The application of Phenylalanine to Tyrosine ratio for Phenylketonuria (PKU) detection using tandem mass spectrometry was first published in 1993. A simple solvent extraction followed by a derivatization method has been used to improve sensitivity and specificity for the detection of amino acids in blood or plasma by MS/MS, which has been modified and customized in labs over the years.
The development of electrospray in mass spectrometry largely improved ionization and facilitated automated analysis. Here, we introduce a Selected Reaction Monitoring method (SRM) with simple Flow Injection Analysis (FIA) via strategically adding internal standards to the clean paper discs prior to the collection of whole blood or plasma samples to achieve a better recovery and reproducibility for amino acids quantitation.
Varying concentrations of the amino acids internal standard mixture in water was pipetted onto clean paper discs either from flat precut paper or using blood sample collection devices (Capitainer) and dried for 30min. 10µl of blood or plasma sample was added and dried overnight at room temperature. The paper discs were removed and extracted with 300µl methanol in a polypropylene tube with shaking for 30min. Following centrifugation, the supernatant was transferred to a clean tube and dried down under nitrogen at 30oC. Next, 50µl of HCl in n-butanol was added, incubated at 65oC for 20min, then the excess butanol was evaporated. Samples were reconstituted using 300µl of 1:1 methanol/water and transferred to LC vial for analysis.
With the addition of labelled amino acids internal standards (IS) prior to the paper matrix before the collection of whole blood or plasma samples, we achieved improved reproducibility compared to the traditional method of adding the internal standards into the extraction solvent. Because the IS was added in the paper, it reflects the extraction efficiency of amino acids from the paper matrix better than adding the IS during the extraction procedure. In addition, it could account for any potential degradation that might occur to the sample before preparing for analysis since degradation would occur equally to target analytes and the internal standards. The ratio of endogenous amino acids to its labeled counterparts were calculated. We observed lower relative standard deviation (RSD) when adding internal standards prior to the collection of samples. As expected, derivatization changed the fragmentation pattern of amino acids, largely improved the ionization efficiency for target analytes, meanwhile, it helped to eliminate background ions that would cause ion suppression in mass spectrometry. Signal intensities detected from dried plasma spots using our SRM-FIA method, based on the measurement of peak areas, were increased 6 to 39-fold for amino acids butyl ester derivatives compared to free amino acids, with reduced RSD values, especially for tyrosine. Our preliminary results show that ion suppression was a major problem which introduced higher error to data analysis because of poor peak shape. Through conducting serial of dilutions, a proper reconstitution volume was chosen for the extraction workflow that reduced overall error.
We utilized quantitative Dried blood spot cards (Capitainer) which deliver a precise volume of whole blood to a separated paper disc in which we added our IS mixture. This unique approach helped to achieve better recovery and reproducibility for the quantitation of amino acids from DXS. The SRM-FIA method was performed via Thermo Scientific TSQ Altis Mass Spectrometer coupled with Vanquish LC system, without any separation and the entire analysis process was conducted automatically with a 1 min per sample analysis rate.
The use of internal standards imprinted on paper for the collection of biofluids in amino acid quantitation provides improved sensitivity and reproducibility in FIA-SRM analysis.