Research Article Development and Validation of RP-HPLC ...F : HPLC-PDA chromatogram of LOD and LOQ...

Hindawi Publishing CorporationISRN SpectroscopyVolume 2013 Article ID 572170 6 pageshttpdxdoiorg1011552013572170

Research ArticleDevelopment and Validation of RP-HPLC Methodfor Azilsartan Medoxomil Potassium Quantitation inHuman Plasma by Solid Phase Extraction Procedure

Paras P Vekariya and Hitendra S Joshi

Department of Chemistry Saurashtra University Rajkot 36005 India

Correspondence should be addressed to Hitendra S Joshi drhsjoshi49gmailcom

Received 21 June 2013 Accepted 14 August 2013

Academic Editors I P Gerothanassis B Liu and S Pandey

Copyright copy 2013 P P Vekariya and H S JoshiThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Simple and rapid reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated usingsolid phase extraction (SPE) technique for the determination of Azilsartan Medoxomil Potassium (AMP) in human plasmadetection was carried out by photo diode array detector Chromatographic separation of the analyte AMP was achieved within75min by Waters symmetry C

18(46 times 250mm 5 120583m) column mobile phase was 25mM ammonium acetate buffer (pH 55)

acetonitrile 55 45 vv flow rate was 10mLmin and the detection was carried out at 254 nm Calibration curve was linear (r2 gt09985) in the range of 10ndash90120583gmL limit of detection (LOD) and limit of quantitation (LOQ)were 0150120583gmL and 0400 120583gmLrespectively and intra- and interday deviations were between 153ndash841 and 178ndash459 respectively The overall mean recoveryof AMP was 9235 No any endogenous constituents were found to interfere at retention time of the analyte This new RP-HPLCmethod was successfully validated and may be applied to conduct bioavailability and bioequivalence studies of AMP

1 Introduction

Azilsartan Medoxomil Potassium is chemically namedas (5-Methyl-2-oxo-13-dioxol-4yl) methyl 2-ethoxy-1-[21015840-(5-oxo-45-dihydro-124-oxadiazol-3-yl) biphenyl-4-yl]met-hyl-1H-benzimidazole-7-carboxylate monopotassium salt(Figure 1) It is a white crystalline powder which is practicallyinsoluble inwater freely soluble inmethanol dimethylsulfox-ide and dimethylformamide soluble in acetic acid slightlysoluble in acetone and acetonitrile and very slightly solublein tetrahydrofuran and 1-octanol [1]

The US Food and Drug Administration (FDA) hasapproved Edarbi tablet (Azilsartan Medoxomil Potassium)on February 25 2011 to treat hypertension in adults It isavailable in 80mg and 40mgdosages with the recommendeddosage set at 80mg once in a day [2]

Angiotensin II hormone plays a vital role in activation ofrenin-angiotensin-aldosterone system as well as in regulationof blood pressure fluid-electrolyte balance and also in patho-physiology of hypertension Activation of type 1 angiotensin

receptor which is a member of G protein coupled receptorefficiently controls the numerous effects of AII which arevasoconstriction secretion of aldosterone and vasopressinand cellular proliferation So blocking of AII receptor willalso block receptor-1 and it will lead to termination of thewhole course of action mentioned above so AII blocker willbe helpful in the management of cardiovascular and renaldiseases as therapeutic agent [3]

The active moiety of AMP is revealed by hydrolysis ofthe medoxomil ester and it converts into Azilsartan which isan active angiotensin II receptor blocker and more effectivein lowering blood pressure within 24 hours as compared tovalsartan and olmesartan [4ndash6]

The literature survey shows that there is only a singlemethod available for quantitation of Azilsartan MedoxomilPotassium and Chlorthalidone in pharmaceutical dosageform [7] As per our findings there is not any methodreported for quantitation of AMP in human plasma thus itis inevitable to develop such a sensitive rapid and accurate

2 ISRN Spectroscopy

OO

O

O

O

OO

O

N

N

N

N

CH3

minus

K+

H3C

Figure 1 Chemical structure of lafutidine hydrochloride salt

method which could be used to investigate further bioavail-ability and bioequivalence studies

2 Experimental

21 Materials and Reagents Pharmacopoeial grade standardof Azilsartan Medoxomil Potassium was gifted by AmiLifesciences Private Limited Baroda LC grademethanol waspurchased from Spectrochem Pvt Ltd Baroda the waterused was purified by milli-Q water assembly analytical gradeammonium acetate was obtained from Spectrochem PvtLtd and solid phase extraction cartridges (Strata-X) werepurchased from Phenomenex

22 Instrumentation The chromatographic system used toperform development and validation of this method wascomprised of an LC-10ATvp binary pump an SPD-M10Avpphoto-diode array detector and a Rheodyne manual injectormodel 7725i with 20120583L loop (Shimadzu Kyoto Japan)connected to a multi-instrument data acquisition and dataprocessing system (LC Solution Shimadzu)

23 Biological Samples Human blank plasma samples for thedevelopment and validation of the method were obtainedfrom government recognise laboratory

24 Chromatographic Conditions Chromatographic analysiswas performed on a reverse phase column purchased fromWaters symmetryC

18(15 cm 46times 250mm 5 120583m)part num-

ber WAT054275 serial number 022638193236 The mobilephase consisted of 25mM ammonium acetate buffer (pH55) methanol 55 45 vv The flow rate of the mobile phasewas adjusted to 10mLmin retention time of analyte was75min and the injection volume was 20120583L Detection wasperformed at 254 nm

25 Preparation of Stock Solution and Spiked Plasma SamplesThe stock solution of AMP was prepared in methanol atconcentration of 10mgmL Working solution of 50120583gmLwas prepared by appropriately diluting the stock solutionof AMP in methanol water 50 50 vv Working solutionof AMP was used to prepare the spiking stock solution forpreparation of nine points of calibration curve (1ndash9120583gmL)and quality control samples at three concentration levels (15

45 and 80 120583gmL) All stock and working solutions werestored in refrigerator (at 2ndash8∘C) when not in use The spikedplasma samples were prepared by adding 25 120583L of respectivespiking stock solution in 475 120583L of blank plasma

26 Sample Preparation and Extraction Procedure Extrac-tion was performed by solid phase extraction procedurewhich is a very efficient technique and the samples extractedby this procedure are untainted compared to those extractedby other techniques Sample extracted by this method showsgood recovery results so it was decided to conduct themethod validation by using SPE technique to achieve moreefficient data of bioavailability and equivalence studies

475 120583L of plasma was spiked with 025120583L of respectiveQC and calibration standard in glass tube mixed with 250120583Lof 10mMammonium acetate buffer and vortexed thoroughlythen mixture was loaded into Strata-X cartridge which waspreconditioned by 2mL methanol followed by 2mL watercartridgewaswashed by 1mLwater two timesThen cartridgewas transferred into clean test tube the analyte was elutedin 10mL methanol and then 20120583L volume is injected intoHPLC system

27 Method Validation The validation was executed as perldquoGuidance for Industry Bioanalytical Method Validationrdquofrom the United States Food and Drug Administration [8]

271 Selectivity The selectivity of themethodwas performedto ensure absolute separation of AMP from the biologicalendogenous components of the human plasma Selectivitywas carried out by analysing seven different lots of blankhuman plasma by solid phase extraction procedure givenabove

272 Linearity Five standard curves of nine different con-centration standards and two blank samples have beenassayed but only nine concentration standards were includedin the calibration curve whereas blank samples were used tocheck interference and contamination Each calibration curveshould meet the following acceptance criteria deviation atLLOQ level must be lower than 20 and not more than 15deviation for the rest of the standards other than LLOQ

273 Precision andAccuracy Interday and intraday precisionand accuracy were evaluated by five spiked samples atanalyzing three different concentrations of AMP (15 45and 80 120583gmL) Within batch precession and accuracy weredetermined by repeated analysis of five spiked samples ofAMP at each QC level Intraday precession and accuracywere determined by repeated analysis of three consecutivedays (5 series per day)The concentration of each sample wasdetermined using standard curves prepared and analysed onthe same day

274 Matrix Effect Matrix effect was evaluated by com-paring peak areas of extracted samples using five differentlots of human plasma in triplicate at three concentrationlevel of analyte (15 45 and 80 120583gmL) percentage RSD

ISRN Spectroscopy 3

(min)

(mAU

)

00 25 50 75 100 125

minus10

minus05

00

05

10

PDA multi 1

(a)

(min)

(mAU

)

00 25 50 75 100 125minus1

0

1

2

3 PDA multi 1

AMP

(b)

Figure 2 HPLC-PDA chromatograms of blank plasma sample and LLOQ (10120583gmL) standard (a) chromatogram of extracted blank plasma(b) Chromatogram of extracted LLOQ sample spiked with 10 120583gmL of calibration standard

(min)00 25 50 75 100 125

PDA multi 1AMP05

00

minus05

minus10

(mAU

)

(a)

(min)

(mAU

)

00 25 50 75 100 125

minus10

minus05

00

05

10

15

PDA maulti 1

AMP

(b)

Figure 3 HPLC-PDA chromatogram of LOD and LOQ samples (a) Chromatogram of LOD sample (b) Chromatogram of LOQ sample

and accuracy were calculated to check interference of matrixeffect on the analyte concentration

275 Recovery Percentage recovery of AMPwas determinedby comparing the AMP peak area obtained from extractedplasma samples with aqueous sample of the same concentra-tion standardThis procedure was followed for three differentconcentration levels of analyte 15 45 and 80120583gmL

3 Results and Discussion

During method development stages we found that theresults obtained by solid phase extractionmethod weremuchbetter than those obtained by protein precipitation extraction(especially mean recovery of analyte) so it was decided toproceed with SPE

31 Sample Preparation To accelerate drug dissociation ofanalyte from plasma different buffers were tried but with25mM ammonium acetate (pH 55) recovery and response

were found better so it was added to plasma sample andmethanol was used as eluent

32 Separation Different mobile phases were investigated byusing methanol acetonitrile and ammonium acetate in vari-ous proportions after several trialsmobile phase acetonitrile25mM ammonium acetate 45 55 vv (pH 55) was finalisedFlow rate selection was based on peak parameters heightasymmetry tailing baseline drift and run time Flow rate wasset at 10mLminThe retention time for the investigated drugwas found at 75min and runtime was 15min

Different columns have been tested with minimal effecton the resolution of the analyte and a Waters C

18column

has been finalised because of its demonstrated smooth-ness and reproducibility in this method Column-to-columnreproducibility was also evaluated only slight variations inretention time were observed when injecting the sample oncolumn from different manufacturers and containing thesame brand of packing material

The optimum wavelength for detection of analyte was254 nm at which much better detector response was

4 ISRN Spectroscopy

Table 1 Results of 5 calibration curves for AMP determination

Added conc 1 2 3 4 5 6 7 8 9

Back calculated conc (120583gmL)

101 198 310 438 497 608 682 805 908104 195 308 400 500 558 722 790 885107 193 307 387 494 611 713 781 885103 201 299 419 537 646 742 836 967108 206 308 355 470 601 676 752 892

Mean 105 198 306 400 500 605 707 793 907Mean accuracy 10453 9923 10215 9994 9994 10081 10098 9910 10081RSD 255 259 140 784 477 520 391 394 383

Table 2 Parameter corresponding to linear regression obtained from 5 calibration curves of AMP

Calibration curve Slope Intercept Correlation coefficient1 307422500 25118611 099852 303792500 14060833 099803 304652500 14870833 099914 305801786 14870833 099955 296609167 17263056 09972Mean 303655690 17236833 09985SD 4167146 4566344 00009SD 13723 264918 00895

obtained For the estimation of AMP a sharp and symmet-rical peak was obtained with good baseline which assists theaccurate measurement of the peak area

33 Assay Selectivity Seven blank samples were assayedto examine selectivity of the method and there was nointerference of endogenous peak observed at the retentiontime of AMP (Figure 2)

34 Linearity Five calibration curves were assayed as men-tioned in Section 25 and all of them met the acceptancecriteria with good regression (1199032 = 09985) The linearityof the method was evaluated by a calibration curve in theconcentration range of 1ndash9120583gmL Calibration curve wasdetermined by peak area versus concentration Concentra-tion of analyte was calculated by using mean calculationformula 119884 = 30365119909 + 1723 Data of calibration curves isgiven in Tables 1 and 2

35 LOD and LOQ Limit of detection and limit of quan-tification (LOD and LOQ) were estimated in accordancewith the baseline to noise ratio it should be 3 and 10 timeshigher than the blank plasma sample respectively LOD andLOQ were estimated to be 0150120583gmL and 0400120583gmLrespectively a representative chromatogram of LOD andLOQ is given in Figure 3

36 Precision and Accuracy RSD and accuracy of intradayprecision were in the range 153ndash841 and 102ndash105 while

for interday it was found to be 178ndash459 and 99ndash113respectivelyThe results revealed good precision and accuracy(Table 3)

37 Matrix Effect Results of matrix effect are given inTable 4 Percentage of RSD and accuracy of the injectedsamples for LQC was 119ndash754 and 9966ndash10478 forMQC 164ndash258 and 10316ndash1084 and for HQC was343ndash711 and 10316ndash10646 respectively Results indi-cate that there is no considerable endogenous compo-nent from blank plasma interferes in the measurement ofanalyte

38 Extraction Recovery The extraction recovery deter-mined for AMP was shown to be consistent precise andreproducible As per result given in Table 5 mean recoveriesof the three concentration levels (15 45 and 80120583gmL)were 9369 9157 and 9164 respectively whereas the meanrecovery between QC level is 9235

39 Stability Table 6 summarizes stability experimentswhich are the bench top 24 hour at room temperature postextraction (at 4ndash8∘C after extraction) freeze thaw stability(three cycle) long-term stability for 30 days (stored atminus20∘C) and post preparative stability data of AMP All theresults showed the stability behaviour during these tests andno stability-related problems occurred during the validationand stability testing The stability of working solutions was

ISRN Spectroscopy 5

Table 3 Intra- and interday precision and accuracy of the method for determination of AMP at three levels of quality control samples

Added conc (120583gmL) Intraday InterdayDetected conca RSD Accuracy Detected conca RSD Accuracy

15 153 (plusmn002) 181 10202 149 (plusmn002) 178 999845 461 (plusmn017) 380 10239 508 (plusmn023) 459 1130380 841 (plusmn033) 394 10509 811 (plusmn031) 394 10127aMean(plusmnSD 120583gmL) intraday 119899 = 5and interday 119899 = 5 series per day

Table 4 Results of matrix effect analysing 5 different lots of blank human plasma at three different levels of quality control samples

LOT 15 120583gmL 45 120583gmL 80 120583gmLMeana RSD Accuracy Meana RSD Accuracy Meana RSD Accuracy

A-1 154 (plusmn007 ) 448 1029 464 (plusmn008) 192 1031 829 (plusmn028) 343 1036A-2 156 (plusmn004) 263 1045 486 (plusmn007) 164 1080 835 (plusmn045) 546 1044A-3 149 (plusmn002) 119 996 472 (plusmn010) 223 1050 837 (plusmn059) 711 1031A-4 152 (plusmn005) 320 1016 475 (plusmn008) 180 1057 851 (plusmn045) 536 1064A-5 157 (plusmn012) 754 1047 472 (plusmn012) 258 1050 849 (plusmn030) 363 1061a(plusmnSD 120583gmL)

Table 5 Absolute recovery of AMP from 05mL of plasma at threedifferent levels of quality control samples

15120583gmL 45 120583gmL 80 120583gmL

Mean recovery within QC level 9369 9157 9164 Mean recovery between QClevel 9235

SD 131 CV 135

Table 6 Stability data of AMP at three levels of quality controlstandard samples

15 120583gmL 45 120583gmL 80 120583gmLAccuracya Accuracya Accuracya

Bench top 9851 (plusmn183) 10471 (plusmn347) 1012 (plusmn145)Process 9812 (plusmn278) 10337 (plusmn424) 10149 (plusmn067)Freeze and thaw 10247 (plusmn433) 10194 (plusmn262) 9894 (plusmn168)Long term 9975 (plusmn259) 10434 (plusmn419) 10175 (plusmn165)aMean (plusmnSD)

tested and on the basis of the results obtained these solutionswere at least stable for 6 h

4 Conclusion

We have developed a suitable method for the determinationof Azilsartan Medoxomil Potassium in human plasma bysolid phase extraction procedure To our knowledge this isthe first description on development and validation of AMPby using RP-HPLC method which gives a good resolutionin short analysis time (lt75min) The method is simplesensitive specific and reproducible and may be used inbioavailability and bioequivalence studies

Acknowledgments

The authors are thankful to Ami Lifesciences Pvt Ltd forsupplying them with the Azilsartan Medoxomil Potassiumfacilities and grants were given under the UGC-SpecialAssistance Program Department of Research Support (SAP-DRS) Department of Science and Technology (DST) NewDelhi Fund for Improvement of Science and Technology(FIST) National Facility forDrugDiscovery andDepartmentof Chemistry Saurashtra University Rajkot 360 005 IndiaThe authors are thankful to them for providing analyticalfacilities

References

[1] European Medicines Agency ldquoEdarbi (Azilsartan MedoxomilPotassium)rdquo Assessment Report Procedure no EMEAHC002293 2011

[2] Press announcement by The US Food and Drug Adminis-tration httpwwwfdagovNewsroomPressAnnouncementsucm244722htm

[3] M Gasparo K J Catt T Inagami J W Wright and T UngerldquoInternational union of pharmacology XXIII The angiotensinII receptorsrdquo Pharmacological Reviews vol 52 no 3 pp 415ndash472 2000

[4] MOjimaH IgataM Tanaka et al ldquoIn vitro antagonistic prop-erties of a new angiotensin type 1 receptor blocker azilsartan inreceptor binding and function studiesrdquo Journal of Pharmacologyand ExperimentalTherapeutics vol 336 no 3 pp 801ndash808 2011

[5] G L Bakris D SicaMWeber et al ldquoThe comparative effects ofazilsartanmedoxomil and olmesartan on ambulatory and clinicblood pressurerdquo Journal of Clinical Hypertension vol 13 no 2pp 81ndash88 2011

[6] W B White M A Weber D Sica et al ldquoEffects of theangiotensin receptor blocker azilsartan medoxomil versusolmesartan and valsartan on ambulatory and clinic blood pres-sure in patients with stages 1 and 2 hypertensionrdquoHypertensionvol 57 no 3 pp 413ndash420 2011

6 ISRN Spectroscopy

[7] K Madhubabu and K Bikshalbabu ldquoReverse phasemdashHplcmethod development and validation of azilsartan medoxomiland chlortalidone in pharmaceutical dosage formrdquo Journal ofAtoms and Molecules vol 2 no 1 pp 117ndash126 2012

[8] ldquoGuidance for industry bio analytical method validationrdquoUS Department of Health and Human Services Food andDrug Administration CDER Rockville Md USA May 2001httpwwwfdagovdownloadsDrugsGuidanceCompliance-RegulatoryInformationGuidancesucm070107pdf

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CatalystsJournal of

2 ISRN Spectroscopy

OO

O

O

O

OO

O

N

N

N

N

CH3

minus

K+

H3C

Figure 1 Chemical structure of lafutidine hydrochloride salt

method which could be used to investigate further bioavail-ability and bioequivalence studies

2 Experimental

21 Materials and Reagents Pharmacopoeial grade standardof Azilsartan Medoxomil Potassium was gifted by AmiLifesciences Private Limited Baroda LC grademethanol waspurchased from Spectrochem Pvt Ltd Baroda the waterused was purified by milli-Q water assembly analytical gradeammonium acetate was obtained from Spectrochem PvtLtd and solid phase extraction cartridges (Strata-X) werepurchased from Phenomenex

22 Instrumentation The chromatographic system used toperform development and validation of this method wascomprised of an LC-10ATvp binary pump an SPD-M10Avpphoto-diode array detector and a Rheodyne manual injectormodel 7725i with 20120583L loop (Shimadzu Kyoto Japan)connected to a multi-instrument data acquisition and dataprocessing system (LC Solution Shimadzu)

23 Biological Samples Human blank plasma samples for thedevelopment and validation of the method were obtainedfrom government recognise laboratory

24 Chromatographic Conditions Chromatographic analysiswas performed on a reverse phase column purchased fromWaters symmetryC

18(15 cm 46times 250mm 5 120583m)part num-

ber WAT054275 serial number 022638193236 The mobilephase consisted of 25mM ammonium acetate buffer (pH55) methanol 55 45 vv The flow rate of the mobile phasewas adjusted to 10mLmin retention time of analyte was75min and the injection volume was 20120583L Detection wasperformed at 254 nm

25 Preparation of Stock Solution and Spiked Plasma SamplesThe stock solution of AMP was prepared in methanol atconcentration of 10mgmL Working solution of 50120583gmLwas prepared by appropriately diluting the stock solutionof AMP in methanol water 50 50 vv Working solutionof AMP was used to prepare the spiking stock solution forpreparation of nine points of calibration curve (1ndash9120583gmL)and quality control samples at three concentration levels (15

45 and 80 120583gmL) All stock and working solutions werestored in refrigerator (at 2ndash8∘C) when not in use The spikedplasma samples were prepared by adding 25 120583L of respectivespiking stock solution in 475 120583L of blank plasma

26 Sample Preparation and Extraction Procedure Extrac-tion was performed by solid phase extraction procedurewhich is a very efficient technique and the samples extractedby this procedure are untainted compared to those extractedby other techniques Sample extracted by this method showsgood recovery results so it was decided to conduct themethod validation by using SPE technique to achieve moreefficient data of bioavailability and equivalence studies

475 120583L of plasma was spiked with 025120583L of respectiveQC and calibration standard in glass tube mixed with 250120583Lof 10mMammonium acetate buffer and vortexed thoroughlythen mixture was loaded into Strata-X cartridge which waspreconditioned by 2mL methanol followed by 2mL watercartridgewaswashed by 1mLwater two timesThen cartridgewas transferred into clean test tube the analyte was elutedin 10mL methanol and then 20120583L volume is injected intoHPLC system

27 Method Validation The validation was executed as perldquoGuidance for Industry Bioanalytical Method Validationrdquofrom the United States Food and Drug Administration [8]

271 Selectivity The selectivity of themethodwas performedto ensure absolute separation of AMP from the biologicalendogenous components of the human plasma Selectivitywas carried out by analysing seven different lots of blankhuman plasma by solid phase extraction procedure givenabove

272 Linearity Five standard curves of nine different con-centration standards and two blank samples have beenassayed but only nine concentration standards were includedin the calibration curve whereas blank samples were used tocheck interference and contamination Each calibration curveshould meet the following acceptance criteria deviation atLLOQ level must be lower than 20 and not more than 15deviation for the rest of the standards other than LLOQ

273 Precision andAccuracy Interday and intraday precisionand accuracy were evaluated by five spiked samples atanalyzing three different concentrations of AMP (15 45and 80 120583gmL) Within batch precession and accuracy weredetermined by repeated analysis of five spiked samples ofAMP at each QC level Intraday precession and accuracywere determined by repeated analysis of three consecutivedays (5 series per day)The concentration of each sample wasdetermined using standard curves prepared and analysed onthe same day

274 Matrix Effect Matrix effect was evaluated by com-paring peak areas of extracted samples using five differentlots of human plasma in triplicate at three concentrationlevel of analyte (15 45 and 80 120583gmL) percentage RSD

ISRN Spectroscopy 3

(min)

(mAU

)

00 25 50 75 100 125

minus10

minus05

00

05

10

PDA multi 1

(a)

(min)

(mAU

)

00 25 50 75 100 125minus1

0

1

2

3 PDA multi 1

AMP

(b)


(min)00 25 50 75 100 125

PDA multi 1AMP05

00

minus05

minus10

(mAU

)

(a)

(min)

(mAU

)

00 25 50 75 100 125

minus10

minus05

00

05

10

15

PDA maulti 1

AMP

(b)










18column



4 ISRN Spectroscopy


Added conc 1 2 3 4 5 6 7 8 9


101 198 310 438 497 608 682 805 908104 195 308 400 500 558 722 790 885107 193 307 387 494 611 713 781 885103 201 299 419 537 646 742 836 967108 206 308 355 470 601 676 752 892













ISRN Spectroscopy 5








15120583gmL 45 120583gmL 80 120583gmL


SD 131 CV 135





4 Conclusion


Acknowledgments


References







6 ISRN Spectroscopy












Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

ISRN Spectroscopy 3

(min)

(mAU

)

00 25 50 75 100 125

minus10

minus05

00

05

10

PDA multi 1

(a)

(min)

(mAU

)

00 25 50 75 100 125minus1

0

1

2

3 PDA multi 1

AMP

(b)


(min)00 25 50 75 100 125

PDA multi 1AMP05

00

minus05

minus10

(mAU

)

(a)

(min)

(mAU

)

00 25 50 75 100 125

minus10

minus05

00

05

10

15

PDA maulti 1

AMP

(b)










18column



4 ISRN Spectroscopy


Added conc 1 2 3 4 5 6 7 8 9


101 198 310 438 497 608 682 805 908104 195 308 400 500 558 722 790 885107 193 307 387 494 611 713 781 885103 201 299 419 537 646 742 836 967108 206 308 355 470 601 676 752 892













ISRN Spectroscopy 5








15120583gmL 45 120583gmL 80 120583gmL


SD 131 CV 135





4 Conclusion


Acknowledgments


References







6 ISRN Spectroscopy












Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

4 ISRN Spectroscopy


Added conc 1 2 3 4 5 6 7 8 9


101 198 310 438 497 608 682 805 908104 195 308 400 500 558 722 790 885107 193 307 387 494 611 713 781 885103 201 299 419 537 646 742 836 967108 206 308 355 470 601 676 752 892













ISRN Spectroscopy 5








15120583gmL 45 120583gmL 80 120583gmL


SD 131 CV 135





4 Conclusion


Acknowledgments


References







6 ISRN Spectroscopy












Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

ISRN Spectroscopy 5








15120583gmL 45 120583gmL 80 120583gmL


SD 131 CV 135





4 Conclusion


Acknowledgments


References







6 ISRN Spectroscopy












Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

6 ISRN Spectroscopy












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

Research Article Development and Validation of RP-HPLC ...F : HPLC-PDA chromatogram of LOD and LOQ...

Documents

Transcript of Research Article Development and Validation of RP-HPLC ...F : HPLC-PDA chromatogram of LOD and LOQ...