Fully Automated Quantitative Measurement of Serum Organic Acids via LC-MS/MS for the Diagnosis of Organic Acidemias: Establishment of an Automation System and a Proof-of-Concept Validation.

Yasushi Ueyanagi,Shinya Matsumoto,Daisuke Kawakami,Taeko Hotta,Yuichi Mushimoto,Daiki Setoyama,Dongchon Kang

doi:10.3390/diagnostics11122195

Yasushi Ueyanagi, Shinya Matsumoto + Show 5 more

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https://doi.org/10.3390/diagnostics11122195

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Abstract

Gas chromatography-mass spectrometry has been widely used to analyze hundreds of organic acids in urine to provide a diagnostic basis for organic acidemia. However, it is difficult to operate in clinical laboratories on a daily basis due to sample pretreatment processing. Therefore, we aimed to develop a fully automated system for quantifying serum organic acids using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pretreatment CLAM-2030 device was connected to an LC-MS/MS system for processing serum under optimized conditions, which included derivatizing serum organic acids using 3-Nitrophenylhydrazine. The derivatized organic acids were separated on a reverse-phase Sceptor HD-C column and detected using negative-ion electrospray ionization multiple reaction monitoring MS. The automated pretreatment-LC-MS/MS system processed serum in less than 1 h and analyzed 19 serum organic acids, which are used to detect organic acidemias. The system exhibited high quantitative sensitivity ranging from approximately 2 to 100 µM with a measurement reproducibility of 10.4% CV. Moreover, a proof-of-concept validation of the system was performed using sera from patients with propionic acidemia (n = 5), methylmalonic acidemia (n = 2), and 3-methylcrotonylglycinuria (n = 1). The levels of marker organic acids specific to each disease were significantly elevated in the sera of the patients compared to those in control samples. The automated pretreatment-LC-MS/MS system can be used as a rapid in-hospital system to measure organic acid levels in serum for the diagnosis of organic acidemias.

Highlights

Newborn screening tests are performed for the early detection of inborn errors in metabolism
We developed an automated LC-MS/MS-based method to differentiate nine types of organic acidemia (Figure 1A)
Derivatization with 3-Nitrophenylhydrazine (3-NPH), which reacts with the carboxylic acids of organic acids in aqueous condition, was chosen to improve the separation and detection sensitivity in our LC-MS/MS system

Summary

Introduction

Newborn screening tests are performed for the early detection of inborn errors in metabolism. Newborn screening tests are capable of detecting amino acid and fatty acid disorders; they are insufficient to distinguish organic acid disorders. Additional analyses are required to detect urinary organic acids. Gas chromatography-mass spectrometry (GC-MS) has been widely used to measure more than a hundred kinds of organic acids in urine samples, providing a diagnostic basis for organic acid disorders [4,5,6]. GC-MS requires complex sample pretreatment, such as the extraction of organic acids from biological samples, drying of extracted solvents, and reaction with derivatizing reagents, which makes it difficult to operate on a daily basis in a clinical laboratory. It takes more than a week to obtain the test results in Japan, since newborn screenings and organic acid analyses are performed in a limited number of facilities. Clinical laboratories require methods to analyze organic acids in a simple, quick, and automated manner while requiring as little human effort as possible

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