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Abstract INTRODUCTION:
Inborn errors of metabolism are a group of rare inherited disorders that can have early onset during infancy. If not diagnosed and treated, these disorders can lead to organ damage, developmental delay and death. Newborn screening detects and quantifies acyl carnitines and amino acids, biomarkers for these disorders, with high-throughput tandem mass spectrometry. Organic acids and acyl glycines are important indicators of multiple inborn errors of metabolism, but are not detected in standard newborn screening and need additional assays for quantification. Second-tier assays such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) may be used to aid in diagnosis and reduce false positives. A single LC-MS/MS assay covering multiple compounds decreases system down-time by eliminating the need to change columns and mobile phases sample-to-sample, minimizes variations in sample preparations and simplifies workflow. However, the diverse physicochemical properties of these biomarkers make the development of a single multiplexed LC-MS/MS assay challenging.
OBJECTIVE:
Our goal is to develop multiplex LC-MS/MS assays for 1) full amino acid profile, 2) acyl carnitines and 3) organic acids and acyl glycines in neonatal dried blood spots, plasma and urine. All assays will use 3-nitrophenylhydrazine (3-NPH) derivatization. 3-NPH alters the chemical structure of analytes, reducing their polarity and making them amenable to reverse-phase liquid chromatography. Once developed, the assays will be validated according to the "ICH M10 on bioanalytical method validation" guideline.
METHODS:
Three LC-MS/MS methods are currently in development. Standards in neat solutions were used to determine provisional MS parameters and screen columns and mobile phases, further development used matrix samples. Upon selecting the column and mobile phases, additional variables such as flow rate, column temperature and capillary voltage were optimized for compound sensitivity.
RESULTS:
Initial screening has revealed a column and mobile phase combination that show acceptable performance for all three methods. Peak shapes for analytes are symmetric and retention of all analytes is successful. Amino acids elute at low organic percentages, thus their gradient is shallow and resolution of alloisoleucine from isoleucine and leucine is achieved with a total analysis time of 10 minutes. Monitoring of phospholipids in plasma indicates minimal ion suppression of amino acids. Acyl carnitines elute linearly in order of increasing chain length with a total analysis time of 5 minutes. Organic acids and acyl glycines are analyzed with a 10 minute method, resolving key isobaric compounds.
DISCUSSION:
While still in development, preliminary results are promising. Amino acids and acyl carnitines are easily observed in endogenous concentrations from non-spiked matrix samples. However, some organic acids need resolution from matrix interferences. Our results indicate the method has the potential to accurately quantify biomarkers for multiple inborn errors of metabolism and give a comprehensive amino acid profile. Current limitations to the method are interferences with organic acids, challenging the clinical application of the organic acid panel to accurately identify and quantify multiple markers for organic acidemias. To resolve these challenges, further optimization of the derivatization, sample cleaning and altered elution gradient may improve resolution of isobaric compounds and interferences. |
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
