Development and Validation of First-Order Derivative Spectrophotometry for Simultaneous Determination of Levocetirizine Dihydrochloride and Phenylephrine Hydrochloride in Pharmaceutical Dosage Form

A simple, precise, accurate, and economical spectrophotometric method has been developed for simultaneous estimation of levocetirizine dihydrochloride (LCT) and phenylephrine hydrochloride (PHE) by employing first-order derivative spectrophotometric method. The first-order derivative absorption at 240 nm (zero crossing point of PHE) was used for quantification of LCT and 283.2 nm (zero crossing point of LCT) for quantification of PHE. The linearity was established over the concentration range of 4–24 μg/mL and 8–48 μg/mL for LCT and PHE with correlation coefficients (r) 0.9964 and 0.9972, respectively. The mean % recoveries were found to be in the range of 99.14%–100.43% for LCT and 98.73%–100.83% for PHE.The proposed method has been validated as per ICH guideline and successfully applied for the simultaneous estimation of LCT and PHE in combined tablet dosage form.

The chemical name of phenylephrine hydrochloride (PHE) is (R)-3-Hydroxy-alpha [(methylamino)methyl] benzenemethanol hydrochloride [3].The structure of PHE is given in Figure 2. PHE is a direct acting sympathomimetic agent.It is a selective  1 adrenoceptor agonist and has negligible  action.It is also a vasoconstrictor, because it has little cardiac action.It is mainly used as a nasal decongestant and for producing mydriasis when cycloplegia is not required.It tends to reduce intraocular tension by constricting ciliary body blood vessels [2].
LCT and PHE in combined dosage form are used as a nasal decongestant.
The review of the literature revealed that various analytical methods involving spectrophotometry, HPLC, HPTLC, and LC-MS have been reported for LCT alone and in combination with other drugs [4][5][6][7][8][9][10][11][12][13][14].Several analytical methods have been reported for PHE alone and in combination with other drugs including spectrophotometry, HPLC, HPTLC, LC-MS/MS, and electrophoresis [15][16][17][18][19][20][21][22][23].However, to the best of our knowledge, no spectrophotometric method is published for the simultaneous determination of LCT and PHE in tablet dosage form.The present work describes the development of a simple, precise, accurate, and reproducible spectrophotometric method for the simultaneous estimation of LCT and PHE in combined dosage forms.The developed method was validated in accordance with ICH guideline and successfully employed for the assay of LCT and PHE in combined tablet dosage form.

Preparation of Standard Stock Solutions.
Accurately weighed 25 mg of LCT and PHE standards was transferred to a separate 25 mL volumetric flask and dissolved in 10 mL distilled water.The flasks were shaken and volume was made up to the mark with distilled water.These solutions are 1000 g/mL LCT and 1000 g/mL PHE respectively.

Selection of Analytical Wavelength.
Working standard solutions of 4-24 g/mL of LCT and 8-48 g/mL of PHE were prepared in distilled water by appropriate dilution, and the spectrum was recorded between 200 and 400 nm, and all zero-order spectrums (D 0 ) were converted to first derivative spectrums (D 1 ) using delta lambda 2 and scaling factor 7.
The overlain first derivative spectrums of LCT and PHE at different concentrations were recorded.The zero crossing point (ZCP) of LCT was found to be 283.2nm, and ZCP of PHE was found to be 240 nm.[24].The proposed method was validated in terms of linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), and reproducibility.The accuracy was expressed in terms of percent recovery of the known amount of the standard drugs added to the known amount of the pharmaceutical dosage forms.The precision (% relative standard deviation-% RSD) was expressed with respect to the repeatability, intraday, and interday variation in the expected drug concentrations.After validation, the developed methods have been applied to pharmaceutical dosage form.quantitation limits.These include visual evaluation, signalto-noise ratio, and the use of standard deviation of the response and the slope of the calibration curve.In the present study, the LOD and LOQ were based on the third approach and were calculated according to the 3.3 /S and 10 /S criterions, respectively, where  is the standard deviation of the -intercepts of the regression lines and  is the slope of the calibration curve.

Method Validation
2.10.Reproducibility.The absorbance readings were measured at a different laboratory for sample solution using another spectrophotometer by another analyst, and the values obtained were evaluated using t-test to verify their reproducibility.

Results and Discussion
First-order derivative spectrophotometric method was developed for determination of LCT and PHE.The overlain first-order derivative spectrum of LCT and PHE at different concentrations revealed that at 283.2 nm a different concentration of LCT possesses zero D 1 absorbance, whereas PHE possesses significant D 1 absorbance (Figure 3).In a similar manner, at 240 nm different concentrations of PHE possess zero D 1 absorbance, whereas LCT possesses significant D 1 absorbance (Figure 4).Considering the above facts, wavelengths 283.2 nm and 240 nm were selected for the estimation of PHE and LCT, respectively.Figure 5 shows overlain D 1 spectra of LCT and PHE at different concentrations.
The proposed method has been extensively validated as per ICH guidelines.Summary of validation parameters for the proposed method is given in Table 1.
Linearity was assessed for LCT and PHE by plotting calibration curves of the D 1 absorbance versus the concentration over the concentration range 4-24 g/mL and 8-48 g/mL, respectively.The correlation coefficients ( 2 ) for LCT and PHE were found to be 0.9964 and 0.9972, respectively (Table 2).The following equations for straight line were obtained for LCT and PHE: linear equation for LCT,  = 0.0152 + 0.0457, linear equation for PHE,  = 0.0045 + 0.0443. ( The % recoveries were found to be 99.14%-100.43%for LCT and 98.73%-100.83%for PHE (Table 3).Precision was determined by repeatability, intraday, and interday variation for both drugs and was expressed as % RSD (Tables 4 and 5).
The proposed spectrophotometric method was successfully applied to LCT and PHE in combined tablet dosage form as shown in Figures 6 and 7 (Table 6).

Conclusion
The proposed first-order derivative method provides simple, specific, precise, and accurate quantitative analysis for simultaneous determination of LCT and PHE in combined tablet dosage form.The method was validated as per ICH guidelines in terms of linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), and reproducibility.The proposed method can be used for routine analysis and quality control assay of LCT and PHE in combined dosage form.

Figure 5 :
Figure 5: Overlain D 1 spectra of LCT and PHE in distilled water.

Table 1 :
Summary of validation parameters.

Table 2 :
Statistical data of LCT and PHE.

Table 4 :
Precision data at 240 nm for LCT.

Table 5 :
Precision data at 283.2 nm for PHE.

Table 6 :
Assay results of marketed formulations.