Quantitative analysis of phenylalanine, tyrosine, tryptophan and kynurenine in rat model for tauopathies by ultra-high performance liquid chromatography with fluorescence and mass spectrometry detection.
We developed and validated a simple and sensitive ultra-high performance liquid chromatography (UHPLC) method for the analysis of phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and kynurenine (Kyn) in rat plasma. Analytes were separated on Acquity UPLC HSS T3 column (2.1 mm×50 mm, 1.8 μm particle size) using a 4 min ammonium acetate (pH 5) gradient and detected by fluorescence and positive ESI mass spectrometry. Sample preparation involved dilution of plasma, deproteinization by trichloroacetic acid and centrifugation. The procedure was validated in compliance with the FDA guideline. The limits of quantification (LOQ) were 0.3 μM for Kyn and from 1.5 to 3 μM for Phe, Tyr, Trp. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 86-108%. The inter-day precision (n=5 days), expressed as % RSD, was in the range 1-13%. The benefit of using UHPLC is a short analysis period and thus, a very good sample throughput. Using this method, we analyzed plasma samples and detected significant changes of Kyn and Phe in transgenic rat model for tauopathies.
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 84510 Bratislava, Slovak Republic; Department of Pharmaceutical analysis and nuclear pharmacy, Faculty of Pharmacy of Comenius University, Odbojarov 10, 832 32 Bratislava, Slovak Republic.
Jaroslav
J
Galba
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 84510 Bratislava, Slovak Republic; AXON Neuroscience SE, Dvorakovo nabrezie 10, 811 02 Bratislava, Slovak Republic.
Alena
A
Michalicova
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 84510 Bratislava, Slovak Republic; AXON Neuroscience SE, Dvorakovo nabrezie 10, 811 02 Bratislava, Slovak Republic.
Vojtech
V
Parrak
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 84510 Bratislava, Slovak Republic; AXON Neuroscience SE, Dvorakovo nabrezie 10, 811 02 Bratislava, Slovak Republic.
Michal
M
Novak
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 84510 Bratislava, Slovak Republic; AXON Neuroscience SE, Dvorakovo nabrezie 10, 811 02 Bratislava, Slovak Republic. Electronic address: andrej.kovac@savba.sk.
Andrej
A
Kovac
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004195:Disease Models, Animal; D006801:Humans; D007737:Kynurenine / Q000097:blood*; D010649:Phenylalanine / Q000097:blood*; D051381:Rats; D011918:Rats, Inbred SHR; D055647:Rats, Transgenic; D053719:Tandem Mass Spectrometry / Q000379:methods*; D024801:Tauopathies / Q000097:blood* / Q000473:pathology; D014364:Tryptophan / Q000097:blood*; D014443:Tyrosine / Q000097:blood*
D007737:Kynurenine; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan
Immune system; Plasma; QDa; Rat model; Tau protein; Tauopathy; UPLC/FLD/MS