Simultaneous Spectrophotometric Estimation of Naphazoline Nitrate and Hydrocortisone in Nasal drops

Jetal Patel¹*, Dhara Patel¹, Sharav Desai²

¹Department of Quality Assurance, Pioneer Pharmacy Degree College, Vadodara, Gujarat, India.

²Department of Pharmaceutical Microbiology and Biotechnology, Pioneer Pharmacy Degree College, Vadodara, Gujarat, India.

*Corresponding Author E-mail: patel.dhara.j@gmail.com

ABSTRACT:

Two new simple, sensitive, accurate, precise and economical spectrophotometric methods developed and validated for the simultaneous estimation of naphazoline nitrate and hydrocortisone in nasal drops. The first method is Q- absorbance ratio method. Q- Absorbance ratio method uses the ratio of absorbances at two selected wavelengths, one which is an isoabsorptive point and other being the λ-max of one of the two components. Naphazoline nitrate and hydrocortisone show an isoabsorptive point at 264 nm in methanol. The second method is based on the derivative spectrophotometric method at zero crossing wavelengths. In this method the zero crossing point of naphazoline nitrate was selected at 277 nm and for hydrocortisone it was 290 nm. The linearity was obtained in the concentration range of 6.25-36.75 μg/ml for naphazoline nitrate and 5-30 μg/ml for hydrocortisone for both the above methods. These methods are simple, accurate and rapid, those require no preliminary separation and can therefore be used for routine analysis of both drugs in quality control laboratories. The suitability of these methods for the quantitative determination of the compounds was proved by validation.

KEY WORDS: Naphazoline nitrate, Hydrocortisone, Q-absorbance ratio method, Derivative spectrophotometric method, Nasal drops, Validation.

INTRODUCTION:

Naphazoline nitrate (NAPH), chemically known as 2-(Naphthalen-1-ylmethyl)-4,5-dihydro-1H-imidazole nitrate (Figure 1) is an adrenergic vasoconstrictor agent used as a nasal decongestant ^[1]. It is official in Indian Pharmacopoeia (IP)^[2] and British Pharmacopoeia (BP) ^[3]. IP and BP describe the potentiometry titration for the estimation of naphazoline nitrate.

A literature survey revealed UV spectrophotometric method, HPLC and miscellar electrokinetic capillary chromatography method of NAPH with other drugs like antazoline, tetra methyl theonine, diphenhydramine, bacitracin, prednisolone, sulphacetamide and phenylephrine^[4-10].

Hydrocortisone (HYD), chemically known as (8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthren-3-one (Figure 2) is a corticosteroids which is secreted by the adrenal cortex, having an glucocorticoids activity with an anti-inflammatory and immunosuppressive property. It is used to treat many immune and allergic disorders such as arthritis, severe psoriasis, severe asthma and crohn’s disease^[1]. It is official in IP, BP and USP (united state Pharmacopoeia)^[11]. IP and BP describe the UV spectroscopy method and USP describe the HPLC method for the estimation of hydrocortisone. A literature survey revealed various UV spectrophotometric, HPLC, HPTLC, NIR spectroscopy, miscellar electro kinetic capillary chromatography, RIA, flourimetry, LC-tandem mass spectroscopy methods for the estimation of HYD alone and with combination of other drugs in pharmaceutical preparations ^[12-24]. The combination of naphazoline nitrate and hydrocortisone is not yet official in any of the pharmacopoeia. Literature survey revealed that there is no reported method for the simultaneous estimation of naphazoline nitrate and hydrocortisone in nasal drops. Therefore, simple, rapid, and reliable method for simultaneous estimation of these drugs in nasal drops seemed to be necessary. Spectrophotometric methods of analysis are more economic and simpler, compared to methods such as chromatography and electrophoresis. The purpose of this study was to determine both drugs concurrently by simple, accurate, rapid and precise Q-absorbance ratio and first derivative spectrophotometric assays for routine analysis.

Figure 1: Chemical structure of naphazoline nitrate

Figure 2: Chemical structure of hydrocortisone

MATERIALS AND METHODS:

Apparatus and instrument:

UV visible double beam spectrophotometer (SHIMADZU -1800, Japan) with software UV Probe 2.33, with spectral slit width of 2 nm, wavelength accuracy of 0.5 nm and pair of 1cm matched quartz cells and digital balance Shimadzu ATX 224, Japan and ultrasonicator were used. Volumetric flasks and pipettes of borosilicate glasses were used in the study.

Chemicals and reagents:

Pure drugs samples of naphazoline nitrate and hydrocortisone were kindly supplied as a gift sample from Sun Pharmaceutical Pvt. Ltds, Vadodara and S-Kant pharma pvt ltd, Vapi, respectively. Methanol (AR Grade) and other reagent were provided by Department of Quality Assurance, Pioneer Pharmacy Degree College, Vadodara, Gujarat, India. Marketed formulation (Nasal drops containing NAPH 0.025 % w/v and HYD 0.02% w/v ) was purchase from local market.

Selection of common solvent:

Methanol of analytical reagent grade was selected as a common solvent for developing spectral characteristics of both drugs. The selection was made after assessing the solubility of both drugs in different solvents like water, chloroform, ether etc.

Preparation of standard stock solutions of NAPH and HYD:

Accurately weighed quantities of NAPH (10 mg) and HYD (10 mg) transferred to separate volumetric flasks (100 ml), dissolved in methanol (small quantity) and diluted up to mark with methanol (100 μg/ml of NAPH and HYD).

Spectrophotometric conditions for Q- absorbance ratio and first order derivative method:

Q-absorbance ratio:

Measurement Mode: Spectrum

Scan speed: Medium

Band width: 1 nm

Wavelength range: 200-400 nm

Absorbance scale: 0.0 A⁰ -2.0 A⁰

Initial baseline: Methanol

First order derivative method:

Measurement Mode: Spectrum

Scan speed: Medium

Band width: 1 nm

Wavelength range: 200-400 nm

Absorbance scale: 0.0 A⁰ -2.0 A⁰

Delta lamda: 10

Scaling factor: 1

Initial baseline: Methanol

METHODOLOGY:

Method 1:Q-absorbance ratio method:

Q-Absorbance ratio method uses the ratio of absorbances at two selected wavelengths, one which is an isoabsorptive point and other being the λ-max of one of the two components. From the overlay spectra of two drugs, it is evident that NAPH and HYD show an isoabsorptive point at 264 nm. The second wavelength used is 248 nm, which is the λ-max of HYD. Working standard solutions having concentration 6.25, 12.50, 18.75, 24.25, 30.50 and 36.75 μg/ml for NAPH and 5, 10, 15, 20, 25 and 30 μg/ml for HYD were prepared in methanol and the absorbances at 264 nm (isoabsorptive point) and 248 nm (λ-max of HYD) were measured and absorptivity coefficients were calculated using calibration curve. The concentration of two drugs in the mixture can be calculated using following equations.

Cx= {(QM-Qy)/ (Qx-Qy)}* (A1/ax1)

Cy= A/ ax1 – Cx

Where,

QM = Absorbance of sample at 248 nm /Absorbance of sample at 264 nm ,

QX = Absorptivity of NAPH at 248 nm/Absorptivity of NAPH at 264 nm,

QY = Absorptivity of HYD at 248 nm /Absorptivity of HYD at 264 nm ,

A = Absorbance of sample at iso-absorptive point

ax1 = Absorptivity of NAPH at iso-absorptive point.

Method 2: First derivative spectroscopic method

The standard solutions of NAPH (12.50μg/ml) and HYD (10μg/ml) were scanned separately in the UV range of 200-400 nm. The zero-order spectra thus obtained was then processed to obtain first-derivative spectra. The two spectra were overlain and it appeared that NAPH showed zero crossing point 277 nm, while HYD showed zero crossing point at 290 nm. At the zero crossing point (ZCP) of NAPH (277 nm), HYD showed an absorbance, whereas at the ZCP of HYD (290 nm), NAPH showed an absorbance. Hence 290 and 277 nm was selected as analytical wavelengths for determination of NAPH and HYD, respectively. The linearity was obtained in the concentration range of 6.25-36.75 μg/ml for NAPH and 5-30 μg/ml for HYD and the absorbance's for both were measured at respective zero crossing point.

Assay of NAPH and HYD from nasal drops:

1ml of solution was taken from formulation having label claim (Nasal drops containing NAPH 0.025 % w/v and HYD 0.02 % w/v) and transferred to 10 ml volumetric flask and dissolved and diluted up to 10 ml with in methanol. 0.1ml of aliquots was taken from stock solution and transferred in to 10ml of volumetric flask and make up with methanol. For Q-absorption ratio method the absorbance of resulting solutions were measured at 248 nm, 264 nm (iso-absorptive point). For First derivative method the absorbance was measured at 290 (ZCP of HYD) and 277 nm (ZCP of NAPH).

Validation of the proposed methods:

The proposed methods were validated according to the International Conference on Harmonization (ICH) guidelines^[25] .

Linearity:

Linearity was studied by preparing standard solutions at different concentration levels. Calibration curves were prepared using the standard solutions of 6.25- 36.75 μg/ml for NAPH and 5-30 HYD in Q-absorption ratio and first derivative method. The calibration curves were plotted over a concentration range of 6.25-36.75 μg/ml for NAPH and 5-30μg/ml for HYD and linear regression analysis was carried out.

Precision (Repeatability):

The precision of the instrument was checked by repeated scanning and measurement of absorbance of solutions (n = 6) for NAPH (12.50 μg/ml) and HYD (10 μg/ml ) without changing the parameter of the proposed spectrophotometry methods.

Intermediate Precision (Reproducibility):

The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of NAPH and HYD (12.50, 18.75, 24.25 μg/ml for NAPH and 10, 15, 20 μg/ml for HYD). The result was reported in terms of relative standard deviation (% RSD).

Accuracy (Recovery Study):

The accuracy of the method was determined by calculating the recoveries of NAPH and HYD by the standard addition method. Known amounts of standard solutions of NAPH and HYD were at added at 80, 100 and 120 % level to prequantified sample solutions of NAPH and HYD (12.50 μg/ml for NAPH and 10 μg/ml for HYD). The amounts of NAPH and HYD were estimated by applying obtained values to the respective regression line equations.

Limit Of Detection And Limit Of Quantification:

The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines.

RESULTS:

In absorbance ratio method (Q-analysis), the primary requirement for developing a method for analysis is that the entire spectra should follow the Beer’s law at all the wavelength, which was fulfilled in case of both these drugs. The two wavelengths were used for the analysis of the drugs were 264 nm (isoabsorptive point) and 248 nm (λ-max of HYD) at which the calibration curves were prepared for both the drugs. The overlain UV absorption spectra of NAPH ( 12.50 μg/ml) and HYD (10 μg/ml ) showing isoabsorptive point (264 nm) in methanol is shown in Figure 3. In First derivative spectroscopy method, The standard solutions of NAPH (12.50 μg/ml) and HYD (10μg/ml) were scanned separately in the UV range of 200-400 nm. The zero-order spectra thus obtained was then processed to obtain first-derivative spectra. The two spectra were overlain( figure 4) and it appeared that NAPH showed zero crossing point 277 nm, while HYD showed zero crossing point at 290 nm. Optical and regression parameters are mentioned in table 1. The validation parameters were studied at all the wavelengths for the proposed method. Accuracy was determined by calculating the recovery and the mean was determined (Table 2). The method was successfully used to determine the amounts of NAPH and HYD present in nasal drops. The results obtained were in good agreement with the corresponding labeled amount (Table 3). The results of assay validation of the proposed methods show that they are accurate and precise according to the RSD values of intraday and interday determinations (Table 4). Based on the results obtained , The proposed procedures can be applied for the simultaneous determination of NAPH and HYD. Moreover, the methods are rapid, accurate, precise and can be used in routine analysis.

Figure. 3: Overlain spectra of NAPH (12.50 μg/ml) and HYD (10 μg/ml)

Figure 4: Overlain first derivative spectra of NAPH (12.50 μg/ml) and HYD(10 μg/ml)

Table 1: Regression analysis data of first derivative results of NAPH and HYD

Parameters	Q-absorbance ratio Spectroscopy				First-derivative UV Spectrophotometry
	NAPH at 264nm	NAPH at 248nm	HYD at 248nm	HYD at 264nm	NAPH at 290 nm	HYD at 277 nm
Concentration range (µg/mL)	6.25-36.75	6.25-36.75	5-30	5-30	6.25-36.75	5-30
Slope	0.0167	0.0086	0.0387	0.0188	0.001	0.0006
Intercept	0.0018	0.0014	0.0192	0.0099	0.0002	0.0007
Correlation coefficient (r^2)	0.9994	0.9996	0.9998	0.9998	0.9991	0.9998

Table 2: Application of the standard addition technique to the analysis of NAPH and HYD in nasal drops by the proposed methods

Proposed methods	Concentration of drug taken (μg/ml)		Concentration of drug added (μg/ml)		Total Concentration of drug (μg/ml)		% Recovery (n^a=3) ± SD^b
	NAPH	HYD	NAPH	HYD	NAPH	HYD	NAPH	HYD
Q- absorbance ratio	12.50	10	10	8	22.50	18	99.83±.50	99.45±0.61
	12.50	10	12.50	10	25	20	100.16±0.78	100.24±0.88
	12.50	10	15	12	27.50	22	98.90±1.53	99.56±1.63
First derivative UV	12.50	10	10	8	22.50	18	99.75±1.22	100.25±0.76
	12.50	10	12.50	10	25	20	100.49±0.41	100.48±1.03
	12.50	10	15	12	27.50	22	99.60 ±1.03	99.35±.0.59

Table 3: Assay results for tablets using the proposed methods

Formulation Nasal Drops	Proposed methods	Label Claim (mg/ml)		Amount of drug found (mg/ml)		% Label claim Assay ± SD
		NAPH	HYD	NAPH	HYD	NAPH	HYD
	Q- absorbance ratio	0.25	0.2	0.197	0.248	98.5 ± 0.0024	99.2 ± 0.026
	First derivative	0.25	0.2	0.198	0.249	99.00 ± 0.0012	99.60 ± 0.0024

Table 4: Summary of validation parameters for the proposed methods

Proposed methods	Drug	Parameters
		LOD^aµg/ml	LOQ^b µg/ml	Repeatability (RSD^c, %)	Interday (n = 3) (RSD^c, %)	Intraday (n^d = 3) (RSD^c, %)
Q- absorbance ratio	NAPH (264 nm)	0.33	1.00	0.14	0.39-1.04	0.37-0.82
	HYD (264nm)	0.30	0.93	0.19	0.27-0.93	0.39-0.50
	NAPH (248 nm)	0.46	1.39	0.53	0.24-0.44	0.76-1.16
	HYD (248 nm)	0.23	0.70	0.15	0.76-1.16	0.24-0.57
First derivative	NAPH (290 nm)	0.313	0.95	0.53	0.48-0.63	0.85-1.63
	HYD (277 nm)	0.59	1.8	1.42	1.49-2.91	0.85-1.89

DISCUSSION:

The proposed spectrophotometric methods were found to be simple, accurate and precise for determination of NAPH and HYD in nasal drops. The method utilizes easily available and cheap solvent for analysis of NAPH and HYD hence the method was also economic for estimation of NAPH and HYD from nasal drops. The common excipients and other additives are usually present in the nasal drops do not interfere in the analysis of NAPH and HYD in method, hence it can be conveniently adopted for routine quality control analysis of the drugs in combined pharmaceutical formulation.

ACKNOWLEDGMENT:

The authors are thankful to Sun Pharmaceuticals. Ltd. Vadodara and S-Kant Pharma Pvt. Ltd, Vapi for providing the gift samples of NAPH and HYD and Pioneer Pharmacy Degree College, Vadodara for providing all the facilities to carry out the research work.

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Received on 27.02.2016 Accepted on 10.03.2016

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2016; 6(2): 61-66

DOI: 10.5958/2231-5691.2016.00011.3