Reproducibility of the Selected Methods for Determination of Phenol in Urine by High Performance Liquid Chromatography

Određivanje fenola u urinu smatra se prikladnim biomarkerom izloženosti benzenu, koji je klasificiran u grupu A kancerogena od strane Međunarodne agencije za istraživanje raka (IARC) i Agencije za zaštitu okoliša (EPA). U ovom radu testirana je ponovljivost nekoliko metoda za analizu fenola u urinu primjenom tekućinske kromatografije visoke djelotvornosti. Publicirane metode uglavnom uključuju dodatnu opremu koja nije standardna (npr. fluorescentni detektor) ili dodatne postupke u pripremi uzoraka kao što su parna destilacija ili derivatizacija, što produljuje vrijeme potrebno za pripremu uzoraka ili se povećava mogućnost gubitka dijela sadržaja analita. Odabrana je metoda koja se pokazala reproducibilnom uz određene modifikacije. Izmjena metode uključivala je promjenu valne duljine, sastava i protoka mobilne faze, te je isključilo primjenu internog standarda u proceduri pripreme uzoraka urina za analizu. Kao mobilna faza upotrijebljena je smjesa destilirane vode/metanola/acetatne kiselina (65 : 34 : 1 v/v %), a upotrijebljena je kolona Selectra C18 (4.6 × 250 mm, 5 µm). Uvjeti kromatografiranja bili su protok 1 ml min–1, temperatura kolone 25 °C, valna duljina 218 i 270 nm i volumen injektiranja 20 µl. Pri valnoj duljini 270 nm metoda je pokazala linearnost u koncentracijskom opsegu 1 – 30 mg l–1 (y = 10290x + 15138; R2 = 0,9932), a iskorištenost metode kreće se u rasponu od 73,8 do 111,2 %. Ovo djelo je dano na korištenje pod licencom Creative Commons Imenovanje 4.0 međunarodna.Determination of phenol in urine is considered as a suitable biomarker of benzene exposure, which is classified as Group A carcinogen by the International Agency for Cancer Research (IARC) and the Environmental Protection Agency (EPA). The aim of this work was to test reproducibility of several published high performance liquid chromatography (HPLC) methods for phenol determination in urine samples. Previous methods mostly included additional equipment (e.g. fluorescence detector) or an additional step in a sample preparation protocol (e.g. derivatization or steam distillation) which is time consuming and can also cause the loss of analyte. The method that was chosen in this work showed good reproducibility with certain modifications in chromatographic conditions and sample preparation protocol. Optimisation of the method included changes in wavelength, constituents, and the flow rate of mobile phase, and excluded the addition of internal standard in sample preparation protocol. Mobile phase consisted of distilled water: methanol: glacial acetic acid (65 : 34 : 1 v/v %), and Selectra C18 (4.6 × 250 mm, 5 μm) column was used. Chromatographic conditions included the flow rate of 1 ml min–1, room temperature, wavelength at 218 nm and 270 nm, and an injection volume of 20 µl. At 270 nm, linearity was shown in the range of concentrations of 1–30 mg l–1 (y = 10290x + 15138; R2 = 0.9932), while the achieved percentage of recovery was between 73.8 % and 111.2 % for the tested concentrations. This work is licensed under a Creative Commons Attribution 4.0 International License