Prevalidation and comparison of methods for determination of nitrite according to Griess

U okviru ovog diplomskog rada provedena je prosudba validacijskih značajki i kontrola kakvoće spektrofotometrijske (SFM1 i SFM2) i mikroanalitičke metode (NO2-Mikro) za određivanje nitrita prema Griessu. Određivanje nitrita po Griessu je postupak koji se temelji na pretvorbi sulfanilne kiseline u diazonijevu sol u prisutnosti nitrita u kiselom mediju, koja zatim reagira s aromatičnim aminom N-(1-naftil)etilendiamin dihidrokloridom, tvoreći crveno-ljubičastu azo boju, koju možemo kvantitativno odrediti mjereći njezinu apsorbanciju na 540 nm. Prosudba i kontrola valjanosti predloženih spektrofotometrijskih postupaka provedena je primjenom cjelovite prevalidacijske strategije koja je uključivala karakterizaciju skupina od 1 do 6, provjeru graničnih skupina 1 i 6, ispitivanje homogenosti, ispitivanje odnosa analit-signal, prepoznavanje bjegunaca i prosudbu graničnih vrijednosti. Izvedbene karakteristike NO2-Mikro postupka pokazale su idealnu linearnu kalibracijsku ( Sˆ = 0,027x), idealnu analitičku evaluacijsku funkciju ( xˆ = 36,7S), vrlo nisku granicu određivanja (LQ = 1,42 μM), značajnu preciznost ( rx s ˆ od ±1,95% do ±13,55%) i točnost ( x od -1,22% do +5,78%). Procijenjena kakvoća spektrofotometrijskih postupaka NO2-SFM1 i NO2-SFM2 je nešto niža. Metoda NO2-SFM1 karakterizirana je nižom preciznošću ( rx s ˆ od ±1,02% do ±22,67%) i točnošću ( x od -5,47% do +13,88%), te lošijom granicom određivanja (LQ = 18,05 μM), dok je metoda NO2-SFM2 također karakterizirana niskom preciznošću ( rx s ˆ od ±2,26% do ±23,58%) i točnošću ( x od -3,04% do +29,2%), ali povoljnijom granicom određivanja (LQ = 6,58 μM) od NO2-SFM1 postupka. Usporedba postupaka je pokazala da se NO2-Mikro postupak odlikuje većom osjetljivošću i povoljnijim analitičkim značajkama s obzirom na spektrofotometrijski postupak. Jednako tako, provedena prevalidacija ukazala je na nedostatke postupaka kao što je nehomogenost slijepih vrijednosti koji utječu na cjelokupnu kakvoću postupaka.In this work, the evaluation of validation characteristics and the quality control of spectrophotometric and microanalytical procedures for the determination of nitrite using the Griess reagent was carried out. This procedure is based on the conversion of sulfanilic acid to a diazonium salt by reaction with nitrite in acid solution. The diazonium salt is then coupled to the aromatic amine N-(1-naphthyl)ethylenediamine, forming a red-violet coloured azo dye that can be spectrophotometrically quantitated based on its absorbance at 540 nm. Quality control and evaluation of the analytical parameters was carried out using a comprehensive prevalidation strategy which included characterization of the groups from 1 to 6, checking of limiting groups, homogeneity testing, the quality of analyte-signal relationship, outlier recognition, and evaluation of limiting values. Performance characteristics of the NO2-Mikro procedure showed an ideal linear calibration ( Sˆ = 0,027x), ideal analytical evaluation function ( xˆ = 36,7S), very low limit of quantitation (LQ = 1,42 μM), significant precision ( rx s ˆ from ±1,95% to ±13,55%) and accuracy ( x from -1,22% to +5,78%). The quality of spectrophotometric procedures NO2-SFM1 and NO2-SFM2 is somewhat lower. The method NO2-SFM1 is characterized with lower precision ( rx s ˆ from ±1,02% to ±22,67%) and accuracy ( x from -5,47% to +13,88%), and higher limit of quantitation (LQ = 18,05 μM); the method NO2-SFM2 is characterized with lower precision ( rx s ˆ from ±2,26% to ±23,58%) and accuracy ( x from -3,04% to +29,2%), but lower limit of quantitation (LQ = 6,58 μM) than NO2-SFM1 procedure. The comparison of the procedures indicated that the NO2-Mikro procedure showed higher sensitivity and greater reliability than the spectrophotometric procedure. Moreover, complete prevalidation pointed out disadvantages of the procedures such as inhomogenity of blank measurements which influence the quality of the procedures