MPPA103

Unit 3 General methods of estimation, analysis for steroids

Trainer: Chandramouli R

PharmaceuticalScope

steroidsandtheirsemisyntheticanalogsfalls intoanumberofdifferentareassuchas:

Determinationofsteroidhormones,the intermediatesoftheirbiosynthesisandtheir metabolites,andthesemisyntheticanalogs andtheirmetabolitesinbiologicalsamples. Thisisofgreatimportanceinclinical biochemistry,diagnostics,therapeuticdrug monitoring

Structureelucidationofsteroidhormone analogs(productsandintermediatesof syntheticdrugresearch,impurities,degradation products,metabolites). Assayofbulkhormonedrugsandimpurity determination. Assayofhormoneformulations,including stabilityassays.

Types...

Steroidhormones: sexhormones

Androgenstestosterone),estrogensestradiol, Gestogensprogestogens,progestinscorpus luteumprogesterone Secretedbyadrenalcortexcortisol

corticosteroidhormones

Egs...

Syntheticanalogs

replacedtoalargeextentbysyntheticor semisyntheticderivativesthatpossesshigher andmoreselectivetherapeuticpotential

MethodsUsedintheAnalysisof Steroids

SpectroscopicMethods

Ultravioletvisiblespectrophotometry

presenceofeitheranunsaturatedoxogroupora phenolicringintheirmolecules,themajorityof steroidhormonesabsorbstronglyintheUV region UsedinPharmacopoeialassayofformulations

UVspectraldataofsomeimportantgroupsofsteroid hormonesandtheirsyntheticanalogs

IR

Classicaltool characterizationofpolymorphicmodifications SamplesprepedbyKbrorNujolMulling

characteristicIRbandsofsteroids

Fluorimetry

estrogens,havingaphenolicring,possess naturalfluorescence(excitation~284nm, emissionintherange310327nm). Theintensityofthisrelativelyweak fluorescenceissufficientforthedetermination ofestrogensincontraceptives LCassayusingfluorescencedetector

Nuclearmagneticresonance(NMR) spectroscopy

themostpowerfultechniqueinstrlelucidation
ApplicationofNOEovercomingthedrawbacksof1DNMR 1Hand13CNMRsignals1H1HCOSY,1H13CCOSY (HETCOR),andvariouslongrangecorrelationtechniques applicationofmodern2Dand3Dtechniquesemploying pulsedfieldgradientsandinversedetectionenablesthe fullassignmentofspectraandthusaidsrapidstructure elucidation. SomeofthesetechniquesaregDQFCOSY,gHMQC,g HMBC,gHSQCTOCSY,andDPFGSENOE.

NMRspectraofnorgestrel.Solvent/reference: CDCl3/TMS.(A)1HNMRspectrum;(B)13CNMR spectrum.

ChromatographicMethods

Thinlayerchromatography

Importanttechnique Theapolarskeletonsandpolarsubstituentsof steroidsgoodmodelsforseparationstudies 4ene3oxoorphenylgroupstronglyabsorb UVradiationdetectionatthenglevel Rapidcharacterizationofsteroidmixturesand checkingofthepurityofbulksteroid hormones

liquidchromatography

carriedoutatambienttemperature,andforthisreasonitis applicablequitegenerallyandwithoutderivatizationor hydrolysis,eveninthosecaseswherethermalinstabilityor lowvolatilityprecludesthedirectapplicationofGC (separationandquantificationofcorticosteroids, conjugates,etc.). AlthoughtheresolvingpowerofLCisgenerallylowerthan thatofcapillaryGC,thegreaterversatilityofLCmaybe exploitedsowellthatsuitablecombinationsofmobileand stationaryphasescanbefoundfortheseparationof practicallyallsteroids

LCenablesevenminorsteroidcomponentsto beobtainedonthesemipreparativeor preparativescale.Thisfacilitatesthe identificationofmetabolites,impurities,and degradationproductsbysubsequent spectroscopicstudies. Althoughcouplingthemassspectrometerwith thehighperformanceliquidchromatograph involvesgreatertechnicalproblemsthanisthe casewithGCMS,whichismorepopularin steroidhormoneanalysis,LCMShasalready beensuccessfullyintroducedintosteroid profiling.

ImmunoassayMethods

Steroidsarenotimmunogenic,butashaptens covalentlyboundtoasuitableproteintheycan beusedtoraiseantisera Antibodiesbindanysteroid,whoseprotein complexhasbeenusedforimmunizationofthe animalsproducingtheantiserum. Thecompetitionbetweenthesteroidtobe determinedandasuitablylabeledanalogforms thebasisofimmunoassay

Inmajorityofcasespolyclonalantibodiesare used. ofmonoclonalantibodieswelldefinedspecificity. Dependingonthemanneroflabeling,various kindsofimmunoassays

RIA, enzymeimmunoassay, Fluorescenceimmunoassay, fluorescencepolarizationimmunoassay, luminescenceimmunoassay.