Validacija analitičke metode za određivanje profila acil-karnitina iz plazme primjenom tandemske spektrometrije masa

master's thesis

• Title (english): Validation of analytical method for acylcarnitine profile analysis in plasma using tandem mass spectrometry
• Author: Nives Antolić
• Mentor: Ksenija Fumić (mentor)
• Committee member: Ksenija Fumić (predsjednik povjerenstva)
• Committee member: Lidija Bach-Rojecky (član povjerenstva)
• Committee member: Olga Gornik (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; Zagreb
• Defense date and country: 2017-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Acil – karnitini produkti su oksidacije masnih kiselina i aminokiselina prisutni u tkivima i tjelesnim tekućinama. Važan su dijagnostički marker za nasljedne bolesti vezane uz oksidacijske procese. Analiza profila acil-karnitina provodi se biokemijskim testovima kao probir za poremećaje u oksidaciji masnih kiselina i metabolizma organskih kiselina. Različiti acil – karnitini javljaju se u obliku izomera i u vrlo niskim koncentracijama stoga je izrazito važno postaviti metodu koja će pouzdano, brzo i efikasno odrediti profil acil – karnitina.
Tijekom posljednjih nekoliko godina dvojni maseni spektrometri (MS/MS) postali su značajni i pouzdani rutinski analitički uređaji koji su omogućili puno osjetljiviju, specifičniju, pouzdanu i usporedivu analizu u odnosu na tradicionalni novorođenački probir. Metodom određivanja profila acil – karnitina iz plazme, omogućit će se pravovremena dijagnostika i praćenje tijeka liječenja u većine bolesnika s nasljednim metaboličkim bolestima. U ovom radu opisana je nova LC – MS/MS metoda za analizu slodobnog karnitina, ali i acil – karnitina duljine lanca C2 – C18 uz ERNDIM vanjsku kontrolu kvalitete.
Nepreciznost metode za određivanje svih analita novom metodom analize profila acil – karnitina iz plazme tandemskom spektrometrijom masa zadovoljava kriterije prihvatljivosti kao i rezultati točnosti koji su unutar zadanih kriterija. ERDNIMQA usporedbom pokazali smo odličnu sljedivost rezultata.
Uspjeli smo uvesti metodu, senzitivnu i robusnu koja omogućuje kvantifikaciju velikog broja acil – karnitina uključujući i izomerne forme pojedinih metabolita vezanih uz metabolizam aminokiselina i oksidacije masnih kiselina, u rutinski rad našeg laboratorija.

Abstract (english)

Acylcarnitines are intermediates of fatty acid and amino acid oxidation found in tissues and body fluids. They are important diagnostic markers for inherited diseases of peroxisomal and mitochondrial oxidation processes. Acylcarnitine profile analysis is performed for the biochemical screening of disorders of fatty acid oxidation and organic acid metabolism. Quantification of acylcarnitine species can become challenging because various species occur as isomers and/or have very low concentrations.
Over the past decade laboratories that test for metabolic disorders have introduced tandem mass spectrometry (MS/MS), which is more sensitive, specific, reliable, and comprehensive than traditional assays, into their newborn screening programs. MS/MS is rapidly replacing these one – analysis, one – metabolite, one – disease classic screening techniques with a one analysis, many – metabolites, many – diseases approach that also facilitates the ability to add new disorders to existing newborn screening panels. Here we described a new LC – MS/MS method for analysis of free carnitine and acylcarnitine species with acyl – chain lengths from C2 to C18 using ERNDIM.
Method validation in plasma showed excellent accuracy and precision with good within – laboratory variation and intermediate inter – laboratory variation. Values obtained from ERNDIMQA, compared to international laboratory results showed good practice and reliable results.
In conclusion, the LC – MS/MS method presented here allows robust quantification of isomeric acylcarnitine species and extends the palette of acylcarnitines with diagnostic potential. All results indicate that described method can be used in routine.

• Keywords: urođene metaboličke bolesti; masne kiseline; poremećaj oksidacije; profil acil – karnitina; validacija; tandemska spektrometrija masa
• Keywords (english): inborn metabolic disorders; fatty acids; oxidation disorder; acylcarnitine profile; validation; tandem mass spectrometry
• Language: croatian
• URN:NBN: urn:nbn:hr:163:468647
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-24 16:44:32