naltrexone and buropion RP-HPLC best research paper award 2014

CH. Naveen Kumar et al., J. Sci. Res. Phar. 2015, 4(1), 19-32
Journal of Scientific Research in Pharmacy 2015, 4(1) 19-32
Journal of Scientific Research in Pharmacy Research Article
Available online through ISSN: 2277-9469
www.jsrponline.com
Stress Degradation studies and Develpoment of Validated Stability Indicating Assay Method by RP-HPLC
for Simultaneous Estimation of Naltrexone and Bupropion in the presence of degradation products as per
ICH Guidelines
CH. Naveen Kumar a, b*, N. Kannappan b, Mahendra Kumar CB c
*a Bright Labs, Kothapet, Dilshuknagar, Hyderabad, Telangana, INDIA.
bDepartment of Pharmacy, Faculty of Engineering and Technology, Annamalainagar, Annamalai University, Chidambaram, Tamilnadu, INDIA.
cDepartment of Pharmacy, St. Mary’s College of Pharmacy, Secunderabad, Telangana, INDIA.
Received on: 23-12-2014; Revised and Accepted on: 02-02-2015
ABSTRACT
The aim of the present study was to establish the inherent stability and stability indicating assay method for simultaneous
determination of naltrexone and bupropion after being subjected to International conference on harmonization stress conditions, such as
hydrolysis, oxidation, heat, and photolysis. The quantification was carried out using Waters symmetry C18 (150x4.6 ID) 3.5 μm column at an Room
temp (20-250 c) and mobile phase comprised of Potassium dihydrogen phosphate buffer pH - 4.5: Acetonitrile in the proportion ratio of (30:70 v/v)
in Isocratic mode. The flow rate was 1.0 ml/min and the effluent detection was performed and monitored at 228 nm using photodiode array
(PDA) detector. The retention time of naltrexone and bupropion were 2.539 & 5.346 mins respectively. The method was validated in terms of
linearity, precision, accuracy, and specificity, limit of detection and limit of quantitation. The method calibration curves were found to be linear
over the concentration range of 5-25µg/ml for naltrexone drug, 56.25-281.25µg/ml for bupropion drug respectively and the range and correlation
coefficient was found to be (r2 = 0.9998). The percentage recoveries of both the drugs were 98.95-101.22%and 98.46-100.98% for naltrexone and
bupropion respectively from the tablet formulation.Limit of detection(LOD)were 0.387 & 1.297μg/mL and limits of quantification (LOQ) were
1.292 and 4.326μg/mL.Degradation products resulting from the stress conditions did not interfere with the detection of naltrexone and bupropion
so The proposed method is suitable for simultaneous determination of naltrexone and bupropion in pharmaceutical dosage form and bulk drugs.
Keywords: Naltrexone, Bupropion, Stability Indicating, Forced Degradation studies, RP-HPLC, Method Validation.
INTRODUCTION
The multidrug therapy is a well-known technique for
administration of two more active drug components in a single
dosage form; it has better patient acceptability due to reduced
number of dosage forms to be taken at a time. There are many
Analytical methods are extensively available for single drug
formulations but due to complexity in multicomponent
formulations, stability Indicating method development for multiple
drugs formulation is a challenge and scope for new developments.
The present research work is focused to develop a proper solvent
system and method development by Reverse Phase High
Performance Liquid Chromatography (RP-HPLC) for the analysis of
multi-drug combination including naltrexone and bupropion and to
validate the developed process as per the ICH guidelines [1].
The stress testing of the new drug substance is carried
out according to stability test guideline Q1A (R2) issued by ICH for
establishing its inherent stability characteristics and for supporting
the suitability of the proposed analytical procedure [2-4].
Naltrexone (NTX) is chemically (5α)-17-
(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6-one
hydrochloride (Fig. 1).The main use of naltrexone is for the
treatment of alcohol dependence [4-6].NTX is used in the treatment of
alcoholism.Naltrexone was approved by the U.S. Food and Drug
Administration (FDA) for the treatment of alcohol dependence in
1994, following publication of the first two randomized, controlled
trials in 1992. Since then a number of studies have confirmed its
efficacy in reducing frequency and severity of relapse to drinking [7].
The multi-centre combine study showed the usefulness of in a
primary care setting without adjunct psychotherapy [8]. BUP is
chemically, (±)-2-(tert-butylamino)-1-(3-chlorophenyl) propan-1-
*Corresponding author:
CH. Naveen Kumar
Bright Labs, Kothapet,
Dilshuknagar, Hyderabad, Telangana, INDIA.
*E-Mail: naveen2626@gmail.com
one (Fig. 2), an atypical antidepressant and smoking cessation aid.
It acts as a norepinephrine and dopamine reuptake inhibitor as well
as α3 β4 nicotinic receptor antagonist [9-11]. Presently, combination
of these two drugs as a controlled release tablet is under clinical
trials for the treatment of obesity Literature survey reveals that a
very few physico-chemical methods appeared in the literature for
the determination of NTX in pharmaceutical formulations. The
methods so far reported include. A LC electro spray Tandem MS
method for the analysis of NTX in canine plasma employing a
molecular model to demonstrate the absence of internal standard
deuterium isotope effects [12]. Nano level detection of NTX in its
pharmaceutical preparation at Au microelectrode in flowing
solutions by fast fourier transforms continuous cyclic voltammetry
as a novel detector [13-15]. However, there were only few validated
HPLC-UV/PDA methods reported so far for the simultaneous
estimation of NTX and BUP in combination but there are no
reported stability indicating methods for NTX and BUP. Hence, the
main objective of the present study was to develop a validated
stability indicating RP-HPLC-PDA method for the simultaneous
estimation of NTX and BUP in bulk and pharmaceutical dosage
forms.
Fig. 1: Chemical structure of naltrexone (NTX)

Fig. 2: Chemical structure of bupropion (BUP)
MATERIALS AND METHODS
1. Experimental:
1.1. Materials and methods:
Pure sample of Naltrexone (NTX) and Bupropion (BUP)
was obtained from KDPL Pharmaceuticals and other reagents such as
Acetonitrile,methanol,ortho phosphoric acid, Potassium dihydrogen
Phosphate and water used were of HPLC and milli-Q grade. All other
chemicals used were of AR grade. Bupropion and
Naltrexone(Contrave) Tablets were purchased from local pharmacy.
1.2 . Instrumentation:
The analysis was performed using waters-2695(Model
alliance) High Performance liquid chromatography waters auto
sampler–PDA detector by using, Empower-software version-2,
analytical balance (MettlerToledo) UV/Visible-Detector (Standard
cell) and data handling system (Autochrome-3000), pH meter (lab
India), Sonicator. The column used is Waters symmetry C18
(150x4.6 ID) 3.5 μm withthe flow rate 0.5ml/min (isocratic).
1.3.Preparation of Buffer solution (pH 4.5):
About1.36 g of Potassiumdihydrogen orthophosphate
was accurately weighed and taken into 250 ml volumetric
flask.Then add 150 ml of HPLC water, dissolve completely to get a
clear solution.Make up the volume up to the mark with water. The
pH was adjusted to 4.5 with Orthophosphoric acid and filtered
through 0.45 μm membrane filter Sonicate for 15 min.
1.4. Diluent( Mobile phase ) Preparation:
Combination of Potassium dihydrogen orthophosphate
buffer (pH-4.5) and Acetonitrile was mixed in the ratio of 30:70(pH
was adjusted to 4.5 with Orthophosphoric acid and filtered through
0.45 μm membrane filter). This prepared solution was used as
mobile phase. This solution was also used for specificity blank
solution.
1.5. Preparation of blank solution:
Combination of Potassium dihydrogen orthophosphate
buffer (pH-4.5) and Acetonitrile was mixed in the ratio of 30:70.
This prepared solution was used as mobile phase. This solution was
also used for specificity blank solution
1.6. Preparation of Placebo Solution:
The placebo Solution was prepared by Dissolving the
Specified amount Excipients in diluent(in house made).
1.7. Preparation of STD stock solution:
Standard solution of Naltrexone and Bupropion were
prepared by dissolving 10 mg of each drug into 10 mL volumetric
flask separately. Then dilution was made by adding 10 mL of the
Diluent solution to 10 mL standard flask and making up the volume
with the Diluent. The final concentration of each drug was found to
be 1000µg/ml.
1.8. Preparation of STD solution:
From the Prepared individual Standard Stock Solution of
Naltrexone and Bupropion take 0.1 ml of Naltrexone and 1.125ml
Bupropion into a 10ml of standard flak to this add 3ml of diluent.
Finally make up the solution upto the mark with diluent. The Final
concentration of the Naltrexone was 10 µg/ml and Bupropion 112.5
µg/ml respectively.
1.9. Preparation of Test solution:
The test solution was prepared by taking an equivalent
amount of Bupropion and Naltrexone tablet powder (In house
made) into a 10ml of volumetric flask make up with diluent,from
that take 1ml into 10 ml of standard flask make up the solution with
diluent. Final concentration of NTX and BUP was 10 µg/ml and
Bupropion 112.5 µg/ml respectively.
1.10. Optimization of HPLC Method:
The HPLC method was optimized and developed with
simultaneous method for NTX and BUP. The mixed standard
solution was injected in HPLC by the following chromatographic
conditions. The chromatographic separation was achived on Waters
symmetry C18 (150x4.6 ID) 3.5 μm, Isocratic mode and the Mobile
phase consists of Potassium dihydrogen phosphate buffer pH - 4.5)
:Acetonitrile (30:70 v/v) was used throughout the analysis and the
pH was adjusted to 4.5 and the flow rate of mobile phase was
1ml/min, run time was 10 min. the column temperature was
maintained at Room temp(20-250 c),volume of injection loop was
20µl.detection was monitored at 228 nm. (Table 1).
1.11. Method validation:
The method validation was done according to the ICH
guidelines. The following validation characteristic parameters
areaccuracy, precision, linearity, and specificity, LOD, LOQ and
robustness.
1.11.1. Linearity and range:
Linearity of the method was studied by the injecting the
mixed standard solutions with the concentration ranges from of 5-
25µg/mL for naltrexone drug, 56.25-281.25µg/mL for bupropion
drug levels of target concentrations were prepared and injected six
times into the HPLCsystem keeping the constant injection volume.
The peak areas were plotted against the concentrations to obtain
the linearity graphs.
1.11.2. Precision:
The precision of the optimized method was evaluated by
carrying out six independent assays of test sample. %RSD of six
assay values was calculated. Intermediate precision was carried out
the samples by using another instrument and with different analyst.
1.11.3. Limit of Detection and Quantification:
The LOD and LOQ procedures were performed on
samples contain very lower concentrations of analytes under the
ICH guidelines. By applying the visual evaluation method, LOD was
expressed by establishing the lowest concentration at which the
analyte can be detected. LOQ was considered as the lowest
concentration of analytes that can be detected and quantified, with
acceptable accuracy and precision.
1.11.4. Robustness:
Robustness was studied by evaluating the effect of small
variations in the chromatographic conditions. The conditions
studied were flow rate altered by ±0.1ml/min, mobile phase
composition with methanol ±5ml. These chromatographic variations
are evaluated for resolution between NTX and BUP.
1.11.5. System suitability:
The system suitability parameters with respect of tailing
factor, theoretical plates, repeatability and resolution between NTX
and BUP peaks were defined.
1.11.6. Specificity:
The specificity of the analytical method is the ability of
the method to estimate the analyte response in the presence of
additional components such as impurities, degradation products
and matrix. The peak purity of NTX and BUP were assessed by
comparing the Retention time of standard NTX and BUP good
correlation was obtained between the Retention time of standard
and sample of NTX and BUP.The specificity method was also
evaluated to ensure that there were no interference products
resulting from forced degradation studies.
1.11.7. Forced degradation study:
Forced degradation or Stress testing of a drug substance
will help to identify the degradation products, which can help to
establish the intrinsic stability of the molecule .All stress
decomposition studies were performed at an initial drug
concentration 200µg/mL of NTX and 20µg/mL of BUP.
The Stability indicating study of NTX and BUP were
undergoes acid, alkali and oxidation degradation, photolysis and
heat condition. Placebo Interference: The placebo (in the present of
excipients in tablet) sample were prepared as per the test method

and analyzed in the HPLC. It expressed there is no additional peaks
at the retention time of NTX and BUP in the chromatograph it
indicates that there is no placebo interference.
Acid Degradation:
Sample was treated with 3ml of 1N hydrochloric acid and
kept for 10hrs. After 10hrs the solution was neutralized with 3ml of
1N sodium hydroxide, made the volume upto the mark with mobile
phase and analyzed using HPLC.
Alkali Degradation:
Sample was treated with 3ml of 1N sodium hydroxide and
kept for 10hr. After 10hr the solution was neutralized with 3ml of
1N hydrochloric acid, made the volume up to the mark with mobile
Oxidative Degradation:
NTX and BUP solutions of 200 and 20μg/ml were mixed
with 3mL of 30%v/v aqueous hydrogen peroxide solution and kept
for 10hrs. After 10hrs made the volume up to the mark with mobile
Photolytic Degradation:
The samples were kept under UV light for different time
intervals (15mins – 7days) and made the volume upto the mark
with mobile phase and analyzed using HPLC. Thermal Degradation:
Samples were heated at 800 C for 15mins -60mins and 2200 C for
2‐5mins and analyzed.
1.11.8. Accuracy:
Accuracy was carried out by applying the method to drug
sample (NTX and BUP combination of tablets) to which known
amounts of NTX and BUP. Standard powder corresponding to
50,100 and 150% of label claim was added, mixed and the powder
was extracted and determined by the system in optimized mobile
phase. The experiment was performed in triplicate and percentage
recovery, % RSD was calculated.
1.11.9. Analysis of marketed formulation:
The marketed formulation was assayed by above
description. The peak areas were monitored at 262nm and
determination of sample concentrations were using by multilevel
calibration developed on the same HPLC system under the same
conditions using linear regression analyzed for NTX and BUP in the
same way as described above.
RESULTS AND DISCUSSIONS
The simultaneous estimation of NTX and BUP was
doneby RP-HPLC and in the optimized method the mobile phase
consists of buffer Potassium dihydrogen phosphate buffer pH -
4.5) :Acetonitrile (30:70 v/v)and the pH was adjusted to be 4.5.
Then finally filtered using 0.45µ membrane filter paper and
degassed in sonicator for 15minutes. The detection is carried out
using PDA detector at 228nm.The solutions are following at the
constant flow rate of 1.0 ml/min.The retention time for NTX and
BUP was 2.539 & 5.346 minutes respectively. Linearity ranges for
NTX and BUP were linear over the concentration range of 5-
25µg/ml for naltrexone drug, 56.25-281.25µg/ml for bupropion
drug respectively and the range and correlation coefficient was
found to be (r2 = 0.9998). The percentage recoveries of both the
drugs were 98.95-101.22%and 98.46-100.98% for NTX and BUP
respectively from the tablet formulation.Limit of detection (LOD)
were 0.387 & 1.297μg/mL and limits of quantification (LOQ) were
1.292 and 4.326μg/mL. All The parameters value of RSD is less than
2.0% indicating the accuracy and precision of the method.
1. Method Development and Optimization:
The HPLC procedure was optimized with a view to
develop a suitable LC method for the analysis of NTX and BUP
infixed dose for bulk and combined dosage form. It was found that
mobile phase consists of buffer Potassium dihydrogen
orthophosphate buffer pH - 4.5) :Acetonitrile (30:70 v/v) gave
acceptable retention time (2.539& 5.346 minutes for NTX and BUP) ,
the theoretical plates, and good resolution for NTX and BUP at the
flow rate of 1.0ml/min (Table. 1; Fig. 2, 3 & 3.1).
Table No. 1: Optimized Chromatographic Conditions
Parameters Method
Stationary phase (column) Waters symmetry C18 (150x4.6 ID) 3.5 μm
Mobile Phase Potassium dihydrogen orthophosphate buffer (pH - 4.5)
: Acetonitrile (30 : 70 v/v)
pH 4.5
Flow rate (ml/min) 1ml/min
Run time (minutes) 10 mins
Column temperature (°C) Room temp(20-250 c)
Volume of injection loop (l) 20µl
Detection wavelength (nm) 228 nm
Drugs RT (min) 2.539&5.346 mins
Fig. 2: Chromatogram of NTX and BUP at 228nm from bulk drug Fig. 3: Chromatogram of NTX and BUP at 228 nm from
pharmaceutical tablet formulation

1. Standard 2. Sample 3.Blank
Fig. 3.1: 3D Chromatogram plots for NTX and BUP by PDA detector
2. Validation of Developed Method:
2.1. Linearity:
The linearity five levels of concentrations with correlation
regression curves are obtained the conc. range of 5-25µg/mL for
NTX, 56.25-281.25µg/mL BUP. The reports of drug were found to be
linear in prepared concn range & a correlation regression equation
of NTX was y = 11680x+15966 with correlation coefficient 0.9994
(Fig. 4) and for BUP was y = 4433x+18678with correlation
coefficient 0.9991 (Fig. 5). Where X was the conc of the drug in
µg/ml & Y was area of the peak in the absorbance unit. The
chromatograms were obtained during the linearity were shown in
the Fig. 6-11 & Table 2.
Table No. 2: Linearity study of NTX and BUP
Linearity level NTX BUP
Conc. (µg/ml) Mean Area Conc. (µg/ml) Mean Area
1 5 72879 56.25 72549
2 10 132699 112.5 308153
3 15 192214 168.75 554068
4 20 253585 225.0 796703
5 25 304428 281.25 1075106
Correlation co-efficient 0.999 0.999
Slope 11680 4433
Intercept 15966 18678
Fig. 4: Linearity curve for standard NTX Fig. 5: Linearity curve for standard BUP
Fig. 6: Overlay linearity Chromatogram for NTX and BUP

Fig. 7: Linearity chromatogram for level-1 Fig. 8: Linearity chromatogram for level-2
Fig. 9: Linearity chromatogram for level-3 Fig. 10: Linearity chromatogram for level-4
Fig. 11: Linearity chromatogram for level-5
2.2. Precision:
Precision of this analysis, as the intraday precision was
evaluated by performing five individual test samples prepared &
calculated the % RSD. Interday precision of this method was
analyzed by the performing same the procedure with the various
days by the person with the same developed environment. The %
RSD values of the intra-day precision & interday precision study was
< 2.0% for NTX and BUP. This is confirmed that method was precise
and overlain chromatogram (Fig. 12) and Resulting data of
precision was given in the (Table 3).
Table No. 3: Precision study of NTX and BUP
Replicate Area of NTX Area of BUP
Intra-day
precision
Inter-day
precision
Intra-day
precision
Inter-day
precision
1 439095 436949 127305 132443
2 435462 435877 128143 130445
3 435350 431699 126372 128713
4 436814 432385 128904 128211
5 436839 433739 127376 132105
Mean 436712 434129.8 127620 130383.4
St. dev. 1510.19585 2242.01008 953.736075 1918.28121
% RSD 0.345 0.516 0.747 1.471

Figure 12: Overlay precision Chromatogram for NTX and BUP
2.3. LOD and LOQ:
Limit of detection (LOD) & the limit of quantifications
(LOQ) are evaluated by the serial dilutions of NTX and BUP stock
solutions in the ordered to be obtaining the signal to the noise ratio
3:1 for the LOD & 10:1 for the LOQ. Then the LOD value for NTX and
BUP were found to be 0.387 & 1.297μg/mL & the LOQ value 1.292
and 4.326μg/mL respectively. The chromatogram of the LOD and
LOQ were shown in the (Figure 13 & 14).
Fig. 13: Chromatogram of LOD study of NTX and BUP Fig. 14: Chromatogram of LOQ study of NTX and BUP
2.4. Specificity:
The specificity is a method for drug establishing by the
verifying for the interferences with drug quantification from
degradation products are formed during forced degradation study
and peak purity for NTX and BUP was found better under the
various conditions. There were no other interferences of any other
peaks degradated product with the drug peaks.
Table No. 4: System suitability parameters for NTX and BUP
System suitability parameters NTX BUP
Retention time (min) 2.539 5.346
Repeatability of retention time;
%R.S.D (n=5)
0.01 0.05
Repeatability of peak area;
%R.S.D= (S.D./Mean)×100
0.7 0.4
Resolution (Rs) - 9.37
Tailing factor (asymmetric factor) 1.32 1.18
USP plate count 2624 4635
LOD (μg/mL) 0.387 1.297
LOQ (μg/mL) 1.292 4.326
2.5. Robustness:
The robustness is studied by the evaluating effects of
small but the deliberate differences in method condition. The
condition is Flow rate (± 0.1/min) and MP composition (altered by ±
5% organic solvent using 40:60 and 20:80v/v buffer: methanol).
The results of robustness for developed methods were started in the
(Table 5). The results are shown the during all the different
conditions of the test solution wasn’t affective & in the accordance
with an actual one. The suitability also found better; hence this
method was conformed as robust. The chromatograms were
obtained during the robustness were shown in the (Figure 15-18).

Table No. 5: Evaluation data of Robustness study of NTX and BUP
Parameters Adjusted to Mean Area a Mean RT SD % RSD
NTX Flow Rate As per method
1.0ml/min
0.9 ml/min 196780 3.015 3685.6 1.87
1.1ml/min 119315 1.982 2183.7 0.92
Mobile Phase (30:70)
(Buffer:Methanol)
40:60 144800 2.584 2557.8 1.17
20:80 154856 2.568 1206.9 0.77
BUP Flow Rate As per method
1.0ml/min
0.9 ml/min 628876 6.607 4623.6 0.73
1.1ml/min 407065 4.027 4072 1.1
Mobile Phase (30:70)
(Buffer:Methanol)
40:60 519570 6.586 8296.3 1.52
20:80 553765 3.962 7714.3 1.13
a = 5 Replicates; a each of the value was indicates for mean of 3 injections
Fig. 15: Chromatogram of NTX and BUP (0.9 ml/min flow rate) Fig. 16: Chromatogram of NTX and BUP (1.1 ml/min flow rate)
Fig. 17: Chromatogram of NTX and BUP Fig. 18: Chromatogram of NTX and BUP
[Buffer: Methanol (40:60v/v)] [Buffer: Methanol (20:80v/v) ]
2.6. Ruggedness:
The ruggedness was studied by evaluating by different
analysts but in the same chromatographic conditions. The results of
ruggedness of developed method are started in the (Table 6). The
results are shown during by different analysts but in the same
chromatographic condition of the test solution wasn’t affected & in
the accordance with the actual. The suitability parameters are also
been found good; hence this method was concluded as rugged.
Chromatograms are obtained during ruggedness was shown in the
(Fig. 19-24).
Table No. 6: Evaluation data of Ruggedness study of NTX and BUP
ID Precisions No. of Injections NALTREXONE DRUG BUPROPION DRUG
Peak Area RT Peak Area RT
ID Precision – 1
1 132443 2.092 436949 4.327
2 130445 2.093 435877 4.33
3 128713 2.094 431699 4.331
ID Precision – 2
1 128211 2.094 432385 4.332
2 132105 2.095 433739 4.333
3 126517 2.096 435272 4.333
MEAN 129738.9 2.094 434319.9 4.331
STDEV 2331.2 0.0014 2058.8 0.00228
% RSD 1.8 0.067 0.5 0.052

Fig. 19: Chromatogram of NTX and BUP [ID Precision-1 (Inj - 1)] Fig. 20: Chromatogram of NTX and BUP [ID Precision-1 (Inj - 2)]
2.7.Solution stability study:
Sample Stability was evaluated by shorting at the ambient
temp & analysis was done in initial time, after 3hrs, 6 hrs, 12 hrs and
24 hrs. The analysis of the reports from all aged solutions was
compared with those of from the freshly prepared solution (initial
solution). (Table 7 & 8) shows results are obtained the stability of
solution study at various intervals for a test preparations and it was
conformed that the test solutions were stable upto the 24hrs at the
ambient temp, because difference in the measured & the original
values were < 2.0 %.
Table No. 7: Evaluation of solution stability for NTX
Replicate Initial Area Area After 3 hrs Area After 6 hrs Area After 12 hrs Area After 24 hrs
1 127651 126947 125987 125364 124568
2 127376 126846 125469 124869 124376
3 128904 126742 125684 124963 124904
4 126372 126372 125368 124853 124372
5 128143 125876 125567 124962 124143
Mean 127689.2 126556.6 125615 125002.2 124472.6
St. dev. 937.58237 438.1299 238.6284 208.5994 284.2953
% RSD 0.734 0.346 0.189 0.166 0.228

Table No. 8: Evaluation data of solution stability for BUP
Replicate Initial Area Area After 3 hrs Area After 6 hrs Area After 12 hrs Area After 24 hrs
1 434309 414256 414327 395478 384309
2 436839 422834 416659 395697 376839
3 436814 423643 426746 386452 376814
4 435350 425357 421467 384685 375350
5 435462 425654 420269 390245 375462
Mean 435754.8 422348.8 419893.6 390511.4 377754.8
St. dev. 1076.72267 4673.794 4770.261 5051.094 3732.336
% RSD 0.247 1.106 1.136 1.293 0.988
2.8. Recovery Studies (Accuracy):
The recovery of NTX and BUP was determined by the 3
various conc. levels. % recovery was found to be 99.86-100.30% for
NTX and 99.99-99.99% for BUP (Table 9). The results are indicating
that this method was accurate. Chromatograms obtained during the
study of accuracy were shown in (Fig. 25-27).
Table No. 9: Accuracy study of NTX and BUP
Brand
Name
Analyst Recovery
levels
Actual Conc.
(μg/mL)
Added Conc.
(μg/mL)
Theoretical
Conc. (μg/mL)
Found Conc.
(μg/mL)
%
Recovery
% RSD % Error a
Contrave
NTX
50 % 15 7.5 22.5 22.58 100.3 0.21 0.35
100 % 15 15 30 29.67 98.9 0.64 -1.1
150 % 15 22.5 37.5 37.45 99.86 0.53 -0.13
BUP 50 % 168.75 84.37 253.12 253.08 99.99 0.24 -0.01
100 % 168.75 168.75 337.5 337.42 99.99 0.19 -0.02
150 % 168.75 253.12 421.87 421.86 99.99 0.35 -0.002
a [found conc. – theoretical conc./theoretical conc.] x 100; Each value was indicates the mean of 3 injections.
Fig. 25: Accuracy chromatogram for NTX and BUP level-1 (50%) Fig. 26: Accuracy chromatogram for NTX and BUP level-2 (100%)
Fig. 27: Accuracy chromatogram for NTX and BUP level-3 (150%)

2.9. Analysis of a commercial formulation:
Experimentally the results for the amount of NTX and
BUP in tablets, expressed as a percentage of label claims were in
good agreement with the label claims thereby suggesting that there
is no interaction from the excipients which are commonly present
informulation of tablets.
2.10. Forced Degradation study:
In a order to the determine whether the analytical
methods were stable NTX and BUP combine tablets are stress on the
different conditions to applied degradation studies. The guidelines
are expressed in ICH Q2A, Q3B, Q2B & FDA 21 CFR section of 211 all
the required for development & for the validation of stability study.
The degradation of a sample was prepared by the transfer
the tablet powder was equivalent to the weight of each tablet was
transfer into 100 ml flask & it was treated under the acidic, alkaline,
thermal, oxidizing and photolytic conditions. When degradation was
complete the solution were left to a equilibrate to the room temp &
dil. with mobile phase to furnish the solutions of a concentration
equivalent to a 15µg/mL of NTXand 168.25µg/mL of BUP. The
specific degradative conditions are described below.
Acid degradation study:
The Acid degradation was done by sample was treated
with 3ml of 1N hydrochloric acid and kept for 10hrs at 60ºC. After
10hrs the solution was neutralized with 3ml of 1N sodium
hydroxide, made the volume up to the mark with mobile phase and
analyzed using HPLC. The degrading drug content was found up to
4.41% in the acidic condition (Fig. 28-30 & Table 10 & 11).
Fig. 28: Chromatogram of acidic forced degradation of NTX and BUP
Fig. 29: Purity Plots for NTX and BUP in acidic forced degradation
Fig. 30: Spectrum index for NTX and BUP in acidic forced degradation
Alkaline degradation:
The Alkaline degradation was done by sample was
treated with 3ml of 1N sodium hydroxide and kept the sample for
10hr. After 10hr solution was neutralized to add 3ml of 1N
hydrochloric acid, made the volume up to the mark with mobile
phase and analyzed using HPLC. In alkali degradation study, it was
found to be 4.48% of the degraded drug (Fig. 31-33 & Table 10 &
11).

Fig. 31: Chromatogram of alkali forced degradation of NTX and BUP
Fig. 32: Purity Plots for NTX and BUP in alkali forced degradation
Fig. 33: Spectrum index for NTX and BUP in alkali forced degradation
Oxidative degradation:
The oxidative degradation was done by sample was
mixed with 3mL of 30%v/v aqueous hydrogen peroxide solution
and kept for 10hrs. After 10hrs made the volume upto the mark with
mobile phase and analyzed using HPLC. In oxidative degradation, it
was found to be 5.41% of the degraded drug (Fig. 34-36 & Table 10
& 11).
Figure 34: Chromatogram of oxidative forced degradation of NTX and BUP

Fig. 35: Purity Plots for NTX and BUP in oxidative forced degradation
Fig. 36: Spectrum index for NTX and BUP in oxidative forced degradation
Photolytic degradation:
The photolytic degradation was done by exposing of drug
content under the UV light for 15mins to 7days. There is 6.47% of
the drug degradation observed in the above specific photolytic
degradation condition (Fig. 37-39 & Table 10 & 11).
Figure 37: Chromatogram of UV-light degradation of NTX and BUP
Fig. 38: Purity Plots for NTX and BUP in UV-light degradation

Fig. 39: Spectrum index for NTX and BUP in Photolytic forced degradation
Thermal degradation:
The Thermal degradation is to be performing by the
exposing the solid drug at the 80°C for 15mins to 60mins and at
220°C for 2-5mins. Resultant chromatogram of thermal degradation
study (Fig. 40-42 & Table 10 & 11) was indicates that the drug was
found to be slightly stable under thermal condition. It was only
11.20% of the drug content were degraded.
Fig. 40: Chromatogram of thermal degradation of NTX and BUP
Fig. 41: Purity Plots for NTX and BUP in thermal degradation
Fig. 42: Spectrum index for NTX and BUP in Thermal forced degradation

Table No. 10: Peak purity results of NTX and BUP
Stress Purity Angle Purity Threshold
Condition NTX BUP NTX BUP
Acid Degradation 0.501 0.158 4.334 0.229
Alkali Degradation 0.572 0.157 4.251 0.230
Oxidative Degradation 0.473 0.129 3.936 0.226
Photolytic Degradation 0.609 0.148 3.959 0.272
Thermal Degradation 0.723 0.636 1.181 1.568
Table No. 11: Percentage of degradation of NTX and BUP
Drug Name Acid Alkali Oxidative Photolytic Thermal
NTX
Std Area 129406
Sample Area 125406 124771 124134 125482 120115
% of Degradation 3.09% 3.58% 4.07% 3.03% 7.17%
BUP
Std Area 435754
Sample Area 424229 426382 424012 405744 400594
% of Degradation 2.64% 2.60% 2.69% 6.88% 8.06%
Average of % Degradation 4.41% 4.48% 5.41% 6.47% 11.20%
CONCLUSION
A new RP-HPLC stability indicating method was
described in this manuscript provides a simple, convenient and
reproducible approach for the simultaneous estimation and
quantification of Naltrexone (NTX) and Bupropion (BUP) in routine
quality control analysis
REFERENCES:
1. ICH,stability testing of new drugs substances and products,
International conference on harmonization, IFPMA, Geneva,
2003.
2. ICH Topic Q 2 (R1) validation of Analytical Procedures:
Textand methodology, note for guidance on validation of
analytical procedures: text and methodology
(CPMP/ICH/381/95), June 1995.
3. International Conference on Harmonization; Draft
Guidanceon specifications: Test Procedures and Acceptance
Criteriafor New Drug Substances and Products: Chemical
Substances, Federal Register (Notices), 2000; 65(251):
83041-83063.
4. FDA, Analytical Procedures and methods
validation:Chemistry, manufacturing, and Controls, Federal
Register (Notices), 2000; 65(169): 52776-52777.
5. Neil M.J.O. “The Merck Index - An Encyclopedia of Chemicals,
Drug, Biological” Merck & Co. Inc, 2007; 14th Edn. 1101.
6. Katzung B. G., Basic and Clinical Pharmacology, Mc. Graw
Hill, singapore, 2005; 9th Edn, 375-7, 512-4
7. Sweetman SC, “Martindale - The complete drug reference,”
Pharmaceutical press, London (UK), 2007; 35th Edn. 1310.
8. The United State Pharmacopeial Convection United State
Pharmacopoeia. NF, asian edn. 2007; 3: 2703-4.
9. Cantwell DP. ADHD through the life span: the role of
bupropion in treatment, Journal of Clinical Psychiatry, 1998;
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10. Lief HI. Bupropion treatment of depression to assist
smoking cessation,. American Journal of Psychiatry, 1996;
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11. The United State Pharmacopoeia, 26th Revision, Rockville
MD: US Pharmacopoeial Convention Inc., 2004; 1277.
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How to cite this article:
CH. Naveen Kumar et al.,: Stress Degradation studies and Develpoment of Validated Stability Indicating Assay Method by RP-
HPLC for Simultaneous Estimation of Naltrexone and Bupropion in the presence of degradation products as per ICH
Guidelines, J. Sci. Res. Phar., 2015; 4(1): 19-32.
Conflict of interest: The authors have declared that no conflict of interest exists.
Source of support: Nil

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