Isoabsorptive UV Spectrophotometry Method development and validation for simultaneous estimation of Eslicarbazepine Acetate and Carbamazepine

Ashwini Pansare*, Aishwarya Pandit, Dr. Hitanshi Darji, Arti Ingole,   Dr. Pravin Sable

Pharmaceutical Science/ S.S.P.S.S.Siddhi College of Pharmacy, Chikhali, Pune-411062/India

*Correspondence: pansareashwini30@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5710         

INTRODUCTION

Epilepsy is one of the most common chronic neurological disorders characterized by recurrent and unprovoked seizures resulting from abnormal electrical activity in the brain. Antiepileptic drugs play a crucial role in controlling seizures and improving the quality of life of patients. Among the various antiepileptic agents, Eslicarbazepine Acetate (ESL) and Carbamazepine (CBZ) are widely used for the management of focal-onset and generalized tonic-clonic seizures. [1,2,3]

Eslicarbazepine Acetate is a third-generation dibenzazepine derivative chemically designated as (S)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide. It acts mainly by stabilizing the inactive state of voltage-gated sodium channels, thereby reducing neuronal hyperexcitability and preventing repetitive neuronal firing. Owing to its favorable pharmacokinetic profile and improved tolerability, ESL has emerged as an effective therapeutic option for epilepsy management. [4,5,6]

Figure 1: Structure of ESL

Carbamazepine, chemically known as 5H-dibenz[b,f]azepine-5-carboxamide, is a widely prescribed anticonvulsant and mood-stabilizing agent. Its mechanism of action involves inhibition of voltage-dependent sodium channels, which suppresses abnormal neuronal discharges and limits the spread of seizure activity. [7,8,9]

Figure 2: Structure of CBZ  

Analytical methods are essential for the quality control and quantitative estimation of pharmaceutical compounds in bulk drugs and dosage forms. Several analytical techniques, including high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC), and liquid chromatography-mass spectroscopy (LC-MS), have been reported for the determination of Eslicarbazepine Acetate and Carbamazepine individually. However, the reported methods are often associated with high operational costs, extensive sample preparation, and longer analysis time. [10-13]

Ultraviolet (UV) spectrophotometry is a simple, rapid, economical, and reliable analytical technique that is widely employed in pharmaceutical analysis. A thorough literature survey indicated that no simple UV spectrophotometric method has been reported for the simultaneous estimation of Eslicarbazepine Acetate and Carbamazepine in combination. [14,15,16] Therefore, the present study aims to develop and validate a simple, accurate, precise, and cost-effective UV spectrophotometric method for the simultaneous estimation of Eslicarbazepine Acetate and Carbamazepine in bulk and pharmaceutical dosage forms in accordance with International Council for Harmonisation (ICH) guidelines. [17]

The combination of ESL (10 mg) and CBZ (10 mg) is in the ratio of 1:1 respectively. Literature survey reveals that, analytical techniques such as UV-Spectrophotometery and HPLC are published for determining ESL and CBZ as individual drugs and for their combinations with other drugs. No methods have been reported for the simultaneous determination of ESL and CBZ. [17,18,19]

METHODOLOGY

Instrumentation

UV-Visible Double Beam Spectrophotometer (Shimadzu 1800) and Precision Balance (Shimadzu ATX224) were used for the study.

Materials

Pure drug samples of ESL and CBZ were procured as a gift sample from Tablets India Ltd (Chennai) and Vav Life Sciences Pvt Ltd (Mumbai) respectively.

Solution Stability

The two original and test solutions were examined for a whole day at room temperature in order to show their stability throughout the evaluation process. A comparison was made between the restored absorbance and the original. The study's findings demonstrated that the absorbances of the two medications by percentage for either solution did not differ significantly. There was no discernible deterioration within the necessary time frame. [10,17]

Stock solution preparation

Standard stock solutions of pure drug containing 100µg/ml ESL                                                                                                                and CBZ were separately prepared in methanol. Standard stock solutions were further diluted with methanol to get working standard solutions in the concentration range of 5-25µg/ml and same for Carbamazepine (CBZ) respectively. [14,17,20,21]

Determination of absorption maxima and wavelength selection

To achieve final concentrations of 10 µg/mL for ESL and CBZ, stock solutions were appropriately diluted separately using a solvent. The maximum absorbance wavelength (λ max) of each of these solutions was determined by scanning them separately in the UV region (400 nm to 200 nm). The isoabsorptive point wavelength was chosen after overlay spectra of ESL and CBZ were produced. [22,23,24]

Isoabsorptive Point method

The Isoabsorptive Point method is a UV spectrophotometric technique used for the simultaneous estimation of two components in a mixture without prior separation. An isoabsorptive point is a wavelength at which two drugs exhibit the same absorptivity (or absorbance). At this wavelength, the absorbance of the mixture is the sum of the absorbances of both components. [25,26,27]

The method uses:

1. Isoabsorptive wavelength (λiso) – where both drugs have equal absorptivity.
2. λmax of one of the components.

Method Validation

The method was confirmed to be in accordance with ICH guideline Q2(R2) for the evaluation of numerous parameters, such as specificity, robustness, LOQ, LOD, accuracy, and precision. [28,29]

Specificity

The capacity to clearly access the analyte in the presence of elements that may be anticipated to be present is known as specificity. To demonstrate the method's specificity, a blank (methanol) and placebo solution were made and scanned between 400 and 200 nm.[30,31,32]

Linearity

In a succession of sets of 20 ml volumetric flasks, stock solutions of ESL and CBZ were appropriately diluted with a solvent to produce 5, 10, 15, 20 and 25 μg/mL solutions for ESL and CBZ separately. The absorbance of both solutions was measured at the isoabsorptive point of 254 nm and at λmax, which is 226 nm. Plotting concentration against absorbance, with each reading being an average of five measurements, allowed for the construction of the linearity curves. [33,34,35]

Precision

Repeatability, intraday precision, and interday precision were used to evaluate the precision of the suggested approach. Six replicates of the sample solution were used to test for repeatability. For intermediate precision, the corresponding responses of six replicates on the same and different days for the test solution containing ESL (15.0 μg/mL) and CBZ (15 μg/mL) alone and in combination were determined. The percentage RSD was used to report the results. [36,37,38]

Accuracy

The usual addition approach was used to conduct recovery investigations. The test solution of ESL and CBZ were spiked with known amounts of standard ESL and CBZ, specifically 80%, 100%, and 120% of the label claim. The percentage RSD was used to report the results. [39,40,41]

LOD and LOQ

The following formulas were used to determine the developed method's LOD and LOQ from the linearity curve:

LOD = 3.3 X σ/S;   LOQ = 10 X σ/S

Where, σ = Standard deviation (y-intercepts of calibration curves)

S= Slopes of linearity curves [42,43,44]

Assay

Weighed accurately 300 mg of lab prepared tablet mixture (Which is equivalent to 10 mg of ESL and 10 mg of CBZ) and transferred to volumetric flask, methanol was added and sonicated for 15 minutes with intermittent shaking after every 5 minutes. Made the volume up to the mark with methanol. Filter the test solution through suitable filter of 0.45micron syringe filter discarding first 3-5 mL of filtrate, diluted filtrate with methanol to get concentration 15.0 μg/mL of ESL and 15 μg/mL of CBZ and record the absorbance at 226 nm and 254 nm. [34,45,46]

Robustness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Determination of variations are: Changes in Extraction time by ±5 minutes for test solution preparation and Change in wavelength (± 2 nm) [47,48,49]

RESULT AND DISCUSSION

Absorption maxima and Isobestic point

Figure illustrates the absorption maxima for ESL and CBZ which were found to be 226 nm and 285 nm, respectively, and the isoelectric point at 254 nm with a distinctive peak. We have chosen 226 nm which is λmax of ESL and 254 nm which is isobestic point.
Therefore, we have chosen λ1 = 226 nm and λ2 = 254 nm.

Figure 3: Overlay UV spectrum of ESL & CBZ for wavelength selection

Specificity

To assess interference at the corresponding wavelengths of ESL and CBZ, blank and placebo solutions were created and scanned between 400 and 200 nm.

Figure 4: UV spectrum of methanol as a blank

Linearity

For ESL and CBZ, the linearity curve was built in the concentration range of 5 - 25 μg/mL, at their respective absorption maxima and at their isobestic point. Below tables provided the linearity data and figure shows the linearity curves. The method of least square regression analysis was used to examine the linear connection between concentration and absorbance. The findings point to a linear association between ESL and CBZ, with correlation coefficients value 0.999.

Table 1 Linearity of ESL at 226 nm and 254 nm

Table 2 Linearity of CBZ at 226 nm and 254 nm

Precision

Table provided the repeatability, intraday, and interday precision data for the test solution comprising ESL 15.0 μg/mL and CBZ 15 μg/mL. The percentage RSD for ESL and CBZ were found to be less than 2 %. The findings indicate that the modified approach was accurate, dependable, and repeatable for the simultaneous estimate of ESL and CBZ.

Table 3 Precision Data

Accuracy

Below table provided the accuracy statistics for ESL and CBZ from sample solutions at three levels (80%, 100%, and 120%) of the standard addition procedure. With an RSD of less than 2%, the mean percentage recovery for ESL was found to be 99.27 (80% level), 99.75 (100% level), and 100.15 (120% level). The results indicate that the CBZ hod was accurate and used for assay and recovery studies. The percentage recovery of CBZ was in the range of 100.69 (80% level), 99.81 (100% level), and 99.25 (120% level), with % RSD less than 2%.

Table 4 Accuracy data

LOD & LOQ

Below table shows the LOD and LOQ that were determined using the standard formula provided in the ICH guideline. For ESL at λ1 and λ2, the LOD was 0.05 µg/mL and 0.07 µg/mL; the LOQ was 0.15 µg/mL and 0.20 µg/mL. For CBZ at λ1 and λ2, the LOD was 0.10 µg/mL and 0.04 µg/mL; the LOQ was 0.31 µg/mL and 0.11 µg/mL.

Table 5 linearity data for LOD and LOQ

Table 6 LOD and LOQ data

Assay

The developed method was applied to both the lab prepared tablet solution and the sample mixed solution.  Table provides a result of assay. According to the marked claim, the percentage assay of the ESL and CBZ were found to be 99.35 and 100.85, respectively.

Table 7 Assay Data

From the above results, it can be concluded that the assay result is within the limit for prepared tablet physical lab mixture sample.

Robustness

Following changes made under Robustness:

A)     Change in Extraction time by ±5 minutes for test sample:

Table 83 Result of Robustness study for Change in Extraction time by minus 5 minutes (10 minutes):

Table 9 Result of Robustness study for Change in Extraction time by plus 5 minutes (20 minutes):

B)      Result of Robustness study for Change in wavelength by ± 2 nm

The % Assay were found to be in acceptance criteria in results remain unaffected by small, deliberate variations ensuring reliable and consistent results. Therefor the developed method is robust.

Table 10 Absorbance at ±2 nm (224 nm and 252 nm)

CONCLUSION

To ascertain ESL and CBZ in dosage form, a UV-Spectroscopic method that has been verified. Method was accurate, precise and reproducible and found to be simple, sensitive and selective for the estimation of ESL and CBZ This approach can simultaneously and independently determine drug combination in multi-component formulation. The method's simplicity allows it to be used in labs without sophisticated, costly, and time-consuming analytical equipment, in contrast to a simple UV method.

ACKNOWLEDGEMENT: We acknowledge our sincere thanks to S.S.P. Shikshan Sanstha’s Siddhi College of Pharmacy, Chikhali, Pune for providing facilities to carryout research work.

DATA AVAILABILITY: Data will be made available on request.

REFERENCES

1.      Fisher RS, Acevedo C, Arzimanoglou A, Bogacz A, Cross JH, Elger CE, et al. ILAE official report: A practical clinical definition of epilepsy. Epilepsia. 2014;55(4):475-482.

2.      Perucca E, Brodie MJ, Kwan P, Tomson T. 30 years of second-generation antiseizure    

medications: Impact and future perspectives. Lancet Neurol. 2020;19(6):544-556.         

3.      Löscher W, Klein P. The pharmacology and clinical efficacy of antiseizure medications:            

From bromide salts to cenobamate and beyond. Neuropharmacology. 2021;199:108677.

4.      Almeida L, Soares-da-Silva P. Eslicarbazepine acetate (BIA 2-093). Neurotherapeutics. 2007;4(1):88-96.

5.      Singh RP, Asconapé JJ. A review of eslicarbazepine acetate for the adjunctive treatment of partial-onset epilepsy. J Cent Nerv Syst Dis. 2011;3:179-187.

6.      Lattanzi S, Brigo F, Cagnetti C, Verrotti A, Zaccara G, Silvestrini M. Eslicarbazepine                

acetate in the treatment of adults with partial-onset epilepsy: an evidence-based review              

of efficacy, safety and place in therapy. Core Evid. 2018;13:21-31.

7.      McNamara JO. Pharmacotherapy of the epilepsies. In: Brunton LL, Lazo JS, Parker KL, editors. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 12th ed. New York: McGraw-Hill; 2011. p. 583–607.

8.      Rogawski MA, Löscher W, Rho JM. Mechanisms of action of antiseizure drugs and the ketogenic diet. Cold Spring Harb Perspect Med. 2016;6(5):a022780.

9.      Patsalos PN. The clinical pharmacology profile of the newer antiepileptic drugs. Epilepsia. 2013;54(Suppl 8):1–2.

10.  Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography. 3rd ed. Hoboken (NJ): John Wiley & Sons; 2010.

11.  International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). ICH Q2(R2): Validation of Analytical Procedures. Geneva: ICH; 2023.

12.  Trivedi HK, Patel MC. Development and validation of a stability-indicating RP-HPLC method for estimation of eslicarbazepine acetate in bulk drug and pharmaceutical dosage form. J Chromatogr Sci. 2016;54(9):1564–1571.

13.  Moffat AC, Osselton MD, Widdop B, Watts J, editors. Clarke's Analysis of Drugs and Poisons. 4th ed. London: Pharmaceutical Press; 2011.

14.  Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry. 4th ed. Part II. New Delhi: CBS Publishers & Distributors; 2002.

15.  Skoog DA, Holler FJ, Crouch SR. Principles of Instrumental Analysis. 7th ed. Boston: Cengage Learning; 2018.

16.  Chatwal GR, Anand SK. Instrumental Methods of Chemical Analysis. 5th ed. Mumbai: Himalaya Publishing House; 2007.

17.  International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). ICH Q2(R2): Validation of Analytical Procedures. Geneva: ICH; 2023.

18.  Almeida L, Soares-da-Silva P. Eslicarbazepine acetate (BIA 2-093). Neurotherapeutics. 2007;4(1):88–96.

19.  Bertilsson L. Clinical pharmacokinetics of carbamazepine. Clin Pharmacokinet. 1978;3(2):128–143.

20.  Harris DC, Lucy CA. Quantitative Chemical Analysis. 10th ed. New York: Macmillan Learning; 2020.

21.  Pedersen-Bjergaard S, Gammelgaard B, Halvorsen TG. Introduction to Pharmaceutical Analytical Chemistry. Chichester: John Wiley & Sons; 2019.

22.  Willard HH, Merritt LL Jr, Dean JA, Settle FA Jr. Instrumental Methods of Analysis. 7th ed. New Delhi: CBS Publishers & Distributors; 1986.

23.  Mendham J, Denney RC, Barnes JD, Thomas MJK. Vogel's Textbook of Quantitative Chemical Analysis. 6th ed. Harlow: Pearson Education Ltd.; 2000.

24.  Sharma BK. Instrumental Methods of Chemical Analysis. 24th ed. Meerut: Goel Publishing House; 2005.

25.  Christian GD, Dasgupta PK, Schug KA. Analytical Chemistry. 7th ed. Hoboken (NJ): John Wiley & Sons; 2014.

26.  Sethi PD. Quantitative Analysis of Drugs in Pharmaceutical Formulations. 3rd ed. New Delhi: CBS Publishers & Distributors; 1997.

27.  Kasture AV, Mahadik KR, Wadodkar SG, More HN. Pharmaceutical Analysis. Vol. II. 17th ed. Pune: Nirali Prakashan; 2008.

28.  Ermer J, Miller JHM, editors. Method Validation in Pharmaceutical Analysis: A Guide to Best Practice. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2005.

29.  Swartz ME, Krull IS. Analytical Method Development and Validation. New York: Marcel Dekker Inc.; 1997.

30.  Ahuja S, Dong MW, editors. Handbook of Pharmaceutical Analysis by HPLC. 2nd ed. Amsterdam: Elsevier Academic Press; 2011.

31.  Kazakevich Y, Lobrutto R. HPLC for Pharmaceutical Scientists. Hoboken (NJ): John Wiley & Sons; 2007.

32.  Watson DG. Pharmaceutical Analysis: A Textbook for Pharmacy Students and Pharmaceutical Chemists. 3rd ed. Edinburgh: Churchill Livingstone Elsevier; 2012.

33.  Miller JN, Miller JC. Statistics and Chemometrics for Analytical Chemistry. 7th ed. Harlow: Pearson Education Ltd.; 2018.

34.  Dong MW. Modern HPLC for Practicing Scientists. 2nd ed. Hoboken (NJ): John Wiley & Sons; 2019.

35.  Kazakevich Y, Lobrutto R. HPLC for Pharmaceutical Scientists. Hoboken (NJ): John Wiley & Sons; 2007.

36.  Eurachem. The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and Related Topics. 2nd ed. Teddington (UK): Eurachem; 2014.

37.  Ribani M, Bottoli CBG, Collins CH, Jardim ICSF, Melo LFC. Validation for chromatographic and electrophoretic methods. Quim Nova. 2004;27(5):771–780.

38.  Green JM. A practical guide to analytical method validation. Anal Chem. 1996;68(9):305A–309A.

39.  Taverniers I, De Loose M, Van Bockstaele E. Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance. TrAC Trends Anal Chem. 2004;23(8):535–552.

40.  Magnusson B, Örnemark U, editors. Eurachem Guide: The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics. 2nd ed. Eurachem; 2014.

41.  Shrivastava A, Gupta VB. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young Sci. 2011;2(1):21–25.

42.  Miller JC, Miller JN. Statistics for Analytical Chemistry. 3rd ed. Chichester: Ellis Horwood Ltd.; 1993.

43.  Long GL, Winefordner JD. Limit of detection: A closer look at the IUPAC definition. Anal Chem. 1983;55(7):712A–724A.

44.  Currie LA. Nomenclature in evaluation of analytical methods including detection and quantification capabilities. Pure Appl Chem. 1995;67(10):1699–1723.

45.  Aulton ME, Taylor KMG, editors. Aulton's Pharmaceutics: The Design and Manufacture of Medicines. 5th ed. London: Elsevier; 2018.

46.  Swarbrick J, editor. Encyclopedia of Pharmaceutical Technology. 4th ed. New York: Informa Healthcare; 2013.

47.  Hubaux A, Vos G. Decision and detection limits for linear calibration curves. Anal Chem. 1970;42(8):849–855.

48.  Rozet E, Marini RD, Ziemons E, Boulanger B, Hubert P. Advances in validation, risk and uncertainty assessment of bioanalytical methods. J Pharm Biomed Anal. 2011;55(4):848–858.

49.  United States Pharmacopeial Convention. United States Pharmacopeia and National Formulary (USP 43–NF 38): General Chapter <1225> Validation of Compendial Procedures. Rockville (MD): United States Pharmacopeial Convention; 2020.