
NLT0.99
0.999
4 Accuracy %Recovery range 98-102 100.19
5 Robustness (flow, mobile phase) System suitability parameters should comply Complies

Table 9. Summary of method validation parameters.

Stability in Analytical Solution

It is important to mention here that the armodafinil API was stable in solution form up to 72 hours at 25°C.

Forced Degradation Studies

Forced degradation studies based on peak purity results, obtained from the analysis of force degradation samples using described method, it can be concluded that the absence of co-eluting peak along with the main peak of armodafinil indicated that the developed method is specific for the estimation of armodafinil in presence of degradation products. The results of the forced degradation studies were given in Table 10 and Figures 11-15.

Mode of degradation Conditions Armodafinil
% Degradation w.r.t. control Purity angle Purity Threshold
Control No treatment - - -
Acid degradation 1N HCl 60°C/30 min 4.04 0.350 0.495
Alkali degradation 0.1N NaOH 60°C/30 min 7.97 0.12 0.30
Peroxide degradation 10% W/V H2O2 60°C/30 min 10.02 0.131 0.353
Thermal degradation 105°C/6 hrs 7.4 0.271 0.456
Photolytic UV/7 days 6.4 0.252 0.492

Table 10. Summary of forced degradation studies.

chemistry-acid-degradation

Figure 11: Chromatogram of Acid Degradation (1N HCL).

chemistry-base-degradation

Figure 12: Chromatogram of base degradation (0.1 N NaOH).

chemistry-oxidation-degradation

Figure 13: Chromatogram of oxidation degradation (10% H2O2).

chemistry-thermal-degradation

Figure 14: Chromatogram of thermal degradation.

chemistry-photolytic-degradation

Figure 15: Chromatogram of photolytic degradation.

Estimation of Armodafinil in Tablet Dosage Form

Assay was performed by using the regression equation (y = 100769x + 27300, R2=0.9991) obtained from the standard curve of temozolomide API. Results obtained are given in Table 5. The assay of containing armodafinil was found to be 99.8% as per the method.

Conclusion

From the above results furnished, the proposed RP-HPLC method was found to be simple, precise, accurate and sensitive for the determination of armodafinil in pharmaceutical dosage form. These are within short analysis time and the low value of RSD in comparison to the previous papers published; indicate that the proposed method is highly precise and accurate. High percentage of recoveries suggests that the proposed method is accurate. Forced degradation studies based on peak purity results, obtained from the analysis of force degradation samples using described method. It can be concluded that the absence of co-eluting peak along with the main peak of armodafinil indicated that the developed method is specific for the estimation of armodafinil in the presence of degradation products.

References

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