UV SPECTRAL STRATEGIES FOR ANALYTICAL METHOD DEVELOPMENT OF MULTICOMPONENT DRUG FORMULATION

UV SPECTRAL STRATEGIES FOR ANALYTICAL METHOD DEVELOPMENT OF MULTICOMPONENT DRUG FORMULATION

A. Naik *, S. Kanolkar, C. Kumbharjuvekar and S. Kudchadkar

Department of Pharmaceutical Quality Assurance, Goa College of Pharmacy, Panaji, Goa, India.

ABSTRACT: This research work aimed at the development and validation of three UV spectrophotometric methods, namely, Ratio Subtraction and Extended Ratio Subtraction (RS-ERS), Ratio Subtraction coupled with Constant Multiplication (RS-CM), and Ratio Subtraction coupled with Spectrum Subtraction (RS-SS) for the simultaneous estimation of Benidipine Hydrochloride (BEN) and Chlorthalidone (CHL) in their combined pharmaceutical dosage form. BEN was detected at 237 nm and CHL at 275 nm. For determination of CHL, BEN at 10 µg/mL was used as the divisor; for determination of BEN, CHL at 18.7 µg/mL was used as the divisor. The methods were validated in accordance with ICH Q2(R2) guidelines for linearity, specificity, accuracy, precision, robustness, LOD, and LOQ. The linearity was established in the concentration ranges of 4-12 µg/mL for BEN and 6.2-18.7 µg/mL for CHL, and good correlation coefficients (r² = 0.9996) were obtained for all methods. Precision studies demonstrated %RSD values below 2% for both intra-day and inter-day analysis, reflecting good repeatability and intermediate precision. The accuracy results were within the acceptable range of 98–102%. The developed methods were found to be robust with %RSD values below 2%. The methods showed adequate sensitivity with LOD and LOQ values of 0.29 µg/mL and 0.87 µg/mL for BEN and 1.10 µg/mL and 3.34 µg/mL for CHL, respectively. The assay of the commercial formulation yielded results within pharmacopeial specifications. The greenness and sustainability of the methods were evaluated using AGREE and ComplexGAPI tools.

Keywords: Chlorthalidone, Benidipine hydrochloride, Extended ratio subtraction, Ratio subtraction, Constant multiplication, Spectrum subtraction, UV spectrophotometry, Method validation, ICH Q2(R2) guidelines, Green analytical chemistry

INTRODUCTION: Chlorthalidone (CHL), chemically (RS)-2-Chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1-yl) benzene-1-sulfonamide, is a thiazide-like diuretic commonly used for the treatment of hypertension and edema. Benidipine Hydrochloride (BEN), chemically 5-O-[(3R)-1-benzylpiperidin-3-yl] 3-O-methyl (4R)-2, 6-dimethyl-4-(3-nitrophenyl)-1, 4-dihydropyridine-3, 5-dicarboxylate hydrochloride, is a calcium channel blocker used for the treatment of hypertension.

The combination of CHL and BEN is available as a fixed-dose tablet formulation and offers enhanced antihypertensive efficacy by acting through complementary mechanisms. Their chemical structures are shown in Fig. (1A) and Fig. (1B). The simultaneous determination of CHL and BEN by UV spectrophotometry is analytically difficult due to the overlapping absorption spectra of both drugs, which makes the direct quantification difficult without prior separation.

To overcome the above limitation, the ratio spectra-based spectrophotometric methods offer a simple, rapid, and cost-effective alternative for the resolution of overlapping spectra. Three ratio spectra-based methods, including Ratio Subtraction and Extended Ratio Subtraction (RS-ERS), Ratio Subtraction coupled with Constant Multiplication (RS-CM), and Ratio Subtraction coupled with Spectrum Subtraction (RS-SS) were developed and validated for the simultaneous estimation of CHL and BEN in bulk and pharmaceutical dosage forms in the present study. The methods allow for the accurate quantification of CHL and BEN without chromatographic separation and thus provide a simple, precise and economical approach for routine quality control analysis.

FIG. 1: MOLECULAR STRUCTURE OF (A) CHLORTHALIDONE AND (B) BENIDIPINE HYDROCHLORIDE

Literature Review: A detailed literature survey revealed that various analytical methods like UV ^4-12 spectrophotometric, HPLC ^11-12, RP-HPLC ^14-18, HPTLC ^19, and methods have been reported for estimation of BEN and CHL individually or in combination with other drugs. However, no reports are available describing the application of Ratio Subtraction coupled with Extended Ratio Subtraction (RS-ERS), Ratio Subtraction coupled with Constant Multiplication (RSCM), and Ratio Subtraction coupled with Spectrum Subtraction (RS-SS) methods for simultaneous estimation of this combination in the available literature. Hence, the present study was undertaken to develop and validate these methods as per the ICH Q2(R2) guidelines along with the evaluation of their greenness and environmental sustainability.

MATERIALS AND METHODS:

Instrumentation: A SHIMADZU UV-2700 double-beam UV-Visible spectrophotometer with 1 cm matched quartz cells was used for spectral measurements. The spectrophotometer was set to a bandwidth of 1 nm, a scan speed of 400 nm/min, and a data interval of 0.5 nm. A Wensar electronic balance (Model MAB 220) was used for sample weighing. Calibrated glassware was used for the entire procedure. A solvent blank was used for baseline correction prior to the scan.

Reagents and Chemicals: Benidipine Hydrochloride (BEN), having a percent purity of 99.51%, and Chlorthalidone (CHL), having a percent purity of 98.93%, were received as gift samples from Ravenbhel Healthcare (P) Ltd, Bari-Brahmana, Jammu, and Acme Foundation, Nalagarh, Himachal Pradesh, respectively. HPLC-grade methanol was purchased from Rankem (Avantor).

Marketed Formulation: BENIDIN CH 6.25 mg, manufactured by Mascot Health Series Pvt Ltd, was purchased from the local market. Each Film-coated bilayered tablet contains 4 mg of Benidipine Hydrochloride and 6.25 mg of Chlorthalidone.

Preparation of Solution:

Standard Stock Solution of CHL (1000 µg/mL): A standard stock solution was prepared by accurately weighing 100 mg of CHL. The weighed drug was transferred into a 100 mL volumetric flask and initially dissolved in approximately 50 mL of methanol. The solution was then diluted up to the mark with the same solvent to obtain a final concentration of 1000 µg/mL.

Working Standard Solution of CHL (100 µg/mL): A 10 mL volume was pipetted from the standard stock solution of CHL 1000 ug/mL and transferred into a 100 mL volumetric flask. The solution was then diluted to the mark with methanol to obtain a final concentration of 100 µg/mL.

Standard Stock Solution of BEN (1000 µg/mL): A standard stock solution was prepared by accurately weighing 100 mg of BEN. The weighed drug was transferred into a 100 mL volumetric flask and initially dissolved in approximately 50 mL of methanol. The solution was then diluted up to the mark with the same solvent to obtain a final concentration of 1000 µg/mL.

Working Standard Solution of BEN (100 µg/mL): A 10 mL volume was pipetted from the standard stock solution of BEN 1000ug/mL and transferred into a 100 mL volumetric flask. The solution was then diluted to the mark with methanol to obtain a final concentration of 100 µg/mL.

Preparation of Tablet Sample Solution for Analysis: Twenty BENIDIN CH 6.25 tablets were accurately weighed (3.84g), and the average tablet weight was calculated (0.192g). Tablets were then crushed into fine powder using a mortar and pestle, and a quantity of tablet powder equivalent to 4 mg of BEN (6.2 mg of CHL) was transferred to a 100 mL volumetric flask. Approximately 50 mL of methanol was added, and the mixture was sonicated for 45 min to ensure complete extraction of the drugs. The volume was then adjusted up to the mark with methanol to obtain a stock solution containing BEN and CHL at concentrations of 4 µg/mL and 6.25 µg/mL, respectively. The resulting solution was centrifuged at 3800 rpm for 15 min, and the supernatant was collected for further analysis.

Note: Methanol was used only for the preparation of stock and working standard solutions of BEN and CHL and for preparation of tablet sample solution. All subsequent dilutions, method development, assay, and validation studies were carried out using the optimized distilled water:methanol (50:50, v/v) solvent system.

Method Development for Simultaneous Estimation of Benidipine Hydrochloride and Chlorthalidone in Water: Methanol (50:50 v/v):

Ratio Subtraction and Extended Ratio Subtraction Method (RS-ERS) ^18-20:  The RS-ERS method is a spectrophotometric technique applicable for the simultaneous estimation of binary drug combinations exhibiting overlapping absorption spectra, wherein one component possesses a more extended spectral profile than the other. In the selected CHL and BEN combination, BEN exhibited a more extended absorption spectrum, while CHL showed a less extended spectrum. Therefore, the RS-ERS method was employed for the recovery and quantification of the individual spectra of CHL and BEN without prior separation.

Ratio Subtraction (RS): For the determination of CHL,

Step 1: The absorption spectrum of the mixture (CHL + BEN) was divided by the spectrum of a selected concentration of BEN (BEN′), used as a divisor.

CHL+BEN) / BEN = CHL / BEN + BEN / BEN = CHL / BEN + Constant (Equation 1)

Step 2: The obtained ratio spectrum consisted of CHL/BEN′ and a constant corresponding to BEN/BEN′. The constant value was measured from the plateau region and subsequently subtracted from the ratio spectrum.

CHL / BEN + Constant – Constant = CHL / BEN (Equation 2)

Step 3: Multiplication of the resulting spectrum by the divisor (BEN′) yielded the original zero-order spectrum of CHL.

CHL / BEN × BEN = CHL   (Equation 3)

The recovered spectrum was used for quantitative estimation of CHL at 275 nm

Determination of CHL by RS:

 
Extended Ratio Subtraction (ERS): For the determination of BEN.

Step 1: The recovered zero-order spectrum of CHL by RS was divided by a spectrum of a selected concentration of CHL (CHL′), used as the divisor to obtain a constant value corresponding to CHL/CHL′.

CHL / CHL = Constant (Equation 4)

Step 2: Subsequently, the spectrum of the mixture (CHL + BEN) was divided by the same divisor (CHL′), producing a ratio spectrum consisting of BEN/CHL′ and the constant obtained previously.

CHL+ BEN / CHL = CHL / CHL + BEN / CHL = BEN / CHL + Constant (Equation 5)

Step 3: The previously obtained constant was subtracted from the ratio spectra of the laboratory-prepared mixtures, which had been divided by the selected CHL divisor concentration (18.7 µg/mL).

BEN / CHL + Constant - Constant = BEN / CHL (Equation 6)

Step 4: The resulting ratio spectra were then multiplied by the selected CHL divisor spectrum (18.7 µg/mL) to regenerate the original zero-order spectra of BEN (Eq. 7).

BEN / CHL × CHL (Equation 7)

Thus, the RS-ERS method enables the selective recovery of the individual zero-order spectra of CHL and BEN from their overlapping spectra, facilitating their simultaneous determination.

Determination of BEN by ERS Method:

Ratio Subtraction Coupled with Constant Multiplication (RS-CM) ^19-21: The RSCM method is a simple and selective spectrophotometric technique employed for the analysis of binary mixtures with overlapping spectra. The method is based on the recovery of the spectrum of the component exhibiting the more extended spectrum through constant multiplication, followed by the determination of the second component by spectrum subtraction.

For the determination of BEN,

Step 1: The absorption spectrum of the mixture (CHL + BEN) was divided by the spectrum of a selected concentration of BEN (BEN′), used as a divisor. The resulting ratio spectrum consisted of CHL/BEN′ and a constant corresponding to BEN/BEN′.

CHL+ BEN / BEN = CHL / BEN + BEN / BEN = CHL / BEN + Constant (Equation 8)

Step 2: The constant value was measured from the plateau region and multiplied by the selected divisor (BEN′) to recover the original zero-order spectrum of BEN (Equation 9).

BEN × BEN / BEN = BEN (Equation 9)

Determination of BEN by RSCM:

FIG. 12: RECOVERED ZERO-ORDER SPECTRA OF BEN (4-12μg/mL)

For the determination of CHL,

Step 3: The recovered zero-order spectrum of BEN was subtracted from the absorption spectrum of the mixture (CHL + BEN) to obtain the original zero-order spectrum of CHL.

(𝐶𝐻𝐿 +𝐵𝐸𝑁)−𝐵𝐸𝑁 =𝐶𝐻𝐿(𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 10)

Determination of CHL by RSCM:

FIG. 13: RECOVERED ZERO-ORDER SPECTRA OF CHL (6.2-18.7 μg/mL)

Ratio Subtraction Coupled with Spectrum Subtraction (RS-SS) ^18-19: CHL was determined using the RS method.

For the determination of BEN,

Step 1: The recovered zero-order spectrum of CHL was subtracted from the absorption spectrum of the mixture, resulting in the recovery of the original zero-order spectrum of BEN.

(𝐶𝐻𝐿 +𝐵𝐸𝑁)−𝐶𝐻𝐿 =𝐵𝐸𝑁 (Equation   11)

FIG. 14: RECOVERED ZERO-ORDER SPECTRA OF BEN (4-12 μg/mL)

Analytical Method Validation: The proposed analytical methods were validated in accordance with the recommendations of the ICH Q2(R2) guidelines. The following parameters, such as linearity and range, specificity, precision, accuracy, LOD, LOQ, robustness, and assay, were analysed.

Linearity and Range: The linearity of BEN and CHL was evaluated over the concentration ranges of 4–12 µg/mL and 6.2–18.7 µg/mL, respectively. Five concentration levels of BEN (4, 6, 8, 10, and 12 µg/mL) and CHL (6.2, 9.3, 12.5, 15.6, and 18.7 µg/mL) were prepared separately in triplicate in 10 mL volumetric flasks. Appropriate aliquots of BEN (0.4, 0.6, 0.8, 1.0, and 1.2 mL) and CHL (0.62, 0.93, 1.25, 1.56, and 1.87 mL) were withdrawn from their respective working standard solutions (100 µg/mL) and diluted to volume with a solvent system comprising distilled water and methanol (50:50, v/v). The UV spectra were recorded using a UV–Visible spectrophotometer over the wavelength range of 200–400 nm.

For the RS-ERS, RSCM, and RS-SS methods, the absorbances of BEN and CHL were measured at 237 nm and 275 nm, respectively. Calibration curves were constructed by plotting concentration versus absorbance, and the corresponding regression equations were obtained for both drugs.

Specificity: The specificity of the proposed methods was evaluated by comparing the spectral characteristics of standard solutions of BEN and CHL at assay concentrations with those obtained from the corresponding pharmaceutical formulation containing the same concentrations.

A laboratory-prepared mixture containing BEN (8 µg/mL) and CHL (12.5 µg/mL) in a ratio of 1:1.56 was prepared by accurately transferring 0.8 mL of BEN working standard solution

(100 µg/mL) and 1.25 mL of CHL working standard solution (100 µg/mL) into a 10 mL volumetric flask and diluting to volume with a solvent system comprising distilled water and methanol (50:50, v/v).

Similarly, a formulation mixture containing BEN (8 µg/mL) and CHL (12.5 µg/mL) in a ratio of 1:1.56 was prepared from BENIDIN CH 6.25 tablets. For all three proposed methods, 2 mL of the tablet solution was accurately transferred to a 10 mL volumetric flask and diluted to volume with a solvent system comprising distilled water and methanol (50:50, v/v), yielding final concentrations of BEN (8 µg/mL) and CHL (12.5 µg/mL).Both the laboratory-prepared mixture and formulation sample solutions were scanned over the wavelength range of 200–400 nm using a UV-Visible spectrophotometer.

Precision: Intra-day and inter-day precision studies were performed at the assay concentration level for both drugs. The study was carried out by analyzing six replicate samples, and the results were expressed as percentage relative standard deviation (%RSD).

For all the proposed methods (RS-ERS, RSCM, and RS-SS), 2 mL of the tablet solution was accurately transferred to a 10 mL volumetric flask and diluted to volume with a solvent system comprising distilled water and methanol (50:50, v/v), yielding final concentrations of BEN (8 µg/mL) and CHL (12.5 µg/mL). The solutions were then analyzed using a UV–Visible spectrophotometer over the wavelength range of 200–400 nm. For the RS-ERS method, CHL was determined using 10 µg/mL BEN as the selected divisor concentration, and absorbance was measured at 275 nm.

Similarly, BEN was determined using 18.7 µg/mL CHL as the selected divisor concentration, and absorbance was measured at 237 nm.

For the RSCM method, BEN was determined using 10 µg/mL BEN as the selected divisor concentration, and absorbance was measured at 237 nm. CHL was determined by subtracting the recovered spectrum of BEN from the absorption spectrum of the mixture, and absorbance was measured at 275 nm.

For the RS-SS method, CHL was determined using 10 µg/mL BEN as the selected divisor concentration, and absorbance was measured at 275 nm. Similarly, BEN was determined by subtracting the recovered spectrum of CHL from the absorption spectrum of the mixture, and absorbance was measured at 237 nm.

Accuracy: The accuracy assessment was carried out at 80%, 100%, and 120% concentration levels. The accuracy studies were performed separately by the standard addition method, and the percent recovery was calculated.

A base solution containing BEN and CHL at concentrations of 4 µg/mL and 6.2 µg/mL, respectively, was prepared by pipetting 1.5 mL of the tablet stock solution into a 10 mL volumetric flask and diluting to volume with a solvent system comprising distilled water and methanol (50:50, v/v). This was then analysed using RS-ERS, RSCM, and RS-SS methods. To this initial concentration of BEN (4 µg/mL) and CHL (6.2 µg/mL) addition of 0.32mL, 0.4 mL, 0.48mL was carried out from working standard solution of BEN and (100µg/mL) and addition of 0.49mL, 0.62 mL, 0.74mL was carried out from Working standard solution  of CHL (100 µg/mL) respectively to obtain 7.2, 8, 8.8µg/mL of concentration of BEN and 11.1, 12.4, and 13.6 µg/mL of concentration of CHL.

For the RS-ERS method, CHL was determined using 10 µg/mL BEN as the selected divisor concentration, and absorbance was measured at 275 nm. Similarly, BEN was determined using 18.7 µg/mL CHL as the selected divisor concentration, and absorbance was measured at 237 nm.

For the RSCM method, BEN was determined using 10 µg/mL BEN as the selected divisor concentration, and absorbance was measured at 237 nm. CHL was determined by subtracting the recovered spectrum of BEN from the absorption spectrum of the mixture, and absorbance was measured at 275 nm.

For the RS-SS method, CHL was determined using 10 µg/mL BEN as the selected divisor concentration, and absorbance was measured at 275 nm. Similarly, BEN was determined by subtracting the recovered spectrum of CHL from the absorption spectrum of the mixture, and absorbance was measured at 237 nm.

Assay of Marketed Formulation: The validated RS-ERS, RS-CM, and RS-SS methods were employed for the quantitative estimation of BEN and CHL in the marketed tablet formulation, Benidin-CH 6.25. The analysis was performed to determine the percentage label claim of both drugs.

For RS-ERS method, CHL and BEN were determined using 10 µg/mL BEN and 18.7 µg/mL CHL as divisor concentrations, respectively, with absorbance measurements performed at 275 nm and 237 nm.

For RSCM method, BEN was quantified using 10 µg/mL BEN as the divisor concentration at 237 nm, while CHL was determined from the recovered spectrum obtained after subtraction of BEN from the mixture spectrum and measured at 275 nm.

For the RS-SS method, CHL was estimated using 10 µg/mL BEN as the divisor concentration at 275 nm, whereas BEN was determined from the recovered spectrum after subtraction of CHL from the mixture spectrum and measured at 237 nm.

RESULTS AND DISCUSSION:

Selection of Solvent: For the selection of a suitable solvent, the Certificate of Analysis (COA) and the monographs of Benidipine hydrochloride (BEN) and Chlorthalidone (CHL) in the Indian Pharmacopoeia (IP) and Japanese Pharmacopoeia (JP) were referred to for information regarding their solubility characteristics. The recommended solvents included methanol and other organic solvents. However, to develop a greener and more sustainable analytical method, different combinations of distilled water and methanol were investigated. Among the solvent systems evaluated, both BEN and CHL were found to be sufficiently soluble and stable in distilled water:methanol (50:50, v/v), whereas BEN exhibited instability in solvent systems containing a higher proportion of water.

Therefore, considering solubility, stability, and green analytical chemistry principles, distilled water:methanol (50:50, v/v) was selected as the solvent system for the development and validation of the proposed spectrophotometric methods.

Determination of Lambda max: The UV absorption spectra of BEN and CHL (10 µg/mL each) were recorded over the range of 200–400 nm, and the overlain spectra are shown in Fig. 17. BEN exhibited a maximum absorbance (λ_max) at 237 nm, whereas CHL showed a λ_max at 275 nm.

These wavelengths were selected for further method development and quantitative analysis.

FIG. 17: OVERLAIN SPECTRA OF BEN (10µG/ML) AND CHL (10 µg/mL) IN DISTILLED WATER AND METHANOL (50:50)

Specificity: Specificity of the developed RS-ERS, RS-SS, and RSCM methods was evaluated by comparing the UV spectra of the laboratory-prepared mixture of BEN and CHL with those obtained from the tablet formulation. The spectra of the laboratory-prepared mixture were found to be closely overlapped with those of tablet formulation, indicating the absence of interference from formulation excipients. No additional spectral features or significant changes in absorbance were observed, confirming the specificity and selectivity of the proposed methods for the simultaneous estimation of BEN and CHL. The corresponding spectra are presented in the figure below.

FIG. 18: OVERLAIN SPECTRA OF LABORATORY-PREPARED MIXTURE AND MARKETED FORMULATION

Selection and Optimization of Divisor Concentration:

TABLE 1: SELECTION OF BEN DIVISOR FOR DETERMINATION OF CHL

TABLE 2: SELECTION OF CHL DIVISOR FOR DETERMINATION OF BEN

The optimum divisor concentration was established during the optimization of the RS-ERS method by evaluating different divisor concentrations of BEN and CHL. CHL (18.7 µg/mL) and BEN (10 µg/mL) exhibited the best spectral quality, minimal baseline noise, excellent recovery, and the highest agreement with the corresponding standard spectra. Since the RS-CM and RS-SS methods are based on the same ratio subtraction principle and utilize identical divisor spectra, these optimized divisor concentrations were adopted for all subsequent methods to ensure consistency in spectral processing and analytical performance.

TABLE 3: ASSAY COMPARISON OF TABLET EXTRACT AND LABORATORY MIXTURE

Note: The assay limit as per JP for Benidipine hydrochloride is NLT 95 % and NMT 105 % than of the stated amount, and the assay limit as per IP for chlorthalidone is NLT 92.5 % and NMT 107.5% of the stated amount.

Placebo studies could not be performed due to the unavailability of the exact excipient composition of the marketed formulation. This represents a limitation of the present study, as interference from individual excipients could not be evaluated using a placebo matrix.

Therefore, specificity was assessed by comparing the marketed tablet formulation with the laboratory prepared mixture, and no significant interference was observed indicating that proposed methods are sufficiently specific for the simultaneous estimation of BEN and CHL. The inclusion of a synthetic placebo containing commonly used tablet excipients or a representative excipient blend would provide a more comprehensive assessment of specificity.

Linearity and Calibration Curve: The linearity of the developed RS-ERS, RS-CM, and RS-SS methods was evaluated using five concentration levels of BEN (4, 6, 8, 10, and 12 µg/mL) and CHL (6.2, 9.3, 12.5, 15.6, and 18.7 µg/mL), prepared from their respective working standard solutions. Calibration curves were constructed by plotting absorbance against concentration.

Both analytes exhibited excellent linearity over the concentration ranges of 4-12 µg/mL for BEN and 6.2-18.7 µg/mL for CHL. The correlation coefficient (r²) was found to be 0.9996 for both BEN and CHL, confirming the linearity of the proposed methods and their suitability for quantitative analysis within the selected ranges.

TABLE 4: LINEARITY STUDY OF BEN AND CHL IN DISTILLED WATER: METHANOL (50:50 V/V)

Note: the same set of calibration standards was employed for the evaluation of linearity in RS-ERS, RSCM and, RS-SS methods, as all three methods are based on mathematical processing of the same zero-order absorption spectra rather than independent instrumental measurements. Consequently, the same concentration ranges, analytical wavelength, and calibration curve were used for all three methods.

Range: The range was established as 4-12 µg/mL for BEN and 6.2-18.7 µg/mL for CHL. The proposed methods were successfully validated throughout these concentration ranges.

Limit of Detection (LOD) and Limit of Quantification (LOQ): LOD and LOQ were determined in accordance with ICH Q2(R2) guidelines based on the standard deviation of the response (σ) and the slope (S) of the calibration curve. The calculations were performed using the following expressions:

LOD = (3.3 σ) / S

LOQ = (10 σ) / S

For BEN, the LOD and LOQ were found to be 0.29 µg/mL and 0.87 µg/mL, respectively. For CHL, the corresponding values were 1.10 µg/mL and 3.34 µg/mL, respectively. The obtained results confirm that the proposed UV spectrophotometric method exhibits adequate sensitivity for the quantification of BEN and CHL at low concentration levels.

Precision: The intra-day and inter-day precision studies were carried out at the assay concentration levels of BEN (8 µg/mL) and CHL (12.5 µg/mL). Six replicate analyses were performed using suitably diluted sample solutions prepared from the marketed formulation. The concentrations of BEN and CHL were determined, and the results were expressed as percentage relative standard deviation (% RSD). The % RSD values obtained for both intra-day and inter-day precision studies were found to be less than 2%, indicating that the proposed methods are precise and reproducible for the simultaneous estimation of BEN and CHL. The results are presented in Table 3.

TABLE 5: PRECISION STUDY OF BEN AND CHL BY RS-ERS, RSCM, AND RS-SS (N = 6)

Accuracy: The accuracy of the developed RS-ERS, RS-CM, and RS-SS methods was assessed using the standard addition method at 80%, 100%, and 120% levels of the assay concentration. Appropriate quantities of BEN and CHL standards were spiked into the pre-analyzed sample solution, and the recovery studies were carried out in triplicate. The percentage recoveries obtained for both analytes were within the range of 98–102%, with % RSD values below 2% across all three levels. The results indicate that the proposed methods are accurate and suitable for the routine quantitative analysis of BEN and CHL in pharmaceutical dosage forms. The detailed results are presented in Table 4, 5, and 6.

TABLE 6: RESULTS OF THE ACCURACY STUDY FOR BEN AND CHL BY RS-ERS

TABLE 7: RESULTS OF THE ACCURACY STUDY FOR BEN AND CHL BY RSCM

TABLE 8: RESULTS OF ACCURACY STUDY FOR BEN AND CHL BY RS-SS

Assay of Marketed Formulation: To assess the applicability of the validated RS-ERS, RS-CM, and RS-SS methods, the marketed tablet formulation Benidin-CH 6.25 containing BEN and CHL was analysed. The assay results were found to be in close agreement with the labelled claim, which indicates that the proposed methods can be effectively utilized for the routine simultaneous estimation of BEN and CHL in pharmaceutical formulations. The obtained results are presented in Table 7.

TABLE 9: ASSAY OF BEN AND CHL

TABLE 10: ROBUSTNESS STUDY OF RS-SS FOR BEN AND CHL

TABLE 11: ROBUSTNESS STUDY OF RS-ERS FOR BEN AND CHL

TABLE 12: ROBUSTNESS STUDY OF RSCM FOR BEN AND CHL

Robustness: The % assay values for BEN and CHL remained within acceptable limits despite deliberate variations in the analytical wavelength, divisor concentration, and the constant region selection.

The %RSD values were below 2% under all the tested conditions, demonstrating that the RS-ERS, RSCM, and RS-SS methods are robust and suitable for routine analysis.

Greenness Assessment: The environmental sustainability of the developed RS-ERS, RS-SS, and RS-CM spectrophotometric methods was evaluated using the Analytical GREEnness (AGREE) metric and the Complex Green Analytical Procedure Index (ComplexGAPI).

The AGREE tool assesses analytical procedures based on the 12 principles of Green Analytical Chemistry, while ComplexGAPI provides a comprehensive evaluation of the environmental impact associated with the entire analytical workflow. The AGREE assessment yielded an overall score of 0.76, indicating excellent compliance with the principles of Green Analytical Chemistry Fig. 19.

The high greenness score was attributed to the use of UV-visible spectrophotometry, which requires minimal sample preparation, low energy consumption, reduced solvent usage, and generates only a small amount of analytical waste.

The ComplexGAPI assessment Fig. 20 further confirmed the environmentally benign nature of the developed methods, with the pictogram predominantly displaying green and yellow zones, reflecting favourable environmental performance throughout the analytical procedure. The calculated E-factor was 0.02, indicating negligible waste generation and minimal environmental impact.

The input parameters used for the ComplexGAPI and AGREE assessments are summarized in Table 13 and 14, respectively. Overall, the results obtained from both greenness assessment tools demonstrate that the proposed analytical methods are environmentally sustainable and suitable for routine quality control analysis.

FIG. 19: GREENNESS ASSESSMENT (RS-ERS, RSCM, AND RS-CM) BY AGREE            

FIG. 20: GREENNESS ASSESSMENT (RS-ERS, RSCM, AND RS-CM) COMPLEX GAP

TABLE 13: INPUT PARAMETERS USED FOR THE COMPLEXGAPI

TABLE 14: INPUT PARAMETERS USED FOR THE AGREE

TABLE 15: COMPARISON OF PROPOSED UV METHODS WITH PREVIOUSLY REPORTED UV/HPLC/HPTLC METHODS FOR SIMULTANEOUS ESTIMATION OF BEN AND CHLORTHALIDONE CHL

CONCLUSION: Three UV spectrophotometric methods, namely RS-SS, RS-ERS, and RS-CM, were developed and validated for the simultaneous estimation of Benidipine Hydrochloride and Chlorthalidone in tablet dosage forms. A methanol:water (50:50 v/v) solvent system was selected, and measurements were carried out at 237 nm for BEN and 275 nm for CHL. The developed methods were validated in accordance with ICH Q2(R2) guidelines and were found to be linear, accurate, precise, robust, and sensitive. The methods were successfully applied to the assay of marketed formulations and demonstrated excellent greenness characteristics as confirmed by AGREE and ComplexGAPI assessments. The proposed methods were compared with previously reported UV, HPLC, and HPTLC methods and were found to offer greater sensitivity and superior greenness compared to the existing methods.

ACKNOWLEDGEMENT: The authors sincerely thank the Almighty for His blessings, guidance, and strength throughout the course of this research work. The authors gratefully acknowledge Goa College of Pharmacy for providing the facilities and resources required for carrying out this study. The authors express their heartfelt gratitude to Dr. Sachi Kudchadkar, Assistant Professor, Department of Pharmaceutical Analysis, Goa College of Pharmacy, for her expert guidance, valuable insights, and continuous support during the execution of this research work. The authors are thankful to Dr. Gopalkrishna Rao, Principal, Goa College of Pharmacy, for his encouragement and support. The authors also acknowledge Dr. Sanjay Pai P. N., Professor and Head, Department of Pharmaceutical Chemistry; Dr. Anand A. Mahajan, Professor; Dr. Adison Fernandes, Assistant Professor; and Mrs. Sweta Borkar, Assistant Professor, Department of Pharmaceutical Analysis, for their constructive suggestions and encouragement throughout the study.

CONFLICT OF INTEREST: The authors declare that they have no conflicts of interest to disclose.

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    How to cite this article:

    Naik A, Kanolkar S, Kumbharjuvekar C and Kudchadkar S: UV spectral strategies for analytical method development of multicomponent drug formulation. Int J Pharm Sci & Res 2026; 17(11): 3550-66. doi: 10.13040/IJPSR.0975-8232.17(11).3550-66.

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