Peroxyl radical

http://www.ncbi.nlm.nih.gov/pubmed/16843819
autosampler
http://www.labmanager.com/product-focus/2010/10/hplc-autosamplersachieving-speed-and-reduced-sample-volumes?fw1pk=2#.VyGdHTbrzOw
methods of validation
http://www.labcompliance.com/tutorial/methods/default.aspx
function of alpha tocopherol
http://healthyeating.sfgate.com/primary-role-alphatocopherol-9516.html
reverse-phase HPLC
https://www.youtube.com/watch?v=MLoitPJQH3g

Selectivity:Toensurenointerferencethereforeblankandspikedliversampleswereextracted
andinjected

Theinjectionsrevealednopeakinterferencewithanyofthetocopherolsandtocotrienols.
AlthoughthealphatocopherolhasawidespreadpresenceinanimaldietssoavitaminEfree
liverwasnotfound,whichresultedinminoralphatocopherolpeaksintheblanksamples.
Linearity:Thestandardsolutionsoftocopherolsandtocotrienolsweretestedforlinearityover8
consecutivedayswith9concentrations(0.025mol/L,0.0625mol/L,0.125mol/L,0.625
mol/L,1.25mol/L,6.25mol/L,12.5mol/L,25mol/L)
ThedetectorresponsesfortheTandT3standardsofratliverwerelinearwithastandard
deviationr^2>0.99
LimitofDetectionandQuantification:LODistheamountoftherespectiveanalyteinjectedinto
thesystemthatcouldbereliabledespitethebackgroundnoise.LLOQandULOQarethelowest
andhighestamountsofanalytesthatcanbequantifiedwiththerequiredaccuracyandprecision
LODwasgenerallyhigherforalphatocopherolbecauseoftheirlowerfluorescent
intensity.DetectorresponseforLODwaslinearwitharangefromlowpicogramuptolow
nanogramamounts.
Stability:Stocksolutionstabilitycomparedfromthedetectorresponsewiththesamesolution
storedatRTfor24hours.Shorttermstabilitieswereevaluatedintheliversamplesspikedwith
0.625mol/Land6.25mol/LafterstorageatRT.Spikedsampleswerecomparedwithsamples
thatwerestoredat80Cfor24hoursandthenthawedout(3times).Postprepsamplesof
samplesspikedat3.125mol/Lweretestedoutofthesamevialat0,6,12,18,and24hours.
StocksolutionsatRTfor24hoursremainedstablewithlessthan5%degradation.The
shorttermstabilitiesspikedwithlowandhighconcentrationstoratliverandstoredatRTfor24
hourswereexceptionalwithlessthan7.5%max.degradation.Thefreezeandthawcycleswere
lessresistanttotherepetitionoffreezingandthawingandhadlossesof1025%.Itwas
suggestedthatthiswasalimitationandthereforeshouldbereducedtofreezingandthawingonce
ratherthanthreetimes.ThepostpreparativestabilityremainedstablewhenthevitaminEwas
extractedfromtheliverandthesampleswerestoredinachilledautosampler.
Recovery:analyteswerequantifiedby5ratliversamples,spikedwithTandT3atlow,medium,
andhighconcentration(0.625,3.125,6.250mol/L)andcomparedtodetectorresponseswith
thesameamountcontainedinstandards
Intradayrecoveries,sorecoveriesdonewithina24hourtimeframe,weredetectedtobe
withinlimitssetbytheFDA.Thealphatocopherolexceededthelimitsatthelowandmedium
concentrationsby23%.Interdayrecoveries,sorecoveriesdoneovera24hourtimeframe,were
alsowithinthelimits.
AccuracyandPrecision:Determinedwithina24hourtimeframeof5ratliversamplesspiked
withlow,medium,andhightocopherolsandtocotrienolsand9standardmixtureswith
concentrationsrangingfrom0.025to25mol/L.Concentrationsoftocopherolsandtocotrienols
werecomparedtosampleconcentrations.Accuracyandprecisionweredeterminedwithlow,
medium,andhighconcentrationsover3consecutivedays,aswell.

IntradayaccuracieswerewithintheFDAlimits,whichisgood.Interdayaccuracieswere
muchbetterthough.Intradayprecisionwasexceptionallygoodforthemajorityofanalytes,
althoughthealphatocopherolexceededtheFDAlimits.Interdayprecisionwasexcellentandmet
alllimits.