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 ArticleAvailable online through ISSN: 2277-9469

www.jsrponline.com

Stress Degradation studies and Develpoment of Validated Stability Indicating Assay Method by RP-HPLCfor 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 simultaneousdetermination of naltrexone and bupropion after being subjected to International conference on harmonization stress conditions, such ashydrolysis, oxidation, heat, and photolysis. The quantification was carried out using Waters symmetry C18 (150x4.6 ID) 3.5 μm column at an Roomtemp (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 oflinearity, precision, accuracy, and specificity, limit of detection and limit of quantitation. The method calibration curves were found to be linearover the concentration range of 5-25µg/ml for naltrexone drug, 56.25-281.25µg/ml for bupropion drug respectively and the range and correlationcoefficient 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 andbupropion respectively from the tablet formulation.Limit of detection(LOD)were 0.387 & 1.297μg/mL and limits of quantification (LOQ) were1.292 and 4.326μg/mL.Degradation products resulting from the stress conditions did not interfere with the detection of naltrexone and bupropionso 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 foradministration of two more active drug components in a singledosage form; it has better patient acceptability due to reducednumber of dosage forms to be taken at a time. There are manyAnalytical methods are extensively available for single drugformulations but due to complexity in multicomponentformulations, stability Indicating method development for multipledrugs formulation is a challenge and scope for new developments.The present research work is focused to develop a proper solventsystem and method development by Reverse Phase HighPerformance Liquid Chromatography (RP-HPLC) for the analysis ofmulti-drug combination including naltrexone and bupropion and tovalidate the developed process as per the ICH guidelines [1].The stress testing of the new drug substance is carriedout according to stability test guideline Q1A (R2) issued by ICH forestablishing its inherent stability characteristics and for supportingthe suitability of the proposed analytical procedure [2-4].Naltrexone (NTX) is chemically (5α)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6-onehydrochloride (Fig. 1).The main use of naltrexone is for thetreatment of alcohol dependence [4-6]. NTX is used in the treatment ofalcoholism.Naltrexone was approved by the U.S. Food and DrugAdministration (FDA) for the treatment of alcohol dependence in1994, following publication of the first two randomized, controlledtrials in 1992. Since then a number of studies have confirmed itsefficacy in reducing frequency and severity of relapse to drinking [7].The multi-centre combine study showed the usefulness of in aprimary care setting without adjunct psychotherapy [8]. BUP ischemically, (±)-2-(tert-butylamino)-1-(3-chlorophenyl) propan-1-*Corresponding author:CH. Naveen KumarBright Labs, Kothapet,Dilshuknagar, Hyderabad, Telangana, INDIA.*E-Mail: [email protected]

one (Fig. 2), an atypical antidepressant and smoking cessation aid.It acts as a norepinephrine and dopamine reuptake inhibitor as wellas α3 β4 nicotinic receptor antagonist [9-11]. Presently, combinationof these two drugs as a controlled release tablet is under clinicaltrials for the treatment of obesity Literature survey reveals that avery few physico-chemical methods appeared in the literature forthe determination of NTX in pharmaceutical formulations. Themethods so far reported include. A LC electro spray Tandem MSmethod for the analysis of NTX in canine plasma employing amolecular model to demonstrate the absence of internal standarddeuterium isotope effects [12]. Nano level detection of NTX in itspharmaceutical preparation at Au microelectrode in flowingsolutions by fast fourier transforms continuous cyclic voltammetryas a novel detector [13-15]. However, there were only few validatedHPLC-UV/PDA methods reported so far for the simultaneousestimation of NTX and BUP in combination but there are noreported stability indicating methods for NTX and BUP. Hence, themain objective of the present study was to develop a validatedstability indicating RP-HPLC-PDA method for the simultaneousestimation of NTX and BUP in bulk and pharmaceutical dosageforms.

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 asAcetonitrile,methanol,ortho phosphoric acid, Potassium dihydrogenPhosphate and water used were of HPLC and milli-Q grade. All otherchemicals used were of AR grade. Bupropion andNaltrexone(Contrave) Tablets were purchased from local pharmacy.1.2 . Instrumentation:The analysis was performed using waters-2695(Modelalliance) High Performance liquid chromatography waters autosampler–PDA detector by using, Empower-software version-2,analytical balance (MettlerToledo) UV/Visible-Detector (Standardcell) and data handling system (Autochrome-3000), pH meter (labIndia), 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 orthophosphatewas accurately weighed and taken into 250 ml volumetricflask.Then add 150 ml of HPLC water, dissolve completely to get aclear solution.Make up the volume up to the mark with water. ThepH was adjusted to 4.5 with Orthophosphoric acid and filteredthrough 0.45 μm membrane filter Sonicate for 15 min.1.4. Diluent( Mobile phase ) Preparation:Combination of Potassium dihydrogen orthophosphatebuffer (pH-4.5) and Acetonitrile was mixed in the ratio of 30:70(pHwas adjusted to 4.5 with Orthophosphoric acid and filtered through0.45 μm membrane filter). This prepared solution was used asmobile phase. This solution was also used for specificity blanksolution.1.5. Preparation of blank solution:Combination of Potassium dihydrogen orthophosphatebuffer (pH-4.5) and Acetonitrile was mixed in the ratio of 30:70.This prepared solution was used as mobile phase. This solution wasalso used for specificity blank solution1.6. Preparation of Placebo Solution:The placebo Solution was prepared by Dissolving theSpecified amount Excipients in diluent(in house made).1.7. Preparation of STD stock solution:Standard solution of Naltrexone and Bupropion wereprepared by dissolving 10 mg of each drug into 10 mL volumetricflask separately. Then dilution was made by adding 10 mL of theDiluent solution to 10 mL standard flask and making up the volumewith the Diluent. The final concentration of each drug was found tobe 1000µg/ml.1.8. Preparation of STD solution:From the Prepared individual Standard Stock Solution ofNaltrexone and Bupropion take 0.1 ml of Naltrexone and 1.125mlBupropion into a 10ml of standard flak to this add 3ml of diluent.Finally make up the solution upto the mark with diluent. The Finalconcentration of the Naltrexone was 10 µg/ml and Bupropion 112.5µg/ml respectively.1.9. Preparation of Test solution:The test solution was prepared by taking an equivalentamount of Bupropion and Naltrexone tablet powder (In housemade) into a 10ml of volumetric flask make up with diluent,from

that take 1ml into 10 ml of standard flask make up the solution withdiluent. Final concentration of NTX and BUP was 10 µg/ml andBupropion 112.5 µg/ml respectively.

1.10. Optimization of HPLC Method:The HPLC method was optimized and developed withsimultaneous method for NTX and BUP. The mixed standardsolution was injected in HPLC by the following chromatographicconditions. The chromatographic separation was achived on Waterssymmetry C18 (150x4.6 ID) 3.5 μm, Isocratic mode and the Mobilephase consists of Potassium dihydrogen phosphate buffer pH - 4.5):Acetonitrile (30:70 v/v) was used throughout the analysis and thepH was adjusted to 4.5 and the flow rate of mobile phase was1ml/min, run time was 10 min. the column temperature wasmaintained at Room temp(20-250 c),volume of injection loop was20µl.detection was monitored at 228 nm. (Table 1).

1.11. Method validation:The method validation was done according to the ICHguidelines. The following validation characteristic parametersareaccuracy, precision, linearity, and specificity, LOD, LOQ androbustness.1.11.1. Linearity and range:Linearity of the method was studied by the injecting themixed standard solutions with the concentration ranges from of 5-25µg/mL for naltrexone drug, 56.25-281.25µg/mL for bupropiondrug levels of target concentrations were prepared and injected sixtimes into the HPLCsystem keeping the constant injection volume.The peak areas were plotted against the concentrations to obtainthe linearity graphs.1.11.2. Precision:The precision of the optimized method was evaluated bycarrying out six independent assays of test sample. %RSD of sixassay values was calculated. Intermediate precision was carried outthe samples by using another instrument and with different analyst.1.11.3. Limit of Detection and Quantification:The LOD and LOQ procedures were performed onsamples contain very lower concentrations of analytes under theICH guidelines. By applying the visual evaluation method, LOD wasexpressed by establishing the lowest concentration at which theanalyte can be detected. LOQ was considered as the lowestconcentration of analytes that can be detected and quantified, withacceptable accuracy and precision.1.11.4. Robustness:Robustness was studied by evaluating the effect of smallvariations in the chromatographic conditions. The conditionsstudied were flow rate altered by ±0.1ml/min, mobile phasecomposition with methanol ±5ml. These chromatographic variationsare evaluated for resolution between NTX and BUP.1.11.5. System suitability:The system suitability parameters with respect of tailingfactor, theoretical plates, repeatability and resolution between NTXand BUP peaks were defined.1.11.6. Specificity:The specificity of the analytical method is the ability ofthe method to estimate the analyte response in the presence ofadditional components such as impurities, degradation productsand matrix. The peak purity of NTX and BUP were assessed bycomparing the Retention time of standard NTX and BUP goodcorrelation was obtained between the Retention time of standardand sample of NTX and BUP.The specificity method was alsoevaluated to ensure that there were no interference productsresulting from forced degradation studies.1.11.7. Forced degradation study:Forced degradation or Stress testing of a drug substancewill help to identify the degradation products, which can help toestablish the intrinsic stability of the molecule .All stressdecomposition studies were performed at an initial drugconcentration 200µg/mL of NTX and 20µg/mL of BUP.The Stability indicating study of NTX and BUP wereundergoes acid, alkali and oxidation degradation, photolysis andheat condition. Placebo Interference: The placebo (in the present ofexcipients in tablet) sample were prepared as per the test method



and analyzed in the HPLC. It expressed there is no additional peaksat the retention time of NTX and BUP in the chromatograph itindicates that there is no placebo interference.Acid Degradation:Sample was treated with 3ml of 1N hydrochloric acid andkept for 10hrs. After 10hrs the solution was neutralized with 3ml of1N sodium hydroxide, made the volume upto the mark with mobilephase and analyzed using HPLC.Alkali Degradation:Sample was treated with 3ml of 1N sodium hydroxide andkept for 10hr. After 10hr the solution was neutralized with 3ml of1N hydrochloric acid, made the volume up to the mark with mobilephase and analyzed using HPLC.Oxidative Degradation:NTX and BUP solutions of 200 and 20μg/ml were mixedwith 3mL of 30%v/v aqueous hydrogen peroxide solution and keptfor 10hrs. After 10hrs made the volume up to the mark with mobilephase and analyzed using HPLC.Photolytic Degradation:The samples were kept under UV light for different timeintervals (15mins – 7days) and made the volume upto the markwith mobile phase and analyzed using HPLC. Thermal Degradation:Samples were heated at 800 C for 15mins -60mins and 2200 C for2‐5mins and analyzed.1.11.8. Accuracy:Accuracy was carried out by applying the method to drugsample (NTX and BUP combination of tablets) to which knownamounts of NTX and BUP. Standard powder corresponding to50,100 and 150% of label claim was added, mixed and the powderwas extracted and determined by the system in optimized mobilephase. The experiment was performed in triplicate and percentagerecovery, % RSD was calculated.

1.11.9. Analysis of marketed formulation:The marketed formulation was assayed by abovedescription. The peak areas were monitored at 262nm anddetermination of sample concentrations were using by multilevelcalibration developed on the same HPLC system under the sameconditions using linear regression analyzed for NTX and BUP in thesame way as described above.RESULTS AND DISCUSSIONS

The simultaneous estimation of NTX and BUP wasdoneby RP-HPLC and in the optimized method the mobile phaseconsists 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 anddegassed in sonicator for 15minutes. The detection is carried outusing PDA detector at 228nm.The solutions are following at theconstant flow rate of 1.0 ml/min.The retention time for NTX andBUP was 2.539 & 5.346 minutes respectively. Linearity ranges forNTX and BUP were linear over the concentration range of 5-25µg/ml for naltrexone drug, 56.25-281.25µg/ml for bupropiondrug respectively and the range and correlation coefficient wasfound to be (r2 = 0.9998). The percentage recoveries of both thedrugs were 98.95-101.22%and 98.46-100.98% for NTX and BUPrespectively from the tablet formulation.Limit of detection (LOD)were 0.387 & 1.297μg/mL and limits of quantification (LOQ) were1.292 and 4.326μg/mL. All The parameters value of RSD is less than2.0% indicating the accuracy and precision of the method.1. Method Development and Optimization:The HPLC procedure was optimized with a view todevelop a suitable LC method for the analysis of NTX and BUPinfixed dose for bulk and combined dosage form. It was found thatmobile phase consists of buffer Potassium dihydrogenorthophosphate buffer pH - 4.5) :Acetonitrile (30:70 v/v) gaveacceptable retention time (2.539& 5.346 minutes for NTX and BUP) ,the theoretical plates, and good resolution for NTX and BUP at theflow rate of 1.0ml/min (Table. 1; Fig. 2, 3 & 3.1).

Table No. 1: Optimized Chromatographic Conditions

Parameters MethodStationary 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/minRun time (minutes) 10 mins

Column temperature (°C) Room temp(20-250 c)Volume of injection loop (l) 20µlDetection 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 frompharmaceutical 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 correlationregression curves are obtained the conc. range of 5-25µg/mL forNTX, 56.25-281.25µg/mL BUP. The reports of drug were found to belinear 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 correlationcoefficient 0.9991 (Fig. 5). Where X was the conc of the drug inµg/ml & Y was area of the peak in the absorbance unit. Thechromatograms were obtained during the linearity were shown inthe Fig. 6-11 & Table 2.

Table No. 2: Linearity study of NTX and BUP

Linearity level NTX BUPConc. (µg/ml) Mean Area Conc. (µg/ml) Mean Area

1 5 72879 56.25 725492 10 132699 112.5 3081533 15 192214 168.75 5540684 20 253585 225.0 7967035 25 304428 281.25 1075106

Correlation co-efficient 0.999 0.999Slope 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 wasevaluated by performing five individual test samples prepared &calculated the % RSD. Interday precision of this method wasanalyzed by the performing same the procedure with the variousdays 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 preciseand overlain chromatogram (Fig. 12) and Resulting data ofprecision was given in the (Table 3).

Table No. 3: Precision study of NTX and BUP

Replicate Area of NTX Area of BUPIntra-dayprecision

Inter-dayprecision

Intra-dayprecision

Inter-dayprecision

1 439095 436949 127305 1324432 435462 435877 128143 1304453 435350 431699 126372 1287134 436814 432385 128904 1282115 436839 433739 127376 132105

Mean 436712 434129.8 127620 130383.4St. 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 stocksolutions in the ordered to be obtaining the signal to the noise ratio3:1 for the LOD & 10:1 for the LOQ. Then the LOD value for NTX andBUP were found to be 0.387 & 1.297μg/mL & the LOQ value 1.292and 4.326μg/mL respectively. The chromatogram of the LOD andLOQ 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 theverifying for the interferences with drug quantification fromdegradation products are formed during forced degradation studyand peak purity for NTX and BUP was found better under thevarious conditions. There were no other interferences of any otherpeaks degradated product with the drug peaks.

Table No. 4: System suitability parameters for NTX and BUP

System suitability parameters NTX BUPRetention time (min) 2.539 5.346

Repeatability of retention time;%R.S.D (n=5)

0.01 0.05Repeatability of peak area;%R.S.D= (S.D./Mean)×100

0.7 0.4Resolution (Rs) - 9.37

Tailing factor (asymmetric factor) 1.32 1.18USP plate count 2624 4635

LOD (μg/mL) 0.387 1.297LOQ (μg/mL) 1.292 4.326

2.5. Robustness:The robustness is studied by the evaluating effects ofsmall but the deliberate differences in method condition. Thecondition 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 differentconditions of the test solution wasn’t affective & in the accordancewith an actual one. The suitability also found better; hence thismethod was conformed as robust. The chromatograms wereobtained 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 % RSDNTX Flow Rate As per method

1.0ml/min0.9 ml/min 196780 3.015 3685.6 1.871.1ml/min 119315 1.982 2183.7 0.92

Mobile Phase (30:70)(Buffer:Methanol)

40:60 144800 2.584 2557.8 1.1720:80 154856 2.568 1206.9 0.77

BUP Flow Rate As per method1.0ml/min

0.9 ml/min 628876 6.607 4623.6 0.731.1ml/min 407065 4.027 4072 1.1

Mobile Phase (30:70)(Buffer:Methanol)

40:60 519570 6.586 8296.3 1.5220:80 553765 3.962 7714.3 1.13a = 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 differentanalysts but in the same chromatographic conditions. The results ofruggedness of developed method are started in the (Table 6). Theresults are shown during by different analysts but in the samechromatographic condition of the test solution wasn’t affected & inthe accordance with the actual. The suitability parameters are alsobeen 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 DRUGPeak Area RT Peak Area RT

ID Precision – 11 132443 2.092 436949 4.3272 130445 2.093 435877 4.333 128713 2.094 431699 4.331

ID Precision – 21 128211 2.094 432385 4.3322 132105 2.095 433739 4.3333 126517 2.096 435272 4.333

MEAN 129738.9 2.094 434319.9 4.331STDEV 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 ambienttemp & analysis was done in initial time, after 3hrs, 6 hrs, 12 hrs and24 hrs. The analysis of the reports from all aged solutions wascompared with those of from the freshly prepared solution (initialsolution). (Table 7 & 8) shows results are obtained the stability ofsolution study at various intervals for a test preparations and it wasconformed that the test solutions were stable upto the 24hrs at theambient temp, because difference in the measured & the originalvalues 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 hrs1 127651 126947 125987 125364 1245682 127376 126846 125469 124869 1243763 128904 126742 125684 124963 1249044 126372 126372 125368 124853 1243725 128143 125876 125567 124962 124143

Mean 127689.2 126556.6 125615 125002.2 124472.6St. 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 hrs1 434309 414256 414327 395478 3843092 436839 422834 416659 395697 3768393 436814 423643 426746 386452 3768144 435350 425357 421467 384685 3753505 435462 425654 420269 390245 375462

Mean 435754.8 422348.8 419893.6 390511.4 377754.8St. 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 3various 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 indicatingthat this method was accurate. Chromatograms obtained during thestudy of accuracy were shown in (Fig. 25-27).

Table No. 9: Accuracy study of NTX and BUP

BrandName

Analyst Recoverylevels

Actual Conc.(μg/mL)

Added Conc.(μg/mL)

TheoreticalConc. (μg/mL)

Found Conc.(μg/mL)

%Recovery

% RSD % Error a

Contrave

NTX50 % 15 7.5 22.5 22.58 100.3 0.21 0.35

100 % 15 15 30 29.67 98.9 0.64 -1.1150 % 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 -0.01100 % 168.75 168.75 337.5 337.42 99.99 0.19 -0.02150 % 168.75 253.12 421.87 421.86 99.99 0.35 -0.002a [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 andBUP in tablets, expressed as a percentage of label claims were ingood agreement with the label claims thereby suggesting that thereis no interaction from the excipients which are commonly presentinformulation of tablets.2.10. Forced Degradation study:In a order to the determine whether the analyticalmethods were stable NTX and BUP combine tablets are stress on thedifferent conditions to applied degradation studies. The guidelinesare expressed in ICH Q2A, Q3B, Q2B & FDA 21 CFR section of 211 allthe required for development & for the validation of stability study.The degradation of a sample was prepared by the transferthe 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 wascomplete the solution were left to a equilibrate to the room temp &dil. with mobile phase to furnish the solutions of a concentrationequivalent to a 15µg/mL of NTXand 168.25µg/mL of BUP. Thespecific degradative conditions are described below.Acid degradation study:The Acid degradation was done by sample was treatedwith 3ml of 1N hydrochloric acid and kept for 10hrs at 60ºC. After10hrs the solution was neutralized with 3ml of 1N sodiumhydroxide, made the volume up to the mark with mobile phase andanalyzed using HPLC. The degrading drug content was found up to4.41% 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 wastreated with 3ml of 1N sodium hydroxide and kept the sample for10hr. After 10hr solution was neutralized to add 3ml of 1Nhydrochloric acid, made the volume up to the mark with mobilephase and analyzed using HPLC. In alkali degradation study, it wasfound 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 wasmixed with 3mL of 30%v/v aqueous hydrogen peroxide solutionand kept for 10hrs. After 10hrs made the volume upto the mark withmobile phase and analyzed using HPLC. In oxidative degradation, itwas 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 drugcontent under the UV light for 15mins to 7days. There is 6.47% of the drug degradation observed in the above specific photolyticdegradation 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 theexposing the solid drug at the 80°C for 15mins to 60mins and at220°C for 2-5mins. Resultant chromatogram of thermal degradationstudy (Fig. 40-42 & Table 10 & 11) was indicates that the drug wasfound to be slightly stable under thermal condition. It was only11.20% 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 ThresholdCondition NTX BUP NTX BUP

Acid Degradation 0.501 0.158 4.334 0.229Alkali Degradation 0.572 0.157 4.251 0.230

Oxidative Degradation 0.473 0.129 3.936 0.226Photolytic Degradation 0.609 0.148 3.959 0.272Thermal 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

NTXStd Area 129406

Sample Area 125406 124771 124134 125482 120115% of Degradation 3.09% 3.58% 4.07% 3.03% 7.17%

BUPStd 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 wasdescribed in this manuscript provides a simple, convenient andreproducible approach for the simultaneous estimation andquantification of Naltrexone (NTX) and Bupropion (BUP) in routinequality control analysisREFERENCES: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 ofanalytical procedures: text and methodology(CPMP/ICH/381/95), June 1995.3. International Conference on Harmonization; DraftGuidanceon specifications: Test Procedures and AcceptanceCriteriafor New Drug Substances and Products: ChemicalSubstances, Federal Register (Notices), 2000; 65(251):83041-83063.4. FDA, Analytical Procedures and methodsvalidation:Chemistry, manufacturing, and Controls, FederalRegister (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. GrawHill, singapore, 2005; 9th Edn, 375-7, 512-47. Sweetman SC, “Martindale - The complete drug reference,”Pharmaceutical press, London (UK), 2007; 35th Edn. 1310.8. The United State Pharmacopeial Convection United StatePharmacopoeia. NF, asian edn. 2007; 3: 2703-4.9. Cantwell DP. ADHD through the life span: the role ofbupropion in treatment, Journal of Clinical Psychiatry, 1998;59(4): 92-94.10. Lief HI. Bupropion treatment of depression to assistsmoking cessation,. American Journal of Psychiatry, 1996;153: 442-442.11. The United State Pharmacopoeia, 26th Revision, RockvilleMD: US Pharmacopoeial Convention Inc., 2004; 1277.12. The Merck Index: An encyclopedia of chemicals, drugs, andbiological. 13th Edition, Merck research laboratories, 2001;1141.13. The United State Pharmacopoeia, 26th Revision, RockvilleMD: US Pharmacopoeial Convention Inc., 2004; 279.14. Indian Pharmacopoeia, Vol - I, 2010; 1748.15. M Sabine; SW Toennes; F Gerold; W Jakel. J. Pharm. Biomed.Anal., 2004; 35: 169-176.

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 ICHGuidelines, J. Sci. Res. Phar., 2015; 4(1): 19-32.Conflict of interest: The authors have declared that no conflict of interest exists.

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naltrexone and buropion RP-HPLC best research paper award 2014

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