Research Article Isocratic High-Performance Liquid ...downloads.hindawi.com/archive/2014/616941.pdf · Isocratic High-Performance Liquid Chromatographic Assay of Olanzapine: Method

Research ArticleIsocratic High-Performance Liquid Chromatographic Assay ofOlanzapine Method Development and Validation

K Basavaiah1 N Rajendraprasad2 and K B Vinay1

1 Department of Chemistry University of Mysore Manasagangotri Mysore 570 006 India2 PG Department of Chemistry JSS College of Arts Commerce amp Science Ooty Road Karnataka Mysore 570 025 India

Correspondence should be addressed to N Rajendraprasad prasadtnpurgmailcom

Received 1 February 2014 Accepted 12 March 2014 Published 3 April 2014

Academic Editors Z Aydogmus A Bouklouze G Erny A Garcia Asuero and W X Misiuk

Copyright copy 2014 K Basavaiah et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

An accurate precise sensitive and rapid isocratic reversed phase high-performance liquid chromatographic (RP-HPLC) methodfor the analysis of olanzapine (OLP) in bulk drug and in tablets has been developed and validated Analysis was performed on a150mm times 46mm 5120583m particle Intersil ODS 3V column with 10mM disodium hydrogen phosphate buffer (pH 74)-acetonitrile(35 65) (vv) as mobile phase at a flow rate of 10mLminminus1 with UV detection at 254 nm the constant column temperaturewas 40∘C The runtime under these chromatographic conditions was less than 8min The calibration plot was linear over theconcentration range of 25ndash200 120583gmLminus1 with limits of detection and quantification values of 50 and 200 ngmLminus1 respectivelyThe precision and accuracy of the method were assessed by determination of validation data for precision (intraday RSD values of011ndash028 interday RSD values of 015ndash046) accuracy (087ndash280 intraday 033ndash180 interday) and specificity in accordancewith the ICH guidelines The stability of standard solution and tablet extract was also studied over a period of 24 h The methodwas applied for the determination of OLP in tablets with satisfactory results

1 Introduction

Olanzapine (OLP) chemically known as 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno-[2 3b][1 5] benzodiazep-ine (Figure 1) is the most commonly prescribed second gen-eration neuroleptic agent for the treatment of schizophreniaand other psychotic disorders

A complete literature survey of analytical methods forOLP is done Titrimetry [1ndash3] visible spectrophotometry[2 4ndash10] kinetic spectrophotometry [11] UV-spectropho-tometry [1 12] and capillary zone electrophoresis and linearvoltammetry [12] have been reported for the quantification ofOLP in pharmaceuticals High-performance thin layer chro-matography (HPTLC)has beenused to quantifyOLP in phar-maceuticals [13ndash15] Several liquid chromatographicmethodshave also been reported for the assay of OLP in phar-maceuticals andor biological materials High performanceliquid chromatography (HPLC) with UV-detection has beenapplied for the determination of the drug in human bloodserum [16ndash18] and blood plasma [19ndash21] The drug in bloodplasmawhole blood has also been assayed by HPLC with

amperometric [22 23] coulometric [24] and MS [25 26]detection OLP in rat brain is reported to have been deter-mined by HPLC with coulometric detection [27] whereas forits assay in breast milk [28] the same technique with elec-trochemical detection has been employed HPLC with UV-detection has earlier been used for the assay of OLP in phar-maceuticals when present alone or in combination with flu-oxetine An RP-HPLC method for the determination of OLPand its tablets was presented by Xuejun and Zhonghua [29]They used ODS C

8

column and 001M KH2

PO4

-methanol-acetonitrile (1 1 1 pH 83) as mobile phase with UV detec-tion at 273 nm The linear range was 001ndash1mg mLminus1 Raggiet al [12] determined OLP in tablets by HPLC with UV-detection at 260 nm using a C

18

column and a mobile phaseconsisting of acetonitrile and aqueous tetramethylammo-nium perchlorate The RSD of the method was better than18 and accuracy was between 999 and 1011 Two rapidand specific reversed phase HPLC methods have beenreported by S Patel and NJ Patel [15] and Pathak and Rajput[30] for the simultaneous determination of OLP and flu-oxetine in their formulations The first report [15] involves

Hindawi Publishing CorporationISRN Analytical ChemistryVolume 2014 Article ID 616941 6 pageshttpdxdoiorg1011552014616941

2 ISRN Analytical Chemistry

NH

NN

N

SCH3

CH3

Figure 1 Structure of OLP

the simultaneous determination of fluoxetine HCl andOLP using reversed-phase liquid chromatography usingacetonitrile methanol 0032M ammonium acetate buffer(45 05 50 vvv) as the mobile phase at a flow rate of15mLminminus1 Quantitation of OLP was achieved with ultra-violet detection at 235 nm over concentration range of 01ndash2 120583gmLminus1 OLP whereas the second report [30] describes theseparation procedure using an Intersil C

18

reversed phasecolumn (150mm times 46mm 5120583m) with a 40 30 30 (vvv)mixture of 95mM NaH

2

PO4

(pH adjusted to 68 plusmn 01 withtriethylamine) acetonitrile and methanol as mobile phaseThe flow rate was 12mLminminus1 with UV-detection at 225 nmThe calibration plot was linear from 25 to 75 120583gmLminus1 forOLPSimultaneous assay of OLP and fluoxetine in tablets byHPLChas also been reported by Reddy et al [31]The separationwasachieved on a Lichrospher 100 RP-180 C

18

column (250mmtimes400mm id 5120583m) using 005M KH

2

PO4

buffer (pH 56adjusted with o-phosphoric acid)-acetonitrile (50 50) as themobile phase at a flow rate of 1mLminminus1 and at ambienttemperature Quantitation was achieved by measuring UVabsorption at 233 nm over the concentration range of 10ndash70 120583gmLminus1The reportedHPLCmethods however are eitherpoorly sensitive [29] or have narrow linear concentrationranges [15 30 31] The present team of workers has [32]reported the oxidative degradation of the OLP degradationproduct and the identification and characterization of thedegradation products The objective of this work was todevelop a simple rapid accurate and precise HPLC methodfor quantitative analysis of OLP in tablets and to validate themethod in accordance with ICH guidelines [33]

2 Methods

21 HPLC Instrumentation and Chromatographic ConditionsHPLC analysis was performed with a Waters HPLC systemequipped with Alliances 2695 series low pressure quaternarygradient pump a programmable variable wavelength UV-visible detector and autosampler Data were collected andprocessed using Waters Empower 2 software

Chromatographic separationwas achieved on a 150mmtimes46mm id 5 120583mparticle Intersil ODS 3V columnThemob-ile phase was a 35 65 (vv) mixture of 10mM disodiumhydrogen phosphate (pH 74)-acetonitrile The flow rate wasfixed as 10mLminminus1 and UV-detection was performed at254 nm Before use the mobile phase was filtered through02 120583m filter The column temperature was maintained at40∘C

22 Chemicals and Reagents A sample of OLP certified to be9988 pure was obtained as gift fromCipla India LtdMum-bai India HPLC-grade acetonitrile disodium hydrogenphosphate and orthophosphoric acid were purchased fromMerk Mumbai India Olanzapine tablets of two differentbrandsOleanz (25 and 75mgOLPper tablet) andOlanex (10and 15mg OLP per tablet) marketed by Sun PharmaceuticalsIndustries LtdMumbai India andRanbaxy Laboratories Ltd(Solus) Haryana India respectively were purchased fromlocal commercial sources HPLC-grade water from Merckwas used to prepare all solutions All other chemicals andreagents used were of analytical grade and purchased fromSD fine chemicals Mumbai India

A buffer solution of pH 74 was prepared by adjusting thepH of 10mMdisodium hydrogen phosphate with orthophos-phoric acid The mobile phase was prepared by mixing thebuffer and acetonitrile in 35 65 (vv) ratio The diluent was amixture of water and acetonitrile (55 45 vv)

A stock solution of OLP (100120583gmLminus1) was prepared inthe diluent Standard solutions were prepared by dilution ofthe stock solution with the diluent solutions to get solution inthe concentration range from 25 to 20 120583gmLminus1 OLP

23 Procedures

231 Calibration Graph Twenty 120583L of working standard sol-utions (25mdash20120583gmLminus1 OLP) was injected automaticallyinto the column in triplicate and the chromatograms wererecorded The calibration graph was prepared by plotting themean peak area versus concentration in 120583g mLminus1

232 Procedure for Tablets Twenty tablets were accuratelyweighed and crushed into a fine powder and mixed using amortar and pestle A quantity of tablet powder equivalent to10mg ofOLPwasweighed accurately into a 100mL calibratedflask 50mL of diluent solution was added and sonicated for20min to complete dissolution of the OLP and the solutionwas then diluted to themark with the diluent andmixed wellA small portion of the tablet solution (say 10mL) was with-drawn and filtered through a 02 120583m filter to ensure the abs-ence of particulate matter The filtrate was appropriatelydiluted with the diluent before injection into the column

3 Results and Discussion

Drug quality control stability metabolism pharmacokinet-ics and toxicity studies all necessitate the determination ofdrugs in pharmaceutical formulations and biological sam-ples Consequently efficient and validated methods are verycrucial requirements for all these investigations

31 Method Development and Optimization Chromatogra-phic parameters were preliminarily optimized to develop thepresent method for the determination of OLP with shortanalysis time (lt8min) because rapid and economic analysisis becoming increasingly important in pharmaceutical anal-ysis to increase the sample throughput In the present casean Intersil ODS 3V column maintained at 40∘C was

ISRN Analytical Chemistry 3

014

012

010

008

006

004

002

000

OLP

000 100 200 300 400 500 700600 800

(min)

(AU)

Figure 2 Typical chromatogram for 125 120583gmLminus1 OLP underoptimized conditions

used for method development The mobile phase 10mMNa2

HPO4

(pH 74)-acetonitrile 35 65 (vv) at a flow rate of10mLminminus1 was selected after several preliminary investi-gatory chromatography runs Under the experimental con-ditions described the peak was well defined and free fromtailing (Figure 2) The retention time (119877

119879

) and asymmetryfactor were 439 plusmn 001 and 12 respectively

32MethodValidation Thedevelopedmethodwas validatedfor linearity accuracy and precision limit of detection(LOD) limit of quantification (LOQ) specificity robustnessand ruggedness in accordance with ICH guidelines

321 Linearity Linearity was studied by preparing standardsolutions of different concentrations from 25 to 20120583g mLminus1plotting a graph of mean peak area three injections againstconcentration and determining the linearity by least-squareregression The calibration plot was linear over the concen-tration range 25ndash200120583g mLminus1 (119899 = 7)

The regression equation was 119910 = 557399119862 minus 1661254where 119910 is the mean peak area and 119909 is concentration of OLPin 120583g mLminus1 with a mean regression coefficient (119903) of 09999The standard error (SE) of slope and intercept were 2884 and48376 respectively

322 Specificity The specificity of an analytical method maybe defined as the ability to unequivocally determine the ana-lyte in the presence of additional components such as impu-rities degradation products and matrix [34ndash36] The speci-ficity was evaluated by injecting the analytical placebo andit was found that the signal measured was caused only bythe analyte A placebo blank containing starch acaciahydroxyl cellulose sodium citrate talc magnesium stearateand sodium alginate was made and its solution was preparedas described under ldquotabletsrdquo and injected The resultingchromatogram is shown in Figure 3 To identify the inter-ference by the inactive ingredients (excipients) a mixture ofplacebo and pure OLP was prepared and a suitable aliquotafter filtration and appropriate dilution was injected into thecolumnThe obtained chromatogram did not show any otherpeaks which confirmed the specificity of themethod In addi-tion the slope of the calibration graph for standardswas com-pared with that prepared from tablet solutions It was foundthat there was no significant difference between the slopeswhich indicated that the excipients did not interfere withOLP

0020

0010

0000

(AU)

000 100 200 300 400 500 700600 800

(min)

0030

0040

0050

Figure 3 Chromatogram obtained for placebo blank under opti-mized conditions

323 Detection and Quantification Limits (LOD and LOQ)The LOD and LOQ were calculated using signal-to-noiseratiomethod [34ndash36] LODwas taken as the concentration ofthe analyte where the signal-to-noise ratio was 3 and it wasfound to be 50 ngmLminus1 LOQ defined as the analyte con-centration at a signal-to-noise ratio of 10 was found to be200 ngmLminus1These values indicate that themethod is suitablefor detection and quantification of OLP over a wide range ofconcentrations

324 Precision Theprecision of themethodwas evaluated interms of intermediate precision (intraday and interday) [34ndash36] Solutions of three different concentrations of OLP wereanalysed in seven replicates during the same day (intradayprecision) and five consecutive days (interday precision)Within each series every solution was injected in triplicateThe RSD values of intraday studies (lt03) showed that theprecision of themethodwas goodThe results of this study aregiven in Table 1 The interday precision was somewhat poorwith the RSD values in the range 015ndash046 (Table 1)

Precision of injection repeatability was also examined byanalyzing seven injections of solution containing 50 100and 150 120583g mLminus1 OLP The RSDs were calculated from thepeak areas and retention times The results of this study arealso complied in Table 1 and the RSD values were found tobe less than 021 and 031 respectively

325 Accuracy The accuracy of an analytical methodexpresses the closeness between the reference value andfound value [34ndash36] Accuracy was evaluated as percentagerelative error (RE) between themeasuredmean concentra-tions and taken concentrationsThe results obtained for threedifferent concentrations are shown in Table 1 from whichthe accuracy is lt3 The accuracy was also assessed by ana-lyzing the synthetic mixture (prepared by adding accuratelyweighed amount of OLP to the placebo) and the calculatedpercent recovery ofOLPwas found to be 9848plusmn064(119899 = 5)(values not mentioned) indicating that the common tabletexcipients like talc starch gum acacia lactose hydroxylmethyl cellulose sodium alginate and magnesium stearatedid not interfere in the assay

326 Robustness and Ruggedness The robustness of themethod was investigated by making small deliberate changes


Table 1 Intraday and interday accuracy and precision

OLP injected120583gmLminus1

Intraday accuracy and precision (n = 7) Interday accuracy and precision (n = 7)OLP founda 120583gmLminus1 RE RSDb RSDc OLP founda 120583gmLminus1 RE RSDb RSDc

50 514 280 011 011 509 18 015 016100 1009 090 028 010 1005 050 028 020150 1513 087 026 021 1505 033 046 031aMean value of seven determinationsbBased on peak areacBased on retention time

Table 2 Results of robustness study (OLP concentration 10 120583gmLminus1)

Chromatographiccondition Alteration Peak area precision Retention time precision

Mean arealowastplusmn SD SE RSD Mean 119877119879

lowast

plusmn SD SE RSD

Mobile phase flowrate (mLminminus1)

09 606358 plusmn 00058 0003 0052 4819 plusmn 0004 0023 007310 547577 plusmn 0000 0000 0000 4364 plusmn 0003 0002 007011 498323 plusmn 0010 0006 0108 3972 plusmn 0002 0002 0038

Column temperature(∘C)


lowastMean value of three injections

Table 3 Results of solution stability studies

Time hour Arealowast (n = 5) RSD 119877119879

RSD 0 547492

0213

4355

00738 545643 435116 546389 435224 548279 4358lowastConcentration of OLP 10 120583gmLminus1

in the chromatographic conditions [33 34] The chromato-graphic conditions selected were flow rate (09 10 and11mL) and the temperature (38 40 and 42∘C)There was nosignificant change in the retention time (119877

119879

) when the flowrate or temperature was changed slightly The values of RSD(Table 2) indicate that the method is robust

The ruggedness [35] of the method was assessed by com-parison of the intraday and interday results for the assay ofOLP performed by three analysts in the same laboratory TheRSD for intraday and interday assay of OLP did not exceed28 indicating the ruggedness of the method

327 Solution Stability To demonstrate the stability of stan-dard solutions and tablet sample solutions during analysisthey were analysed over a period of 24 h The results showedthat for both the solutions the 119877

119879

and peak area of OLPremained almost unchanged (RSD lt 0073 and 0213resp) and no significant degradation was observed duringthis period suggesting that both the solutions were stable forat least 24 h which was sufficient for the whole analytical pro-cess The results of this study are shown in Table 3

328 Application of theMethod for the Analysis of CommercialTablets The developed and validated method was applied to

the determination of OLP in two brands of tablets containingOLP in four strengths (25 75 10 and 15mg per tablet) whichwere available in the local market Quantification was per-formed using the regression equation The results obtainedare presented in Table 4 and are in fair agreement with thelabel claimThe same tablet powder used for assay by the pro-posed method was used for assay by a literature method [2]for comparison and the method consisted of the visual titra-tion of the acetous solution of the tablet with acetous perchlo-ric acid in acetic acid medium The results were comparedstatistically by applying the Studentrsquos 119905-test for accuracy and119865-test for precision As shown by the results compiled inTable 4 the calculated 119905-test and 119865 values did not exceed thetabulated values of 277 and 639 for four degrees of freedomat the 95 confidence level suggesting that the proposedHPLC method and the reference method do not differ sig-nificantly with respect to accuracy and precision

The accuracy and validity of the proposed HPLCmethodwere further ascertained by performing recovery experi-ments Preanalyzed tablet powder was spiked with pure OLPat three different concentration levels and the total was foundby the proposed method Each determination was repeatedthree timesThe recovery of pure drug addedwas quantitative(Table 5) and revealed that coformulated substances did notinterfere in the determination

4 Conclusions

A simple rapid accurate precise and sensitive HPLCmethodwithUV-detection was developed for the determina-tion of olanzapine in bulk drug and in tablets The methodwas validated for linearity LOD and LOQ specificity accu-racy and precision and robustness and ruggedness as per the


Table 4 Results of assay of tablets

Tablet brand name Nominal amount mg Foundlowast (percent plusmn SD)Reference method Proposed method Studentrsquos t-value (277) F value (639)

Oleanz 25 1014 plusmn 058 1007 plusmn 036 236 259Oleanz 75 9836 plusmn 072 9884 plusmn 028 152 661Olanex 100 9775 plusmn 082 9816 plusmn 052 097 249

150 1023 plusmn 042 1016 plusmn 064 209 232lowastMean value of five determinationsFigures in the parenthesis are the tabulated values for four degrees of freedom at 95 confidence level

Table 5 Results of recovery study

Tablet studied OLP in tablet 120583gmLminus1 Pure OLP added 120583gmLminus1 Total found 120583gmLminus1 Pure OLP recoveredlowast (percent plusmn SD)

Oleanz (75mg)988 50 1486 9960 plusmn 12988 75 1746 1011 plusmn 086988 100 2010 1022 plusmn 054

Olonex (15mg)1016 50 1534 1036 plusmn 0731016 75 1775 1012 plusmn 0921016 100 2072 1056 plusmn 048

lowastMean value of three experiments

ICH guidelines The retention time of lt5min enables rapiddetermination of drug which is important in routine analy-sis The other advantages of the method are high sensitivity(LOD 50 ngmLminus1) and wide linear dynamic range comparedto other reported HPLC methods for the drug The methodcan also be used to study the stability of solutions Themethod sounds suitable for the quality control in pharmaceu-tical industry because of its simplicity sensitivity selectivityand high accuracy and precision besides being robust andrugged

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to Cipla India Ltd Mumbia Indiafor providing the pure sample of olanzapine as gift Oneof the authors (N Rajendraprasad) thanks the JSS Maha-vidyapeeta for providing platform and support to pursueresearch work

References

[1] S Firdous T Aman and A-U Nisa ldquoDetermination of olan-zapine by UV spectrophotometry and non-aqueous titrationrdquoJournal of the Chemical Society of Pakistan vol 27 no 2 pp163ndash167 2005

[2] K Basavaiah and S A M Abdulrahman ldquoSensitive and selec-tive methods for the determination of olanzapine in pharma-ceuticals using N-bromosuccinimide and two dyesrdquo Interna-tional Journal of ChemTech Research vol 2 no 1 pp 660ndash6682010

[3] K Basavaiah N Rajendraprasad and K B Vinay ldquoMicrotitri-metric determination of a drug content of pharmaceuticals con-taining olanzapine in non-aqueousmediumrdquoChemical Industryand Chemical Engineering Quarterly vol 15 no 2 pp 77ndash812009

[4] A Jasinska and E Nalewajko ldquoBatch and flow-injection meth-ods for the spectrophotometric determination of olanzapinerdquoAnalytica Chimica Acta vol 508 no 2 pp 165ndash170 2004

[5] A Krebs B Starczewska H Puzanowska-Tarasiewicz and JSledz ldquoSpectrophotometric determination of olanzapine by itsoxidation with N-bromosuccinimide and cerium(IV)sulfaterdquoAnalytical Sciences vol 22 no 6 pp 829ndash833 2006

[6] N Rajendraprasad K Basavaiah K Tharpa and K B VinayldquoQuantitative determination of olanzapine in tabletswith visiblespectrophotometry using cerium(IV)sulphate and based onredox and complexation reactionsrdquo Eurasian Journal of Analyt-ical Chemistry vol 4 pp 193ndash203 2009

[7] N Rajendraprasad and K Basavaiah ldquoHighly sensitive spec-trophotometric determination of olanzapine using cerium(IV)and iron(II) complexes of 110-phenanthroline and 221015840-bipyridylrdquo Journal of Analytical Chemistry vol 65 no 5 pp482ndash488 2010

[8] K Basavaiah AM A Sameer and K B Vinay ldquoNew extractivespectrophotometric methods for the determination of olanza-pine in pharmaceutical formulatins using bromocresol greenrdquoJordan Journal of Chemistry vol 5 pp 101ndash117 2010

[9] N Rajendraprasad and K Basavaiah ldquoDetermination of olan-zapine by spectrophotometry using permanganaterdquo BrazilianJournal of Pharmaceutical Sciences vol 45 no 3 pp 539ndash5502009

[10] K Basavaiah K Tharpa N Rajendraprasad S G HiriyannaandK B Vinay ldquoSpectrophotometric determination of antipsy-chotic drug olanzapine in pharmaceuticalsrdquo Jordan Journal ofChemistry vol 4 no 1 pp 65ndash76 2009

[11] A A Mohamed ldquoKinetic and maximum-absorbance spec-trophotometric methods for the determination of olanzapinerdquoMonatshefte fur Chemie vol 139 no 9 pp 1005ndash1010 2008

[12] M A Raggi G Casamenti R Mandrioli G Izzo and EKenndler ldquoQuantitation of olanzapine in tablets byHPLCCZE


derivative spectrometry and linear voltammetryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 23 no 6 pp 973ndash981 2000

[13] R B Patel M R Patel K K Bhari and B G Patel ldquoDevelop-ment and validation of an HPTLC method for determinationof olanzapine in formulationsrdquo Journal of AOAC Internationalvol 93 no 3 pp 811ndash819 2010

[14] C Shah B Suhagia N Shah D Patel and N Patel ldquoStability-indicating simultaneous HPTLC method for olanzapine andfluoxetine in combined tablet dosage formrdquo Indian Journal ofPharmaceutical Sciences vol 70 no 2 pp 251ndash255 2008

[15] S Patel and N J Patel ldquoSimultaneous RP-HPLC and HPTLCestimation of fluoxetine hydrochloride and olanzapine in tabletdosage formsrdquo Indian Journal of Pharmaceutical Sciences vol71 no 4 pp 477ndash480 2009

[16] O V Olesen and K Linnet ldquoDetermination of olanzapinein serum by high-performance liquid chromatography usingultraviolet detection considering the easy oxidability of thecompound and the presence of other psychotropic drugsrdquoJournal of Chromatography B Biomedical Applications vol 714no 2 pp 309ndash315 1998

[17] H Weigmann S Hartter S Maehrlein et al ldquoSimultaneousdetermination of olanzapine clozapine anddemethylatedmeta-bolites in serum by on-line column-switching high-perform-ance liquid chromatographyrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 759 no 1 pp 63ndash712001

[18] O V Olesen B Poulsen and K Linnet ldquoFully automated on-line determination of olanzapine in serum for routine therapeu-tic drug monitoringrdquoTherapeutic Drug Monitoring vol 23 no1 pp 51ndash55 2001

[19] C DrsquoArrigo G Migliardi V Santoro and E Spina ldquoDetermi-nation of olanzapine in human plasma by reversed-phase high-performance liquid chromatographywith ultraviolet detectionrdquoTherapeutic Drug Monitoring vol 28 no 3 pp 388ndash393 2006

[20] L J Dusci L P Hackett L M Fellows and K F Ilett ldquoDeter-mination of olanzapine in plasma by high-performance liquidchromatography using ultraviolet absorbance detectionrdquo Jour-nal of Chromatography B Analytical Technologies in the Biomed-ical and Life Sciences vol 773 no 2 pp 191ndash197 2002

[21] M A Raggi G Casamenti R Mandrioli and V Volterra ldquoAsensitive high-performance liquid chromatographic methodusing electrochemical detection for the analysis of olanzapineand desmethylolanzapine in plasma of schizophrenic patientsusing a new solid-phase extraction procedurerdquo Journal of Chro-matography B Biomedical Sciences and Applications vol 750no 1 pp 137ndash146 2001

[22] M A Raggi G Casamenti R Mandrioli S Fanali DDe Ronchi and V Volterra ldquoDetermination of the novelantipsychoticz drug olanzapine in human plasma using HPLCwith amperometric detectionrdquo Chromatographia vol 51 no 9-10 pp 562ndash566 2000

[23] M A Raggi R Mandrioli C Sabbioni N Ghedini S Fanaliand V Volterra ldquoDetermination of olanzapine and desmethy-lolanzapine in the plasma of schizophrenic patients bymeans ofan improved HPLC method with amperometric detectionrdquoChromatographia vol 54 no 3-4 pp 203ndash207 2001

[24] M A Saracino A Koukopoulos G Sani M Amore andM ARaggi ldquoSimultaneous high-performance liquid chromato-graphic determination of olanzapine and lamotrigine in plasmaof bipolar patientsrdquoTherapeutic Drug Monitoring vol 29 no 6pp 773ndash780 2007

[25] E Choong S Rudaz A Kottelat D Guillarme J-L Veutheyand C B Eap ldquoTherapeutic drug monitoring of seven psy-chotropic drugs and four metabolites in human plasma byHPLC-MSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 50 no 5 pp 1000ndash1008 2009

[26] M Josefsson M Roman E Skogh and M-L Dahl ldquoLiquidchromatographytandemmass spectrometry method for deter-mination of olanzapine and N-desmethylolanzapine in humanserum and cerebrospinal fluidrdquo Journal of Pharmaceutical andBiomedical Analysis vol 53 no 3 pp 576ndash582 2010

[27] MA Saracino OGandolfi R DallrsquoOlio L Albers E Kenndlerand M A Raggi ldquoDetermination of Olanzapine in rat brainusing liquid chromatography with coulometric detection and arapid solid-phase extraction procedurerdquo Journal of Chromatog-raphy A vol 1122 no 1-2 pp 21ndash27 2006

[28] S C Kasper E L Mattiuz S P Swanson J A Chiu J T John-son and C O Garner ldquoDetermination of olanzapine in humanbreast milk by high-performance liquid chromatographywith electrochemical detectionrdquo Journal of Chromatography BBiomedical Sciences and Applications vol 726 no 1-2 pp 203ndash209 1999

[29] X Xuejun and T Zhonghua ldquoDetermination of olanzapine andits tablets by HPLCrdquo Zhongguo Yiyao Gongye Zazhi vol 35 pp46ndash48 2004

[30] A Pathak and S J Rajput ldquoDevelopment of a stability-indicat-ing HPLC method for simultaneous determination of olan-zapine and fluoxetine in combined dosage formsrdquo Journal ofChromatographic Science vol 47 no 7 pp 605ndash611 2009

[31] B V Reddy K V N S Reddy J Sreeramulu and G VKanumula ldquoSimultaneous determination of olanzapine and flu-oxetine byHPLCrdquoChromatographia vol 66 no 1-2 pp 111ndash1142007

[32] C R Shah N J Shah B N Suhagia and N M PatelldquoSimultaneous assay of olanzapine and fluoxetine in tablets bycolumn high-performance liquid chromatography and high-performance thin-layer chromatographyrdquo Journal of AOACInternational vol 90 no 6 pp 1573ndash1578 2007

[33] S G Hiriyanna K Basavaiah P S K Goud V Dhayanithi KRaju and H N Pati ldquoIdentification and characterization ofolanzapine degradation products under oxidative stress condi-tionsrdquo Acta Chromatographica vol 20 no 1 pp 81ndash93 2008

[34] International Conference on Hormonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse ICHHarmonised Tripartite Guideline Validation of Analyt-ical Procedures Text and Methodology Q2(R1) ComplementaryGuideline on Methodology dated November 1996 ICH Lon-don UK 2005

[35] M Zeaiter J-M Roger V Bellon-Maurel and D N RutledgeldquoRobustness of models developed by multivariate calibrationPart I the assessment of robustnessrdquo Trends in AnalyticalChemistry vol 23 no 2 pp 157ndash170 2004

[36] M Mulholland ldquoRuggedness testing in analytical chemistryrdquoTrends in Analytical Chemistry vol 7 no 10 pp 383ndash389 1988

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


NH

NN

N

SCH3

CH3

Figure 1 Structure of OLP

the simultaneous determination of fluoxetine HCl andOLP using reversed-phase liquid chromatography usingacetonitrile methanol 0032M ammonium acetate buffer(45 05 50 vvv) as the mobile phase at a flow rate of15mLminminus1 Quantitation of OLP was achieved with ultra-violet detection at 235 nm over concentration range of 01ndash2 120583gmLminus1 OLP whereas the second report [30] describes theseparation procedure using an Intersil C

18

reversed phasecolumn (150mm times 46mm 5120583m) with a 40 30 30 (vvv)mixture of 95mM NaH

2

PO4

(pH adjusted to 68 plusmn 01 withtriethylamine) acetonitrile and methanol as mobile phaseThe flow rate was 12mLminminus1 with UV-detection at 225 nmThe calibration plot was linear from 25 to 75 120583gmLminus1 forOLPSimultaneous assay of OLP and fluoxetine in tablets byHPLChas also been reported by Reddy et al [31]The separationwasachieved on a Lichrospher 100 RP-180 C

18

column (250mmtimes400mm id 5120583m) using 005M KH

2

PO4

buffer (pH 56adjusted with o-phosphoric acid)-acetonitrile (50 50) as themobile phase at a flow rate of 1mLminminus1 and at ambienttemperature Quantitation was achieved by measuring UVabsorption at 233 nm over the concentration range of 10ndash70 120583gmLminus1The reportedHPLCmethods however are eitherpoorly sensitive [29] or have narrow linear concentrationranges [15 30 31] The present team of workers has [32]reported the oxidative degradation of the OLP degradationproduct and the identification and characterization of thedegradation products The objective of this work was todevelop a simple rapid accurate and precise HPLC methodfor quantitative analysis of OLP in tablets and to validate themethod in accordance with ICH guidelines [33]

2 Methods

21 HPLC Instrumentation and Chromatographic ConditionsHPLC analysis was performed with a Waters HPLC systemequipped with Alliances 2695 series low pressure quaternarygradient pump a programmable variable wavelength UV-visible detector and autosampler Data were collected andprocessed using Waters Empower 2 software

Chromatographic separationwas achieved on a 150mmtimes46mm id 5 120583mparticle Intersil ODS 3V columnThemob-ile phase was a 35 65 (vv) mixture of 10mM disodiumhydrogen phosphate (pH 74)-acetonitrile The flow rate wasfixed as 10mLminminus1 and UV-detection was performed at254 nm Before use the mobile phase was filtered through02 120583m filter The column temperature was maintained at40∘C

22 Chemicals and Reagents A sample of OLP certified to be9988 pure was obtained as gift fromCipla India LtdMum-bai India HPLC-grade acetonitrile disodium hydrogenphosphate and orthophosphoric acid were purchased fromMerk Mumbai India Olanzapine tablets of two differentbrandsOleanz (25 and 75mgOLPper tablet) andOlanex (10and 15mg OLP per tablet) marketed by Sun PharmaceuticalsIndustries LtdMumbai India andRanbaxy Laboratories Ltd(Solus) Haryana India respectively were purchased fromlocal commercial sources HPLC-grade water from Merckwas used to prepare all solutions All other chemicals andreagents used were of analytical grade and purchased fromSD fine chemicals Mumbai India

A buffer solution of pH 74 was prepared by adjusting thepH of 10mMdisodium hydrogen phosphate with orthophos-phoric acid The mobile phase was prepared by mixing thebuffer and acetonitrile in 35 65 (vv) ratio The diluent was amixture of water and acetonitrile (55 45 vv)

A stock solution of OLP (100120583gmLminus1) was prepared inthe diluent Standard solutions were prepared by dilution ofthe stock solution with the diluent solutions to get solution inthe concentration range from 25 to 20 120583gmLminus1 OLP

23 Procedures

231 Calibration Graph Twenty 120583L of working standard sol-utions (25mdash20120583gmLminus1 OLP) was injected automaticallyinto the column in triplicate and the chromatograms wererecorded The calibration graph was prepared by plotting themean peak area versus concentration in 120583g mLminus1

232 Procedure for Tablets Twenty tablets were accuratelyweighed and crushed into a fine powder and mixed using amortar and pestle A quantity of tablet powder equivalent to10mg ofOLPwasweighed accurately into a 100mL calibratedflask 50mL of diluent solution was added and sonicated for20min to complete dissolution of the OLP and the solutionwas then diluted to themark with the diluent andmixed wellA small portion of the tablet solution (say 10mL) was with-drawn and filtered through a 02 120583m filter to ensure the abs-ence of particulate matter The filtrate was appropriatelydiluted with the diluent before injection into the column

3 Results and Discussion

Drug quality control stability metabolism pharmacokinet-ics and toxicity studies all necessitate the determination ofdrugs in pharmaceutical formulations and biological sam-ples Consequently efficient and validated methods are verycrucial requirements for all these investigations

31 Method Development and Optimization Chromatogra-phic parameters were preliminarily optimized to develop thepresent method for the determination of OLP with shortanalysis time (lt8min) because rapid and economic analysisis becoming increasingly important in pharmaceutical anal-ysis to increase the sample throughput In the present casean Intersil ODS 3V column maintained at 40∘C was


014

012

010

008

006

004

002

000

OLP

000 100 200 300 400 500 700600 800

(min)

(AU)



HPO4


119879






0020

0010

0000

(AU)

000 100 200 300 400 500 700600 800

(min)

0030

0040

0050















lowast

plusmn SD SE RSD








RSD 0 547492

0213

4355



119879




119879





4 Conclusions















Acknowledgments


References
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


014

012

010

008

006

004

002

000

OLP

000 100 200 300 400 500 700600 800

(min)

(AU)



HPO4


119879






0020

0010

0000

(AU)

000 100 200 300 400 500 700600 800

(min)

0030

0040

0050















lowast

plusmn SD SE RSD








RSD 0 547492

0213

4355



119879




119879





4 Conclusions















Acknowledgments


References
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









lowast

plusmn SD SE RSD








RSD 0 547492

0213

4355



119879




119879





4 Conclusions















Acknowledgments


References
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of














Acknowledgments


References
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Isocratic High-Performance Liquid ...downloads.hindawi.com/archive/2014/616941.pdf · Isocratic High-Performance Liquid Chromatographic Assay of Olanzapine: Method