A high-performance liquid chromatographic analysis of erythromycin in human serum and urine with UV detection at 200 nm is presented. The method involves a solid-phase extraction procedure followed by a simple phase separation step and chromatography on a reversed-phase column. The method has sensitivity limits of 0.25 and 1.0 g/mL in serum and urine, respectively, and is sufficiently sensitive to monitor concentrations of erythromycin in human serum and urine after the administration of a single 500-mg erythromycin stearate tablet

Haigh, J M

Kanfer, I

Stubbs, C

Stubbs, Christopher

Haigh, John M

Kanfer, Isadore

South East Academic Libraries System (SEALS)

Reprintedfrom the Journal of PharmaceuticalSciences.Vol. 74,No. 10,October1985.@ 1985by theAmericanPharmaceuticalAssociation.Determination of Erythromycin in Serum and Urine by High-Performance Liquid Chromatography with UltravioletDetectionC. STUBBS, J. M. HAIGH, AND I. KANFERxReceived September 13,1984, from the School of Pharmaceutical Sciences, Rhodes University, Grahamstown 6140, South Africa.July 3, 1985.Abstract D A high-performanceliquidchromatographicanalysisoferythromycinin humanserumandurinewithUV detectionat200nmispresented.The methodinvolvesa solid-phaseextractionprocedurefollowedbya simplephaseseparationstepandchromatographyon areversed-phasecolumn.The methodhas sensitivitylimitsof 0.25and1.0JLg/mLin serumandurine,respectively,and is sufficientlysensitiveto monitorconcentrationsof erythromycinin humanserumand urineaftertheadministrationof a single500-mgerythromycinstearatetablet.Erythromycin, a macrolide antibiotic used mainly againstgram-positive bacteria, has been in clinical use since 1952.1Previous pharmacokinetic data published on this antibiotichave been derived predominantly from microbiological assaytechniques. These techniques, however, are relatively impre-cise as well as nonspecific and tedious to perform.In 1969, Stephens et al,2 used a two-step paper chromato-graphic-bioautographic technique to determine erythromy-cin in blood, whereas Easterbrook and Hersey3 described aTLC technique also used in conjunction with bioautography.These procedures, although enabling the determination offree base from the parent ester form of the drug, suffer fromthe same disadvantages associated with microbiologicalmethods.Tsuji4 described a fluorometric determination of erythro-mycin, including erythromycin ethyl succinate and somemajor metabolites and degradation products, in serum byhigh-performance liquid chromatography (HPLC) and on-stream post-column derivatization and extraction. Althoughthis method appears to be highly sensitive, it suffers from thedisadvantage of requiring complex and expensive equipment.In addition, erythromycin is not well resolved from itsmetabolites and degradation products present in serum.A recent paper by Chen and Chiou5 described the HPLCanalysis of erythromycin in biological fluids by electrochemi-cal detection.A novel dual-electrode electrochemical detec-tor, operated in the oxidative screen mode, was used. Thisprocedure has the main advantage of high sensitivity, allow-ing the use of extremely small samples. To reduce back-ground currents and enhance detector stability, however, itis necessary to maintain the HPLC system in continuousoperation, even when it is not in use. Furthermore, theirchoice of erythromycin B as an internal standard may lead toanomalies during pharmacokinetic studies, since variousamounts of this compound are usually present in batches ofraw material.6 A further recent report by Duthu,7 who usedthe same detector system as Chen and Chiou, described theanalysis of erythromycin in serum only, under differentchromatographic conditions.The objective of the present study was to develop a sensi-tive, reproducible, and rapid method to quantitatively deter-mine erythromycin in serum and urine by HPLC with UVdetection by employing a conventional variable-wavelengthUV detector. The success of this method was largely due to1126 / Journal of Pharmaceutical SciencesVol. 74, No. 10, October 1985Acceptedthe efficiency of the solid-phase extraction procedure em-ployed, which resulted in a clean sample extract. This en-abled the utilization of a detection wavelength of 200 nm.Experimental SectionApparatus-The modular high-performanceliquid chromato-graphconsistedofan M 6000Aconstantflowpumpanda WispnOBautomatedsample injector (Waters Associates),a variable-wave-lengthUV detector(model769;Kratos Analytical Instruments),and'a strip chartrecorder(model561;Hitachi, Japan). The mobilephasewas constantlydeaeratedwith an in-line vacuumdeaeratingunit(modelERC-3510;Erma Optical Works, Japan). The separationwasperformedona 15cm x 3.9mmLd. columncontainingmicroparticu-late bonded5-JLmoctadecylsilane(C1s)material (Novapak;WatersAssociates).The temperatureofthe columnwasmaintainedat 35°Cwith the aid of a modelLC-22 temperaturecontroller(BioanalyticalSystems).Reagents and Materials-Reagents were of at least analyticalgrade.UV-grade acetonitrilewas obtainedfrom Burdick and Jack-son Laboratories.Erythromycin baseand oleandomycinphosphatewere obtainedfrom Abbott Laboratories and Pfizer Laboratories,respectively.Erythromycin B, theanhydrometaboliteoferythromy-cin, erythralosamine,and erythromycinenol ether were obtainedfromAbbott Laboratories,and thede-N-methylmetaboliteoferyth-romycinwasobtainedfromLilly ResearchLaboratories.The HPLC-gradewaterusedin themobilephasewaspurifiedthrougha Milli-Qsystem(Millipore Corp.).Chromatographic Conditions-The mobile phasewas preparedby mixing acetonitrile(300mL) with 0.05M phosphatebuffer(700mL). The phosphatebuffer was prepared by adding 3.2 mL ofphosphoricacidto 1 L of water. Sodiumhydroxidewasthen usedtoadjust the solution to pH 6.30.The solventmixture (pH 7.00)wasdeaeratedand filtered through a 0.6-JLmfilter (typeBD; MilliporeCorp.).This mobilephasewas usedat a flow rate of 1.0mLimin for theanalysisofbothserumandurine samples,with a resulting pressureof 100bar (1 x 104kPa). The wavelengthof detectionwas 200nm,with a detectorsensitivity of 0.01AUFS and a time constantof 2 s.Extraction of Serum and Urine-Samples of serum (2 mL) orurine (1 mL) were mixedwith 0.25or 0.5mL, respectively,of a 12-JLg/mLaqueousoleandomycinphosphateinternal standardsolution.Addition of 1mL of acetonitrile,followedbyvortexmixing for 1minandcentrifugationfor 5min at 1600xg,resultedin deproteinizationof the biological samples.The supernatant was transferred to aculture tube containing 8 mL of water for direct loading onto aprewashed, 1-mL, disposable octadecyl extraction column (J. T.Baker Chemical Co.) which had been prewashedunder 10-15mmlHgvacuum(Baker 10Extraction Systemcoupledtoa modelA2-S Aspirator; Rikakikai Co., Japan) by successivelypassing3 mL ofacetonitrileand3mL ofwaterthrougheach.The dilutedsamplewasthen addedto the extractioncolumnwith the aid of 15-mLsamplereservoir (J. T. Baker Chemical Co.). The extractioncolumn waswashedwith 5mL of waterand thenwith 5 mL of acetonitrile:water(1:1),with the vacuummaintaineduntil dry. Erythromycinand theinternal standard were eluted into 2-mL taperedcollectiontubeswith twosuccessive500-JLLaliquotsofacetonitrile:0.05M phosphatebuffer (1:1).The samplewas then dried under vacuumin a rotaryvacuumcentrifuge(SpeedVac Concentrator;Savant Instruments).The residuein thecollectiontubewasreconstitutedin 20JLLofwater0022-3549/85/1000-1126$01.00/0@ 1985,AmericanPharmaceuticalAssociationandvortexedfor1min.When25tLLofacetonitrilewasadded,twolayersformed uetothehighconcentrationofsaltsin theaqueouslayer.Thismixturewasvortexedfor1minandthencentrifugedfor1minat1600xg.An aliquot(15-20tLL)oftheacetonitrilephasewasthentransferredto a WISP limitedvolumeinsert(WatersAsso-ciates)with a microsyringe.A 5-tLIaliquotof this samplewasinjectedontothecolumn.RelevantchromatogramsaredepictedinFigs.1and2.ClinicalStudy-A pilottrial inwhichonevolunteerwasusedwasconductedto checkthe effectivenessof the analyticalmethodtomeasureconcentrationsof erythromycinbaseand any possiblemetabolitesor degradationproductsin serumandurineaftertheoraladministrationofa commerciallyavailableerythromycinstea-rateformulation(Erythrocin500;AbbottLaboratories).Thesubjectreceivedonefilm-coatedtabletcontaining500mgoferythromycinstearateafteranovernightfast,accompaniedby 250mL ofwaterandimmediatelyfollowedbya lightbreakfast.Bloodsamplesweredrawnat0,0.25,0.5,0.75,1,2,3,4,6,8,10,and12h afteringestionofthemedication.Theserumwasseparatedby centrifugationandfrozenuntilanalyzed.Urinesampleswerecollecteduringthetimeintervals0-2,2-4,4-6,6-8, 8-12,and12-24h,andrepresentativesampleswerefrozenuntilanalyzed.ResultsLinearity-Calibrationcurves,determinedwith fivedif-ferentconcentrationsof erythromycinbasein serumandurine,wereobtainedbyplottingtheratioofthepeakheight~Ibl (al!!j~Figure 1-(a) Chromatogramofblankserumextract.(b)Chromatogramofanextractofserumcontainingoleandomycin(peak1)anderythromy-cin (peak2).~(bl (aluw~u~~Figure 2-(a) Chromatogramof blankurineextract.(b)Chromatogramof anextractof urinecontainingoleandomycin(peak1)anderythromy-cin (peak2).oferythromycinbasetothatoftheinternalstandardversustheir respectiveconcentrations,and werelinearovertheconcentrationranges tudied.Thecalibrationcurveinserum(0.25-5.00p,g/mL)hada slopeof0.2938anday interceptof0.0310,witha correlationcoefficientof0.9997,whereasthecurveinurine(5.00-50.00p,g/mL)hadaslopeof0.0686anday interceptof0.0127,withacorrelationcoefficientof0.9997.Precisionand Accuracy-Within-runprecisionwasas-sessedby extractingsix spikedserumandurinesampleseachat the upperand lower limits of the concentrationranges tudied.Therelativestandard eviationswerefoundtobe6.12%at0.50p,g/mLand2.10%at2.00p,g/mLforserum.and4.2%at5.00p,g/mLand1.40%at50.00tLg/mLforurine.Spikedserumsampleswerefoundtobestableforatleast1monthandurinesamplesfor2weekswhenstoredfrozenat-lOoC. Extractedurinesampleswerefoundtobestableat4°Cfor3dayswhenleftontheextractioncolumnsandfor1weekwhenelutedandstoreddryat4°Cpriortoreconstitu-tion.ExtractionEfficiency-Spikedserumandurinesampleswereassayedin triplicateat fourdifferentconcentrations.All sampleswereextractedaspreviouslydescribed,exceptthattheinternalstandardwasincorporatedintothe25-p,Lacetonitrilealiquotusedin thefinalreconstitutionandphaseseparationstep.Themeanrecoveryvaluesof81%and80%thusobtainedforserumandurine,respectively,donottakeintoaccounttheverysmallpercentageofdrugremaininginthe aqueouslayer.Partitionstudiesof the distributionoferythromycinbetweenthetwophasesobtainedonreconsti-tution indicatedthat approximately10%of the drugre-mainedin the aqueouslayer.The overallrecoveryvaluesmaythusbeadjustedby 10%.SensitivityandDetectionLimit-Under theconditionsofthis assayandbasedon a signal-to-noiseratio of 3, thedetectionlimit for erythromycinbasewas 0.25p,g/mLinserumand1 p,g/mLin urine.Thedetectionlimit in serummaybeincreasedbyusinga largerinjectionvolumeof8-10p,Lorby usinga detectorwith a 0.005AUFS setting.Thiswasachievedin ourlaboratorywiththeaidofanamplifier-filter(model1021A;SpectrumScientificCorp.),whichdou-bledthedetectoroutput.Serum and Urine Profiles-The serumconcentration-timeprofileandfractionalurinaryexcretionplotfromtheclinicalstudyaredepictedin Figs.3 and4,respectively.DiscussionThe methoddescribedinvolvesa rapid, accurate,andhighlyreproduciblesystemforthedeterminationoferythro-mycinin serumandurineaftera single500-mgoraldose.Duringthe initial stagesof selectionof a suitablemobile:::J6EC;,'=5z0;::.<t0::~3uz82:1:::>~1V14 6HOURS10Figure 3-Erythromycin serumconcentration-timeprofilein a humanvolunteerafter the administrationof a single 500-mgerythromycinstearatetablet.Journal of Pharmaceutical Sciences / 1127Vol.74,No. 10,October198512;;'10.§:iJs~6xOJ106HOURS12Figure 4-Fractionalurinaryexcretionplot of erythromycinafter theadministrationof a single500-mgdose of erythromycinstearateto ahumanvolunteer.phasetoseparaterythromycinbyreversed-phaseHPLC, itwasestablishedthat the drugdid not elutefromthe C18columnwhena buffer-freemobilephasewasused.Hence,whenasolid-phasextractioncolumnwasused,erythromy-cin couldbe selectivelyseparatedandretainedwhile un-wantedcomponentswerewashedoffwithwaterandacetoni-trile:water,andthenthedrugwaselutedfromtheextractioncolumnwith an acetonitrile:buffermixture.Utilizationofthis solid-phasextractiontechniquein the conventionalmanneras describedin previousreportsfor variouscom-pounds8.9resultsin reasonablycleanextractsandallowstheir determinationandquantitation,providedthat highsensitivityisnotrequired.In thecaseoferythromycin,whichhasa relativelylowUV molarabsorptivity,furthersampleconcentrationwasnecessary.Thiswasaccomplishedbyeva-poratingtheextractioncolumneluate,addinga 20-p.Lali-quotofwatertodissolvethedrugandresidualbuffersalts,and thenselectivelyconcentratingthe erythromycinandinternalstandardintoa 25-p.Lacetonitrilelayer.This layercouldberemovedforinjectionasa resultofsuccessfulphaseseparationduringthisfinalstep.Theresultingcleansampleextractenabledthesuccessfulutilizationof a conventionalUV detectorsetat200nm.Themanyinterferencesnormallyseenwhenmonitoringbiologicalextractsatsuchlowwave-lengthswereovercome,andselectivitywasenhancedas aresultofthisphase-separationstep.Injectionoftheaqueouslayer of reconstitutedextractsindicatedextremelyhighlevelsof interference,in contrasto theacetonitrilelayer,whichallowedtheunhinderedquantitationof erythromy-1128 I Journal of Pharmaceutical SciencesVol. 74, No. 10, October 1985cin.Retentiontimesunderthechromatographicconditionsofthis assaywereas follows:erythromycinbase,6.2 min;oleandomycinphosphate,4.2min;de-N-methylmetaboliteoferythromycin,4.5min;erythromycinB,8.2min;anhydroer-ythromycin,11.0min;erythromycinenolether,22.4min;erythralosamine,13.0min.Despitesimilarretentiontimesobtainedforbotholeandomycinandthede-N-methylmetab-olite of erythromycin,serumandurine samplesobtainedfromtheclinicalstudyandprocessedwithouttheadditionoftheinternalstandardoleandomycinshowednoevidenceofthede-N-methylmetaboliteoferythromycin.In viewofthelowUV molarabsorptivityoferythromycinat conventionallyuseddetectionwavelengths(>220nm),HPLC withUV detectionhasnotbeenconsideredfeasibletodate.Wavelengthsbelow220nm,andespeciallyat200nm,wherethe molarabsorptivityis considerablygreater,areassociatedwith additionalproblems,suchas interferingspeciesco-extractedfrombiologicalsamples.Our methodovercomestheseproblemsasaresultofsystemoptimizationandthesuccessfulapplicationofahighlyspecific,solid-phaseextractionprocedure,followedbya simplephaseseparationsteptoquantitativelydeterminerythromycini serumandurine.References and Notes1. McGuire, J. M.; Burch, R. L.; Anderson, R. C.; Baaz, H. E.;Flynn, H. E.; Powell, H. M.; Smith, J. W. Antibiot. Cherrwther.1952,2, 281.2. Stephens,V. C.; Pugh, C. T.; Davis, N. E.; Hoehn, M. M.; Ral-ston,S.;Sparks,M. C.; Thompkins,L. J. Antibiot. 1969,22,551-557.3. Easterbrook,S. M.; Hersey,J. A. J. Chromatogr.1976,121,390-394.4. Tsuji, K. J. Chromatogr.1978,158,337-348.5. Chen, M.-L.; Chiou, W. L. J. Chromatogr.1983,278,91-100.6. Tsuji, K.; Roberts~:m,J. Anal. Chem.1971,43,818-821.7. Duthu, G. S. J. Llq. Chromatogr.1984,7, 1023-1032.8. Good,T. J.; Andrews, J. S. J. Chromatogr.Sci. 1981,19, 562-566.9. Goto,J.; Goto,N.; Nambara,T. J. Chromatogr.1982,239,559-564.AcknowledgmentsThe authors gratefully acknowledgefinancial support from theSouthAfrican Council for ScientificandIndustrial ResearchandtheSouthAfrican Medical ResearchCouncil.

Determination of erythromycin in serum and urine by high performance liquid chromatography with ultraviolet detection

Rhodes Repository (SEALS)

Reprinted from the Journal of Pharmaceutical Sciences.Vol. 74, No. 10, October 1985. @ 1985 by the American Pharmaceutical Association.Determination of Erythromycin in Serum and Urine by High-Performance Liquid Chromatography with UltravioletDetectionC. STUBBS, J. M. HAIGH, AND I. KANFERxReceived September 13,1984, from the School of Pharmaceutical Sciences, Rhodes University, Grahamstown 6140, South Africa.July 3, 1985.Abstract D A high-performance liquid chromatographic analysis oferythromycin in human serum and urine with UV detection at 200 nm ispresented. The method involves a solid-phase extraction procedurefollowed by a simple phase separation step and chromatography on areversed-phase column. The method has sensitivity limits of 0.25 and1.0 JLg/mLin serum and urine, respectively, and is sufficiently sensitiveto monitor concentrations of erythromycin in human serum and urineafter the administration of a single 500-mg erythromycin stearate tablet.Erythromycin, a macrolide antibiotic used mainly againstgram-positive bacteria, has been in clinical use since 1952.1Previous pharmacokinetic data published on this antibiotichave been derived predominantly from microbiological assaytechniques. These techniques, however, are relatively impre-cise as well as nonspecific and tedious to perform.In 1969, Stephens et al,2 used a two-step paper chromato-graphic-bioautographic technique to determine erythromy-cin in blood, whereas Easterbrook and Hersey3 described aTLC technique also used in conjunction with bioautography.These procedures, although enabling the determination offree base from the parent ester form of the drug, suffer fromthe same disadvantages associated with microbiologicalmethods.Tsuji4 described a fluorometric determination of erythro-mycin, including erythromycin ethyl succinate and somemajor metabolites and degradation products, in serum byhigh-performance liquid chromatography (HPLC) and on-stream post-column derivatization and extraction. Althoughthis method appears to be highly sensitive, it suffers from thedisadvantage of requiring complex and expensive equipment.In addition, erythromycin is not well resolved from itsmetabolites and degradation products present in serum.A recent paper by Chen and Chiou5 described the HPLCanalysis of erythromycin in biological fluids by electrochemi-cal detection. A novel dual-electrode electrochemical detec-tor, operated in the oxidative screen mode, was used. Thisprocedure has the main advantage of high sensitivity, allow-ing the use of extremely small samples. To reduce back-ground currents and enhance detector stability, however, itis necessary to maintain the HPLC system in continuousoperation, even when it is not in use. Furthermore, theirchoice of erythromycin B as an internal standard may lead toanomalies during pharmacokinetic studies, since variousamounts of this compound are usually present in batches ofraw material.6 A further recent report by Duthu,7 who usedthe same detector system as Chen and Chiou, described theanalysis of erythromycin in serum only, under differentchromatographic conditions.The objective of the present study was to develop a sensi-tive, reproducible, and rapid method to quantitatively deter-mine erythromycin in serum and urine by HPLC with UVdetection by employing a conventional variable-wavelengthUV detector. The success of this method was largely due to1126 / Journal of Pharmaceutical SciencesVol. 74, No. 10, October 1985Acceptedthe efficiency of the solid-phase extraction procedure em-ployed, which resulted in a clean sample extract. This en-abled the utilization of a detection wavelength of 200 nm.Experimental SectionApparatus-The modular high-performance liquid chromato-graph consisted of an M 6000A constant flow pump and a Wisp nOBautomated sample injector (Waters Associates), a variable-wave-length UV detector (model 769; Kratos Analytical Instruments), and'a strip chart recorder (model 561; Hitachi, Japan). The mobile phasewas constantly deaerated with an in-line vacuum de aerating unit(model ERC-3510; Erma Optical Works, Japan). The separation wasperformed on a 15 cm x 3.9 mm Ld. column containing microparticu-late bonded 5-JLm octadecylsilane (C1s) material (Novapak; WatersAssociates). The temperature of the column was maintained at 35°Cwith the aid of a model LC-22 temperature controller (BioanalyticalSystems).Reagents and Materials-Reagents were of at least analyticalgrade. UV-grade acetonitrile was obtained from Burdick and Jack-son Laboratories. Erythromycin base and oleandomycin phosphatewere obtained from Abbott Laboratories and Pfizer Laboratories,respectively. Erythromycin B, the anhydro metabolite of erythromy-cin, erythralosamine, and erythromycin enol ether were obtainedfrom Abbott Laboratories, and the de-N-methyl metabolite of eryth-romycin was obtained from Lilly Research Laboratories. The HPLC-grade water used in the mobile phase was purified through a Milli-Qsystem (Millipore Corp.).Chromatographic Conditions-The mobile phase was preparedby mixing acetonitrile (300 mL) with 0.05 M phosphate buffer (700mL). The phosphate buffer was prepared by adding 3.2 mL ofphosphoric acid to 1 L of water. Sodium hydroxide was then used toadjust the solution to pH 6.30. The solvent mixture (pH 7.00) wasdeaerated and filtered through a 0.6-JLm filter (type BD; MilliporeCorp.).This mobile phase was used at a flow rate of 1.0 mLimin for theanalysis of both serum and urine samples, with a resulting pressureof 100 bar (1 x 104 kPa). The wavelength of detection was 200 nm,with a detector sensitivity of 0.01 AUFS and a time constant of 2 s.Extraction of Serum and Urine-Samples of serum (2 mL) orurine (1 mL) were mixed with 0.25 or 0.5 mL, respectively, of a 12-JLg/mL aqueous oleandomycin phosphate internal standard solution.Addition of 1 mL of acetonitrile, followed by vortex mixing for 1 minand centrifugation for 5 min at 1600xg, resulted in deproteinizationof the biological samples. The supernatant was transferred to aculture tube containing 8 mL of water for direct loading onto aprewashed, 1-mL, disposable octadecyl extraction column (J. T.Baker Chemical Co.) which had been prewashed under 10-15mmlHg vacuum (Baker 10 Extraction System coupled to a model A2-S Aspirator; Rikakikai Co., Japan) by successively passing 3 mL ofacetonitrile and 3 mL of water through each. The diluted sample wasthen added to the extraction column with the aid of 15-mL samplereservoir (J. T. Baker Chemical Co.). The extraction column waswashed with 5 mL of water and then with 5 mL of acetonitrile:water(1:1), with the vacuum maintained until dry. Erythromycin and theinternal standard were eluted into 2-mL tapered collection tubeswith two successive 500-JLL aliquots of acetonitrile:0.05 M phosphatebuffer (1:1). The sample was then dried under vacuum in a rotaryvacuum centrifuge (Speed Vac Concentrator; Savant Instruments).The residue in the collection tube was reconstituted in 20 JLLof water0022-3549/85/1000-1126$01.00/0@ 1985, American Pharmaceutical Associationand vortexed for 1 min. When 25 tLLof acetonitrile was added, twolayers formed due to the high concentration of salts in the aqueouslayer. This mixture was vortexed for 1 min and then centrifuged for 1min at 1600xg. An aliquot (15-20 tLL)of the acetonitrile phase wasthen transferred to a WISP limited volume insert (Waters Asso-ciates) with a microsyringe. A 5-tLI aliquot of this sample wasinjected onto the column. Relevant chromatograms are depicted inFigs. 1 and 2.Clinical Study-A pilot trial in which one volunteer was used wasconducted to check the effectiveness of the analytical method tomeasure concentrations of erythromycin base and any possiblemetabolites or degradation products in serum and urine after theoral administration of a commercially available erythromycin stea-rate formulation (Erythrocin 500; Abbott Laboratories). The subjectreceived one film-coated tablet containing 500 mg of erythromycinstearate after an overnight fast, accompanied by 250 mL of waterand immediately followed by a light breakfast. Blood samples weredrawn at 0, 0.25, 0.5, 0.75,1,2,3,4,6,8,10, and 12h after ingestionof the medication. The serum was separated by centrifugation andfrozen until analyzed. Urine samples were collected during the timeintervals 0-2, 2-4, 4-6, 6-8, 8-12, and 12-24 h, and representativesamples were frozen until analyzed.ResultsLinearity-Calibration curves, determined with five dif-ferent concentrations of erythromycin base in serum andurine, were obtained by plotting the ratio of the peak height~Ibl (al!!j~Figure 1-(a) Chromatogram of blank serum extract. (b) Chromatogramof an extract of serum containing oleandomycin (peak 1)and erythromy-cin (peak 2).~(bl (aluw~u~~Figure 2-(a) Chromatogram of blank urine extract. (b) Chromatogramof an extract of urine containing oleandomycin (peak 1) and erythromy-cin (peak 2).of erythromycin base to that of the internal standard versustheir respective concentrations, and were linear over theconcentration ranges studied. The calibration curve in serum(0.25-5.00 p,g/mL)had a slope of 0.2938 and a y intercept of0.0310, with a correlation coefficient of 0.9997, whereas thecurvein urine (5.00-50.00p,g/mL)had a slopeof0.0686and ay intercept of 0.0127, with a correlation coefficient of 0.9997.Precision and Accuracy-Within-run precision was as-sessed by extracting six spiked serum and urine sampleseach at the upper and lower limits of the concentrationranges studied. The relative standard deviations were foundto be 6.12% at 0.50 p,g/mLand 2.10% at 2.00 p,g/mLfor serum.and 4.2%at 5.00p,g/mLand 1.40%at 50.00tLg/mLfor urine.Spiked serum samples were found to be stable for at least 1month and urine samples for 2 weeks when stored frozen at-lOoC. Extracted urine samples were found to be stable at4°C for 3 days when left on the extraction columns and for 1week when eluted and stored dry at 4°C prior to reconstitu-tion.Extraction Efficiency-Spiked serum and urine sampleswere assayed in triplicate at four different concentrations.All samples were extracted as previously described, exceptthat the internal standard was incorporated into the 25-p,Lacetonitrile aliquot used in the final reconstitution and phaseseparation step. The mean recovery values of 81% and 80%thus obtained for serum and urine, respectively, do not takeinto account the very small percentage of drug remaining inthe aqueous layer. Partition studies of the distribution oferythromycin between the two phases obtained on reconsti-tution indicated that approximately 10% of the drug re-mained in the aqueous layer. The overall recovery valuesmay thus be adjusted by 10%.Sensitivity and Detection Limit-Under the conditions ofthis assay and based on a signal-to-noise ratio of 3, thedetection limit for erythromycin base was 0.25 p,g/mL inserum and 1 p,g/mL in urine. The detection limit in serummay be increased by using a larger injection volume of 8-10p,L or by using a detector with a 0.005 AUFS setting. Thiswas achieved in our laboratory with the aid of an amplifier-filter (model 1021A; Spectrum Scientific Corp.), which dou-bled the detector output.Serum and Urine Profiles-The serum concentration-time profile and fractional urinary excretion plot from theclinical study are depicted in Figs. 3 and 4, respectively.DiscussionThe method described involves a rapid, accurate, andhighly reproducible system for the determination of erythro-mycin in serum and urine after a single 500-mg oral dose.During the initial stages of selection of a suitable mobile:::J6EC;,'=5z0;::.<t0::~3uz82:1:::>~1V14 6HOURS10Figure 3-Erythromycin serum concentration-time profile in a humanvolunteer after the administration of a single 500-mg erythromycinstearate tablet.Journal of Pharmaceutical Sciences / 1127Vol. 74, No. 10, October 198512;;'10.§:iJs~6xOJ106HOURS12Figure 4-Fractional urinary excretion plot of erythromycin after theadministration of a single 500-mg dose of erythromycin stearate to ahuman volunteer.phase to separate erythromycin by reversed-phase HPLC, itwas established that the drug did not elute from the C18column when a buffer-free mobile phase was used. Hence,when a solid-phase extraction column was used, erythromy-cin could be selectively separated and retained while un-wanted components were washed offwith water and acetoni-trile:water, and then the drug was eluted from the extractioncolumn with an acetonitrile:buffer mixture. Utilization ofthis solid-phase extraction technique in the conventionalmanner as described in previous reports for various com-pounds8.9 results in reasonably clean extracts and allowstheir determination and quantitation, provided that highsensitivity is not required. In the case of erythromycin, whichhas a relatively low UV molar absorptivity, further sampleconcentration was necessary. This was accomplished by eva-porating the extraction column eluate, adding a 20-p.L ali-quot of water to dissolve the drug and residual buffer salts,and then selectively concentrating the erythromycin andinternal standard into a 25-p.Lacetonitrile layer. This layercould be removed for injection as a result of successful phaseseparation during this final step. The resulting clean sampleextract enabled the successful utilization of a conventionalUV detector set at 200 nm. The many interferences normallyseen when monitoring biological extracts at such low wave-lengths were overcome, and selectivity was enhanced as aresult of this phase-separation step. Injection of the aqueouslayer of reconstituted extracts indicated extremely highlevels of interference, in contrast to the acetonitrile layer,which allowed the unhindered quantitation of erythromy-1128 I Journal of Pharmaceutical SciencesVol. 74, No. 10, October 1985cin. Retention times under the chromatographic conditions ofthis assay were as follows: erythromycin base, 6.2 min;oleandomycin phosphate, 4.2 min; de-N-methyl metabolite oferythromycin, 4.5 min; erythromycin B, 8.2 min; anhydroer-ythromycin, 11.0 min; erythromycin enol ether, 22.4 min;erythralosamine, 13.0 min. Despite similar retention timesobtained for both oleandomycin and the de-N-methyl metab-olite of erythromycin, serum and urine samples obtainedfrom the clinical study and processed without the addition ofthe internal standard oleandomycin showed no evidence ofthe de-N-methyl metabolite of erythromycin.In view of the low UV molar absorptivity of erythromycinat conventionally used detection wavelengths (>220 nm),HPLC with UV detection has not been considered feasible todate. Wavelengths below 220 nm, and especially at 200 nm,where the molar absorptivity is considerably greater, areassociated with additional problems, such as interferingspecies co-extracted from biological samples. Our methodovercomes these problems as a result of system optimizationand the successful application of a highly specific,solid-phaseextraction procedure, followed by a simple phase separationstep to quantitatively determine erythromycin in serum andurine.References and Notes1. McGuire, J. M.; Burch, R. L.; Anderson, R. C.; Baaz, H. E.;Flynn, H. E.; Powell, H. M.; Smith, J. W. Antibiot. Cherrwther.1952,2, 281.2. Stephens, V. C.; Pugh, C. T.; Davis, N. E.; Hoehn, M. M.; Ral-ston, S.; Sparks, M. C.; Thompkins, L. J. Antibiot. 1969,22,551-557.3. Easterbrook, S. M.; Hersey, J. A. J. Chromatogr. 1976,121,390-394.4. Tsuji, K. J. Chromatogr. 1978, 158, 337-348.5. Chen, M.-L.; Chiou, W. L. J. Chromatogr. 1983,278, 91-100.6. Tsuji, K.; Roberts~:m,J. Anal. Chem. 1971,43, 818-821.7. Duthu, G. S. J. Llq. Chromatogr. 1984, 7, 1023-1032.8. Good, T. J.; Andrews, J. S. J. Chromatogr. Sci. 1981, 19, 562-566.9. Goto, J.; Goto, N.; Nambara, T. J. Chromatogr. 1982,239, 559-564.AcknowledgmentsThe authors gratefully acknowledge financial support from theSouth African Council for Scientific and Industrial Research and theSouth African Medical Research Council.

http://vital.seals.ac.za:8080/vital/access/services/Download/vital:6428/SOURCEPDF

Determination of erythromycin in serum and urine by high performance liquid chromatography with ultraviolet detection

Abstract

Similar works

Full text

Available Versions

South East Academic Libraries System (SEALS)

Rhodes Repository (SEALS)