Razvoj LC-MS metode za določitev askorbinske in eritorbinske kisline

Nikolić, Mina (author), Kočar, Drago (mentor), Šala, Martin (comentor)

Razvili smo metodo tekočinske kromatografije visoke ločljivosti sklopljene s tandemsko masno spektrometrijo za določitev askorbinske kisline in njenega izomera, eritorbinske kisline. Za separacijo smo uporabili Aminex HPX-87H kolono in izokratsko elucijo z 0,1 % mravljično kislino v vodi. Kvantifikacijo smo izvedli z uporabo ESI ionizacije v negativnem načinu s prekurzorskim ionom m/z 174,9 in produktnim ionom m/z 115 za obe kislini. Da se izognemo lažno pozitivnim rezultatom, smo pri vsaki meritvi spremljali še produktni ion m/z 87, ki je služil kot kontrola. Študirali smo stabilnost standardov, linearnost, meje zaznave in ponovljivost instrumenta. Ugotovili smo, da standardi na sobni temperaturi zelo hitro razpadajo in se odločili za uporabo reducenta TCEP. Območje linearnosti je bilo od meje zaznave do 2000 ppb, meja zaznave za askorbinsko kislino 69 ppb, za eritorbinsko pa 61 ppb. RSD meritev je bil pod 10 %. Preučevani parametri metode so bili v sprejemljivih območjih kar je vodilo do zanesljive določitve askorbinske in eritorbinske kisline. Metodo smo ovrednotili na različnih realnih vzorcih: šumečih in navadnih tabletah ter na sokovih. Rezultati analize tablet so odstopali od deklarirane vrednosti največ ±10 %. Preverili smo tudi homogenost tablet in ugotovili dobro homogenost tako znotraj tablet kot med tabletami. Pri vseh analiziranih vzorcih je bila koncentracija eritorbinske kisline pod mejo zaznave, kar je zelo pomembno zaradi njene nizke aktivnosti v primerjavi z askorbinsko kislino.

• Language: Slovenian
• Keywords: tekočinska kromatografija visoke ločljivosti, masna spektrometrija, vitamin C, prehranska dopolnila, tablete
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-137367
• COBISS.SI-ID: 112018691
• Publication date in RUL: 15.06.2022

Secondary language

• Language: English
• Title: Development of a LC-MS method for determination of ascorbic and erythorbic acid

Abstract

A high-performance liquid chromatography coupled with tandem mass spectrometry method for determination of ascorbic acid and its isomer, erythorbic acid was developed. The separation was performed on Aminex HPX-87H column using isocratic elution with 0,1 % formic acid in water. Quantification was done using ESI ionization in negative mode with m/z 174,9 as a precursor ion and m/z 115 as a product ion for both acids. In order to avoid false positive results, the product ion m/z 87 was also recorded at each measurement and was used as a confirmation. The stability of standards, linearity, limits of detection and instrument repeatability were studied. The standards were found to degrade very quickly at room temperature. Because of that, TCEP was used as a reducing agent. The linear range was from the limit of detection to 2000 ppb while the limits of detection were 69 ppb and 61 ppb for ascorbic and erythorbic acid, respectively. RSD of measurements was below 10 %. The studied parameters were within the acceptable ranges which lead to the reliable determination of ascorbic and erythorbic acid. The method was evaluated on different real samples: effervescent and ordinary tablets and on juices. The results for tablets deviated from the value on the label by a maximum of ±10 %. The tablets were found to be homogeneous with good homogeneity both within and between tablets. The concentration of erythorbic acid was below the limit of detection in all the analyzed samples which is very important due to its low activity compared to ascorbic acid.

• Keywords: high-performance liquid chromatography, mass spectrometry, vitamin C, dietary supplements, tablets