STABILITY-INDICATING SPECTROPHOTOMETRIC DETERMINATION OF NALBUPHINE HYDROCHLORIDE USING FIRST DERIVATIVE OF RATIO SPECTRA AND RATIO DIFFERENCE METHODS

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El-Olemy et al. European Journal of Biomedical and Pharmaceutical Sciences

STABILITY-INDICATING SPECTROPHOTOMETRIC

DETERMINATION OF NALBUPHINE HYDROCHLORIDE USING

FIRST DERIVATIVE OF RATIO SPECTRA AND RATIO

DIFFERENCE METHODS

Khalid Abdel-Salam Attia, Mohammed Wafaa Nassar, Ahmed El-Olemy *

Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar

University, 11751, Nasr City, Cairo, Egypt.

Article Received on 10/05/2014 Article Revised on 03/06/2014 Article Accepted on 28/06/2014

ABSTRACT

Spectrophotometric stability-indicating procedures are described for

determination of nalbuphine hydrochloride (NAL) in pure and dosage

forms. First derivative of ratio spectra (1DD) and ratio difference

methods are proposed for determination of nalbuphine in presence of

its oxidative degradate. Beer’s law was obeyed in the concentration

range of 1-20 µg/ml for both methods. The proposed methods can

selectively analyse the drug in presence of up to 80 % of its degradate

with mean recoveries of 101.26± 0.48 and 98.85±0.61 for derivative

ratio and ratio difference methods respectively. These methods were validated and

successfully applied for the determination of NAL in its commercial preparation and the

obtained results were statistically compared with those of the reported method by applying

t-test and F-test at 95% confidence level and no significant differences were observed

regarding accuracy and precision.

Keywords: Nalbuphine; stability-indicating; derivative ratio; ratio difference.

*Correspondence for

Author

Dr. Ahmed El-Olemy

Pharmaceutical Analytical

Chemistry Department,

Faculty of Pharmacy, Al-

Azhar University, 11751,

Nasr City, Cairo, Egypt. [email protected]

EuropEan Journal of BiomEdical AND

Pharmaceutical sciences http://www.ejbps.com

ISSN 2349-8870 Volume: 1

Issue: 1 Page No: 01-11

Year: 2014

Research Article ejbps, 2014, Volume1, Issue 1, 01-11.

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INTRODUCTION

Nalbuphine hydrochloride (Fig. 1) is (56 (-17-) Cyclobutylmethyl)-4,5-epoxymorphinan-

3,6,14-triol hydrochloride [1].It is a phenanthrene derivative opioid analgesic. It has mixed

opioid agonist and antagonist activity. It is used for the relief of moderate to severe pain,

including that associated with myocardial infarction, and as an adjunct to anaesthesia[2].

Figure 1: Structural formula of nalbuphine hydrochloride.

Few analytical methods have been reported for analysis of nalbuphine including

spectrophotometric[3, 4], spectrofluorimetric[4, 5], electro-chemical[6] and chromatographic

methods[7-13].

Under computer-controlled instrumentation, first derivative of ratio spectra (1DD) and ratio

difference methods are playing a very important role in the analysis of binary mixtures

without previous separation by UV–VIS spectrophotometry[14-19].

In this work, both first derivative of ratio spectra and ratio difference methods were applied

to the determination of nalbuphine in presence of its oxidative degradate. The proposed

procedures were successfully applied for determination of nalbuphine in bulk powder and in

its pharmaceutical dosage form.

MATERIALS AND METHODS

Apparatus

Shimadzu UV-Vis. 1650 Spectrophotometer, (Tokyo, Japan), equipped with 10 mm

matched quartz cells.

Hot plate, Torrey pines scientific, USA.

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Materials and reagents

Pure sample

Nalbuphine hydrochloride was kindly supplied by Amoun Pharmaceutical Company, Cairo,

Egypt. The purity was assigned as 99.25%.

Pharmaceutical preparation

NALUFIN® ampoules, each ampoule (1 ml) claimed to contain 20 mg nalbuphine

hydrochloride (B.No. 369, manufactured by Amoun Pharmaceutical Company), purchased

from local market. Reagents and solvents

All chemicals and reagents used throughout the work were of analytical grade.

Water used throughout the procedures was freshly double distilled.

Methanol (Sigma–Aldrich, USA).

Ethanol (Riedell-detlean, Germany).

Hydrogen peroxide (50%) (El Nasr Co., Egypt). Standard solutions

Standard Solution of Intact NAL

A standard solution of NAL (100 µg/ml) was prepared by dissolving 10 mg of NAL in 50 ml

of water and complete to 100 ml with water. This solution was stable for one month when

kept in the refrigerator [3]. Standard Solution of Degraded Sample

100 mg of pure NAL powder were dissolved in 45 ml distilled water and transferred to a 100-

ml round bottomed flask to which 5 ml of 50% H2O2 was added. The solution was heated

under reflux for 6 hours and evaporated to dryness under vacuum. The obtained residue was

extracted with ethanol (2 × 10 ml), filtered into a 100-mL volumetric flask and diluted to

volume with ethanol to obtain a stock solution labeled to contain degradate derived from 1

mg/ml of NAL[3].This solution was diluted with water when needed. Procedures

Construction of the calibration curve (General procedure)

Different aliquots of NAL standard solution ranging from (10–200) µg were transferred to a

10-ml volumetric flasks and completed to volume with water. The absorption spectra (from

200 to 400 nm) of these solutions were recorded using water as a blank, and then divided by

the spectrum of NAL degradate solution (12 µg/ml).

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a. First derivative of ratio spectra method

The first derivative corresponding to each ratio spectrum was recorded, using Δλ = 2 nm. The

amplitude values at 214.6 nm were measured. The measured amplitude values versus the

final concentrations in µg/ml were plotted to get the calibration graph. Alternatively, the

regression equation was derived.

b. Ratio difference method

The difference in the peak amplitudes (ΔP) at the ratio spectra was measured at 206.8 and

229.2 nm (ΔP 206.8-229.2 nm). The measured ΔP values versus the final concentrations in µg/ml

were plotted to get the calibration graph. Alternatively, the regression equation was derived.

Analysis of pharmaceutical preparation

Contents of 10 NALUFIN® ampoules (each containing 20 mg NAL) were mixed well. A

volume equivalent to 10 mg of NAL was transferred into 100-mL volumetric flask and

completed to volume with water to obtain a solution labeled to contain 100 µg/ml of NAL.

Transfer aliquots covering the working concentration range into 10 ml volumetric flasks.

Proceed as described under “General Procedure” for each method. Determine the content of

the ampoules either from the calibration curve or using the corresponding regression

equation.

RESULTS AND DISCUSSION

Degradation of NAL: Stressed degradation of NAL was studied by refluxing the drug using

different media; aqueous, 1M NaOH, 1M HCl and 50% H2O2 for different time intervals. No

degradation took place using aqueous, acidic or basic conditions, whereas complete

degradation was attained when the drug was refluxed with 50% H2O2 for 6 hours[3](Fig. 2).

Figure (2): proposed degradation pathway of nalbuphine.

The zero-order absorption spectra of NAL and its oxidative degradate (Fig. 3) show severe

overlap, which does not permit direct determination of NAL in presence of its degradate.In

O

N

O H

H O

H O

R e f lu x 6 h o u rs

w it h 5 0 % H 2 O 2

O

N

O H

H O

O

N a lb up h in eO x id at i v e d eg ra d a te

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this work we develop two spectrophotometric methods which allow direct determination of

NAL in presence of its degradate without previous separation.

Spectral characteristics and optimization of the methods

First derivative of ratio spectra method

In this method, the absorption spectra of the drug were divided by a suitable absorption

spectrum of the degradate (divisor) to get the ratio spectra. By application of the first-

derivative to these ratio spectra, NAL can be quantitatively determined at 214.6 nm without

any interference from its degradation product, as shown in Figs. 4, 5. Careful choice of the

divisor and the working wavelength were of great importance so different concentrations of

degradation product were tried as a divisor (4, 8, 12, 16 and 20 µg/ml); the best one was 12

µg/ml as it produced minimum noise and gave better results in accordance with selectivity. Ratio difference method

In this method, the absorption spectra of the drug were divided by a suitable absorption

spectrum of the degradate (divisor) to get the ratio spectra. The difference in peak amplitudes

between two selected wavelengths in the ratio spectra is proportional to the concentration of

the drug without interference from its degradate (divisor), as shown in Fig. 6. The method

comprises two critical steps, the first is the choice of the divisor, the selected divisor should

compromise between minimal noise and maximum sensitivity. The divisor concentrations of

12 µg /ml gave the best results. The second critical step is the choice of the wavelengths at

which measurements are recorded. Any two wavelengths can be chosen provided that they

exhibit different amplitudes in the ratio spectrum and good linearity is present at each

wavelength individually. The selected wavelengths are 206.8 and 229.2 nm (ΔP 206.8-229.2 nm)

which gave the best results.

Figure (3): Absorption spectra of nalbuphine (16 µg/ ml) and its oxidative degradate (16 µg/ ml).

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Figure (4): First derivative of the ratio spectra of nalbuphine (16 µg/ml) and its

oxidative degradate (16 µg/ml) using 12 µg/ml of degradate as a divisor.

Figure (5): First derivative of the ratio spectra of nalbuphine at various concentrations

(1, 4, 8, 10, 12, 16 and 20 µg/ml) using 12 µg/ml of degradate as a divisor.

Figure (6): Ratio spectra of nalbuphine at various concentrations (1, 4, 8, 10, 12, 16 and

20 µg/ml) using 12 µg/ml of degradate as a divisor.

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Validation of the methods

Linearity and range

Under the described experimental conditions, the calibration graphs for the methods were

constructed by plotting either the amplitudes of the first derivative of the ratio spectra (for

ratio derivative method) or the differences in peak amplitudes between the two selected

wavelengths in the ratio spectra (for ratio difference method) versus concentrations in µg/ml.

The regression plots were found to be linear over the range of 1-20 µg/ml for the two

methods.Linearity ranges, regression equations, intercepts, slopes and correlation coefficients

for the calibration data were presented in table 1.

Limits of detection and quantitation

The limit of detection (LOD) and the limit of quantitation (LOQ) were calculated according

to ICH guidelines [20] from the following equations:

LOD = 3.3 Sa / slope

LOQ = 10 Sa / slope

Where Sa is the standard deviation of y-intercepts of regression lines.

LOD were found to be 0.287 and 0.114 µg/ml, while LOQ were found to be 0.869 and 0.345

µg/ml for ratio derivative and ratio difference methods respectively. The small values of LOD

and LOQ indicate good sensitivity.

Accuracy and precision

According to the ICH guidelines [20], three replicate determinations of three different

concentrations of NAL in pure form within linearity range for each method were performed

in the same day (intra-day) and in three successive days (inter-day).Accuracy as recovery

percent (R%) and precision as percentage relative standard deviation (RSD%) were

calculated and results are listed in table 2. The small values of RSD% indicates high precision

of the methods.Morever, the good R% confirms excellent accuracy.

Specificity

The specificity of the proposed methods were assured by applying the laboratory prepared

mixtures of the intact drug together with its degradation product. The proposed methods were

adopted for the specific determination of intact NAL in presence of up to 80 of its degradate

with mean recoveries of 101.26±0.48 and 98.85±0.61 for derivative ratio and ratio difference

methods respectively (table 3).

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Pharmaceutical Applications

The proposed methods were applied to the determination of the studied drug in NALUFIN®

ampoules. The results were validated by comparison to a previously reported method[4]. No

significant difference was found by applying t-test and F-test at 95% confidence level[21],

indicating good accuracy and precision of the proposed methods for the analysis of the

studied drug in its pharmaceutical dosage form (table 4).

Table (1): Spectral data for determination of nalbuphine by the proposed methods.

Parameters Ratio derivative Ratio difference Wavelength (nm) 214.6 nm 206.8 and 229.2 nm Linearity range (µgml-1) 1 — 20 1 — 20 LOD (µgml-1) 0.287 0.114 LOQ (µgml-1) 0.869 0.345 Regression equation* Slope (b) Intercept (a)

0.0275 0.0032

0.2224 0.0158

Correlation coefficient (r2) 0.9997 1.0000 * y= a + bx where y is the response and x is the concentration.

Table (2): Intraday and interday accuracy and precision for the determination of

nalbuphine by the proposed methods.

Met

hod

Conc g.ml-1 Intraday Interday

Found Conc. + SD

Accuracy (R%)

Precision (RSD%)

Found Conc. + SD

Accuracy (R%)

Precision (RSD%)

Rat

io

deriv

ativ

e 4 3.99 ± 0.036 99.79 0.910 4.00 ± 0.041 100.06 1.023 10 9.96 ± 0.091 99.63 0.918 10.01 ± 0.117 100.11 1.167 16 16.01 ± 0.147 100.04 0.917 15.99 ± 0.151 99.97 0.946

Rat

io

diff

eren

ce

4 4.01 ±0.052 100.29 1.293 3.98 ±0.029 99.5 0.741 10 10.13 ±0.049 101.32 0.487 9.99 ±0.095 99.85 0.947 16 15.87 ±0.144 99.19 0.909 15.91 ±0.109 99.41 0.683

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Table (3): Determination of intact nalbuphine in mixtures with its oxidative degradate

by the proposed methods.

Method Intact (µg ml-1)

Degradate (µg ml-1)

Degradate %

Intact found (µg ml-1)

Recovery % of Intact

Rat

io

deriv

ativ

e

18 2 10 18.25 101.39 16 4 20 16.11 100.69 12 8 40 12.20 101.67 8 12 60 8.06 100.80 4 16 80 4.07 101.75

Mean ± RSD% 101.26+0.48

Rat

io

diff

eren

ce

18 2 10 17.88 99.33 16 4 20 15.85 99.06 12 8 40 11.78 98.17 8 12 60 7.96 99.44 4 16 80 3.93 98.25

Mean ± RSD% 98.85+0.61 Table (4): Determination of nalbuphine in NALUFIN® ampoules by the proposed and

reported methods.

Parameters Ratio derivative

Ratio difference

Reported method[4]

N* 5 5 5 X‾ 100.29 99.92 99.61 SD 0.98 0.96 1.03

RSD% 0.98 0.96 1.03

t** 1.07 (2.31)

0.49 (2.31) ——

F** 1.11 (6.39)

1.15 (6.39) ——

* No. of experimental.

** The values in the parenthesis are tabulated values of t and F at (p= 0.05).

CONCLUSION

The proposed methods are simple, rapid, accurate and precise and can be used for the

analysis of NAL in pure form and in pharmaceutical dosage form (either alone or in the

presence of its degradation product).

ACKNOWLEDGMENT

I am deeply thankful to ALLAH, by the grace of whom this work was realized. I wish to

express my indebtedness and gratitude to staff members Pharmaceutical Analytical

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Chemistry Department, Faculty of Pharmacy Al-Azhar University, Cairo, Egypt for their

valuable supervision, continuous guidance, and encouragement throughout the whole work.

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STABILITY-INDICATING SPECTROPHOTOMETRIC DETERMINATION OF NALBUPHINE HYDROCHLORIDE USING FIRST DERIVATIVE OF RATIO SPECTRA AND RATIO DIFFERENCE METHODS

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