Zero-Order Derivative and Area Under Curve UV-Spectrophotometric Methods for Determination of Calcium Levofolinate in Bulk and Pharmaceutical Dosage Form

Rahul D. Rathod¹, Sopan N. Nangare²*, Vikas R. Patil¹, Harshada S. Rathod¹, Mayuri M. Shitole³, Rahul S. Tade²

¹Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur- 425405 Dist: Dhule (MS) India.

²Department of Pharmaceutical Chemistry, H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur- 425405 Dist: Dhule (MS) India.

³Research Scientist, ProcessDevelopment Lab Department, Murli Krishna Pharma Pvt. Ltd.

Ranjangaon- 412209 Dist: Pune (MS) India.

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

ABSTRACT:

Simple, accurate, and precise UV-Spectrophotometric methods were developed for the estimation of Calcium levofolinate (CLF) in bulk and parenteral dosage form by Zero Order (Method A), Area Under Curve (AUC) (Method B). The solution of standard and sample was prepared in 0.1N sodium hydroxide (NaOH) and further dilution was carried out using Double Distilled Water (DDW). The quantitative determination of the drug was carried out by using the zero-order derivative values measured at 282±2nm (method A) and the AUC method values measured at 270.00nm - 297.80nm (method B). Calibration graphs constructed at their respective wavelength of determination were linear in the concentration range of CLF using 3-18µg/mL (r²=0.999) for zero-order and AUC spectrophotometric methods. Moreover, all these methods proposed were thoroughly validated with ICH guidelines. To summarize, developed spectrophotometric methods in this research are simple, precise, accurate, and sensitive to the assay of CLF in injections.

KEYWORDS: Calcium levofolinate; UV-spectrophotometer; zero-order; Area Under Curve; validation; ICH guidelines.

INTRODUCTION:

From its inception drugs play a crucial role in the men’s development through the treatment of health issues¹. Calcium levofolinate (CLF) is folinic acid calcium salt. Chemically, CLF a calcium 2-[[4-[(2-amino-5-formyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl)methylamino] benzoyl]amino]pentanedioate (Figure 1)². CLF is the anti-neoplastic, Lveo isoform of leucovorin calcium³. CLF is an active metabolite of a folic acid metabolite that doesn’t necessitate the dihydrofolate reductase metabolism². Besides, the toxic effects of other folic acid derivatives are counteracted by this compound, which rescues patients, while permitting folate antagonist antitumor action. Also, CFL activates the fluorouracil and its different derivatives by stabilizing the metabolite binding to the target enzymes and thereby prolonging drug action². A plentiful literature survey revealed that only a few methods are reported for the determination of CLF and most of the methods are used for the determination of CLF in individuals. Hence, only analytical methods reported for the quantitative determination of CLF in pharmaceutical formulations such as reverse phase HPLC³, capillary electrophoresis⁴, etc.

No UV spectrophotometric method to determine CLF in bulk and injections using zero-order and AUC has been documented so far. Therefore, efforts have been undertaken to improve the spectrophotometric method zero-order and AUC⁵ to estimate CLF in bulk and pharmaceutical parenteral formulations with good accuracy, precision, and economy. Moreover, the proposed work has been validated as per the International Conference of Harmonization (ICH) guidelines⁶.

Calcium levofolinate²

Molecular formula²: C₂₀H₂₁CaN₇O₇

Molecular weight²: 511.5 g/mol

Figure 1: Chemical Structure of Calcium levofolinate

MATERIAL AND METHODS:

Chemicals and reagents:

The reference standard of CLF active pharmaceutical ingredient was supplied as a gift sample from Pawar Supplier, Karad (MS), India. All chemicals and reagents used were of analytical grade and purchased from Qualigens Fine Chemicals, Mumbai, India. CLF injection sample (Leucovorin Calcium Injection IP, Mfg by RMPL Pharma LLP.) with label claim 10mg/mL were purchased from the local market, Shirpur.

Instrumentation:

A Shimadzu UV-2450 Shimadzu, Japan double beam spectrophotometer with 1 cm matched quartz cell, Deuterium Lamp, Wavelength range from 200 - 400nm was used for all spectral measurements. The electronic balance (Shimadzu AUX – 120) was used for weighing purposes. Calibrated volumetric glassware was used for the validation study.

Selection of Solvents⁷:

In this study, 0.1N NaOH was chosen as a common solvent for dissolving of CLF. The solvent selection was carried out based on the CLF solubility in a different solvent.

Preparation of Standard Stock Solutions of CLF:

CLF Stock Solution:

The standard stock solution of 100µg/mL of CLF was prepared by 0.1N NaOH using the addition method, for zero-order and AUC spectrophotometric analysis. An accurately weighed quantity of CFL of 10mg was taken in a 100mL volumetric flask and dissolved in 0.1N NaOH using ultrasonication (5 min). Finally, the volume was made up to the mark using 0.1N NaOH to gel CLF standard stock solution.

Atenolol Working Standard Solution:

The working standards were prepared by dilution of the standard stock solution with DDW in a concentration range of 3-18μg/ml for zero-order and AUC spectrophotometric methods. DDW was used as a blank solution.

Determination of λ Max of Individual Component:

The resultant solution was scanned in the UV range (200-400nm) in a 1 cm cell against the solvent blank (DDW). Initially, the λ max of CLF was found to be 282 nm. In method A, the absorbance at 282 nm was considered for analysis (Figure 2). While in method B, the 270.00nm to 297.80nm two wavelengths were selected for the integration of AUC (Figure 3).

Linearity Study for CLF:

For linearity study, six solutions of CLF of different concentrations (3, 6, 9, 12, 15, and 18µg/ml) were prepared using the stock standard solution and analyzed by using proposed methods. The obtained data were utilized to further process. The UV spectrum linearity was reported in Figure 4. The calibration curves of zero-order (Figure 5) and AUC (Figure 6) was a plot using the absorbance of respective concentration (Table 1) to check the linearity for both methods⁸ (Table 3).

Analysis of Marketed Formulation⁸ (Injection):

A Vial containing 50mg/5mL CLF injection was opened and further diluted with DDW to obtain concentration 9 µg/mL of CLF. Then, the AUC was recorded in between the selected wavelengths (270nm - 297.80nm) using a UV spectrophotometer. Besides, the concentrations of CLF in solutions were determined by using the respective linear regression equation. The analysis was repeated six times to achieve accurate results (Table 2).

Validation:

The zero-order and AUC methods were validated in terms of accuracy, precision, ruggedness, sensitivity, repeatability, etc by using a UV spectrophotometric method^9-12.

Accuracy:

To study the accuracy of the proposed methods (method A and B) and to check the interference in dosage forms, recovery experiments were carried out by using the standard addition method¹³. The accuracy for analytical methods was evaluated at 80%, 100%, and 120% levels ¹⁴ of 9µg/mL standard solutions. The AUC for both methods was measured in the wavelength range of 270 nm - 297.80 and for zero-order derivatives at 282nm. The results of the experiments were obtained in terms of percent recovery. Three determinations at each level were carried out and %RSD was calculated for each level.

Precision (Intra-day and Inter-day precision):

To determine the precision of methods, CLF solutions at a 6, 9, and 12µg/mL were analyzed every three times for both methods using spectrophotometric methods. In brief, the precision was determined as intra-day and inter-day variations^15-17. The Intra-day precision was determined by analyzing the 6, 9, and 12µg/mL of CLF solution (n=3) on the same day. Inter-day precision was determined by analyzing 6, 9, and 12µg/mL of CLF drug solution daily for three consecutive days for a week (Table 3).

Repeatability:

Repeatability of methods was determined by analyzing the 9µg/mL of CLF (n=6) by both methods (A and B) using a UV spectrophotometer.

Ruggedness:

The ruggedness of the proposed method was determined by two analysts using the same operational and environmental conditions.

Sensitivity:

The limit of detection (LOD)¹⁸ and Limit of Quantification (LOQ)¹⁷ was calculated using formulae “LOQ = 10 ×N/B” and “LOD = 3.3 ×N/B,” where “N” is the average standard deviation of the absorbance or peak areas of the CLF (n = 3), taken as a measure of noise, and “B” is the slope^14,19.

RESULT AND DISCUSSION:

The zero-order and AUC spectra for CLF were recorded at the wavelength of 282 nm (Figure 2) and 270-297.80nm (Figure 3) respectively. The graph obtained in the zero-order and AUC showed a linear relationship under the experimental conditions mentioned (Figure 4). The regression analysis was made for the slope, correlation coefficient, intercept values. Based on this, the regression equations of calibration curves were y = 0.047x+0.017 ((r2=0.999) at 282nm for zero-order spectrophotometric analysis (Figure 5). Besides, y = 0.166x+0.107 at 270nm-297.80nm for AUC spectrophotometry (6). The range was found to be 3-18 µg/mL for both zero-order and AUC spectrophotometric methods. Analysis of marketed formulation was carried out by using both methods. There was no interference from the different excipients used in the parenteral dosage form. Percent recovery (% recovery) of CLF was found to be 98.20-99.03% and 98.70 -99.57% for zero-order derivative and AUC spectrophotometric method. The low % RSD value indicated that the suitability of this method for routine analysis of CLF in the parenteral formulation (Table 3). The LOD and LOQ were found to be 3.56 and 10.81 for zero order. The LOD and LOQ were found to be 3.73 and 11.31 for AUC methods respectively. The repeatability study was revealed that both methods showed the acceptable reparability with % RSD < 2. Moreover, the ruggedness of the zero-order and AUC demonstrated less than 2 % RSD, which concludes that the method having excellent ruggedness at the same operational and environmental conditions. The summery of both methods are depicted in Table 3.

Figure 2: UV-spectrum of CLF (Method - A)

Figure 3: UV-spectrum of PB (Method - B)

Figure 4: UV-spectrum Linearity

Figure 5: Zero Order Linearity (Method - A)

Figure 6: Zero Order AUC (Method - B)

Table 1: Regression characteristics of CLF

Parameters	Value for Method A	Value for Method B
Correlation Coefficient (r)	0.999	0.999
Regression equation
Slope	0.047	0.166
Intercept	0.017	0.107

Table 2: Analysis of injection (CLF)

Parameter	CLF
Parameter	Method A	Method B
Label claim (mg/mL)	10	10
Concentration (µg/mL)	9	9
% Percent amount Found (n=6)	98.57	98.10
SD	0.267	0.823
% RDS	0.271	0.839

Table 3: Summary of validation parameters

Parameter	CLF
Parameter	Method A	Method B
Working wavelength (nm)	282	270 - 297.80
Linearity range (µg/mL)	03-18	03-18
Precision (% RSD)
Inter-day (n=3)	0.29-0.83	0.28-1.03
Inter-day (n=3)	0.50-1.35	0.71-0.89
Repeatability (n=6)	0.48	0.57
Ruggedness (% RSD)
Analyst (I)	0.67	0.86
Analyst (II)	0.42	0.77
% Recovery (n=9)
Injection	98.20-99.03	98.70-99.57
% RSD	0.14-0.76	0.33-0.90

CONCLUSION:

No zero-order and AUC spectrophotometric methods have been described for the determination of CLF. Therefore, simple, fast, and reliable zero-order and AUC spectrophotometric methods were developed for the routine determination of CLF. At the conclusion, this developed method can be sensitive, accurate, and precise and for future perception, it can be easily applied to the pharmaceutical formulation.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Bhangale SM, Hiwale VS, Rane SS, Chaudhari RY, Patil VR. Simultaneous spectrophotometric estimation of atenolol and losartan in tablet dosage form. Research Journal of Pharmacy and Technology. 2016; 9: 501-505.

2. National Library of Medicine. National Center for Biotechnology Information. Calcium levofolinate. Available from URL: https://pubchem.ncbi.nlm.nih.gov/compound/Calcium-levofolinate.

3. Patel NC, Sakhreliya B, Patel MB. Development and validation of analytical method for estimation of leucovorin calcium in it’s marketed dosage form. Journal of Pharmaceutical Science and Bioscientific Research. 2016; 6: 207-210.

4. Suss F, Harang V, Sänger‐van de Griend CE, Scriba GK. Development and validation of a robust capillary electrophoresis method for impurity profiling of calcium levofolinate including the (6r,2's)‐diastereomer using statistical experimental design. Electrophoresis. 2004; 25: 766-777.

5. Mali AD, Bathe R, Tamboli A. Zero Order and Area under Curve spectrophotometric methods for determination of domperidone in pharmaceutical formulation. Asian Journal of Pharmacy and Technology. 2015; 5: 182-187.

6. International Conference of Harmonization (ICH) 1997 Q2R1: Validation of Analytical Procedure Methodology. USFDA Federal Register.1997; 62: 27463.

7. Jumle R, Mundhey A, Wate S, Dangare S, Ramteke U. UV-Spectrophotometric method development and validation for estimation of tizanidine and aceclofenac in tablet formulation. Asian Journal of Pharmaceutical Analysis.2012; 2: 101-103.

8. Mali A, Mali S, Bathe R, Patil M, Tamboli A. Zero-order and Area under Curve spectrophotometric methods for determination of riboflavin in pharmaceutical formulation. Asian Journal of Pharmaceutical Analysis. 2016; 6: 35-40.

9. Mali A, Kekan V, Dongare R, Gholve S, Bathe R. Simultaneous UV Spectrophotometric methods for estimation of carvedilol and hydrochlorothiazide in bulk and tablet dosage form. Asian Journal of Pharmaceutical Research.2016; 6: 15-20.

10. Mali A, Gorad R, Quazi A, Bathe R, Tamboli A. Development and estimation of pyridoxine hcl in bulk and formulation by Second Order Derivative Area under Curve UV-Spectrophotometric methods. Asian Journal of Pharmacy and Technology. 2016; 6: 106-112.

11. Shelke A, Shinde M. Mogal R. Sable R, Jadhav A. Application of UV spectrophotometric methods for simultaneous estimation of emtricitabine and tenofovir alafenamide fumarate in bulk. Asian Journal of Pharmacy and Technology. 2018; 8: 103-107.

12. Nagamallika J, Mahesh A. Development and validation of spectrophotometric method for the estimation of aripiprazole in tablet dosage form. Asian Journal of Pharmaceutical Analysis. 2011;1: 46-49.

13. Kumar PR, Spandana P, Shareef A, Sahithi K, Babu NR. Development and validation of difference spectrophotometric method for quantitative estimation of gemcitabine hydrochloride injection. Asian Journal of Pharmaceutical Analysis. 2019; 9: 219-223.

14. Mali AD, Bathe R, Patil M, Tamboli A. Zero Order and Area Under Curve spectrophotometric methods for determination of levocetirizine in pharmaceutical formulation. International Journal of Advances in Scientific Research. 2015;1, 270-275.

15. Gupta SK, Kumar B, Sharma PK. Development and validation of a spectrophotometric method for estimation of triamcinolone in solid dosage form. Asian Journal of Pharmaceutical Analysis. 2013; 3: 42-43.

16. Annareddy GR, Singh A, Akkenapally S, Akiful HM, Bakshi V, Boggula N. Comparative studies of apixaban in bulk and its formulations by Uv-Spectroscopy (Zero Derivatives and Area Under Curve). International Journal of Pharmacy and Biological Sciences. 2018;8(4): 1002-1008.

17. Pramod K, Ansari SH, Ali J. Development and validation of UV spectrophotometric method for the quantitative estimation of eugenol. Asian Journal of Pharmaceutical Analysis.2013; 3: 58-61.

18. Daharwal S. Development and validation of UV spectrophotometric method for simultaneous estimation of diazepam and propranolol in bulk drug and its formulations. Asian Journal of Pharmaceutical Analysis. 2013; 3: 20-23.

19. Patil SD, Chaure SK, Kshirsagar S. Development and validation of uv spectrophotometric method for simultaneous estimation of empagliflozin and metformin hydrochloride in bulk drugs. Asian Journal of Pharmaceutical Analysis. 2017; 7: 117-123

Received on 14.04.2020 Modified on 16.05.2020

Asian J. Pharm. Ana. 2020; 10(3):129-133.

DOI: 10.5958/2231-5675.2020.00023.X