LC-MS/MS Analysis of Cerebrospinal Fluid Metabolites in the Pterin Biosynthetic Pathway

Abstract

The analysis of (6R)-5,6,7,8-tetrahydrobiopterin (BH4) and neopterin in cerebrospinal fluid (CSF) is often used to identify defects in the pterin biosynthetic pathway affecting monoamine metabolism that can lead to pediatric neurotransmitter diseases. Low levels of BH4 and neopterin alone may not be sufficient to determine the defect, and further testing is often required. We have developed a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of BH4, 7,8-dihydrobiopterin (BH2), neopterin, and sepiapterin in CSF, which provides a more comprehensive evaluation of the pterin pathway. The method utilizes labeled stable isotopes as internal standards and allows for a fast 10-minute analysis by LC/MS/MS over a linear working range of 3 to 200 nmol/L. Total analytical imprecision is less than 14.4% for all pterin metabolites. Accuracy for BH4 and neopterin was determined by comparing data obtained by an alternative method using HPLC with EC and fluorescence detection. Excellent correlation was demonstrated for BH4 (r = 0.9646, 1/slope = 0.9397; n = 28; concentration range 3 to 63 nmol/L) and neopterin (r = 0.9919, 1/slope = 0.9539; n = 13; concentration range 5 to 240 nmol/L). CSF specimens from patients diagnosed with inborn errors of sepiapterin reductase (SR), 6-pyruvoyl-tetrahydropterin synthase (PTPS), dihydropteridine reductase (DHPR), and guanosine triphosphate cyclohydrolase (GTPCH) have been analyzed, and distinct pterin metabolite patterns were consistent with the initial diagnosis. This method differentiates patients with DHPR and SR deficiency from other pterin defects (GTPCH and PTPS) and will be useful for the diagnosis of specific defects in the pterin biosynthetic pathway.

Single-Step Rapid Diagnosis of Dopamine and Serotonin Metabolism Disorders

“…The first one is devoted to the quantification of dopamine and serotonin metabolites and the second one to the quantitation of pterins. 10 − 19 …”

“… 10 − 17 More recently, some liquid chromatography–mass spectrometry (LC–MS) methods have also been implemented. 19 , 20 On the other hand, the CSF pterin concentrations are in the low nM range at basal levels. Thus, their direct quantification does require an HPLC method coupled to fluorescence or MS detections.…”

“…Thus, their direct quantification does require an HPLC method coupled to fluorescence or MS detections. 1 , 10 , 11 , 17 , 19 …”

Intestinal microbiota as a tetrahydrobiopterin exogenous source in hph-1 mice

“…Determination of BH4 in tissue and bacterial pellets was performed by LC-MS/MS as previously described35 with modifications to sample preparation. Briefly, analysis was performed on an AB Sciex 5500QTRAP mass spectrometer (Foster City, CA, USA) coupled with a Shimadzu Nexera ultrahigh pressure liquid chromatograph system (Kyoto, Japan).…”

Tetrahydrobiopterin Concentrations in Normal and Coronary Artery Diseased Heart Tissue

“…Heart tissue BH4 and BH2 were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described with slight modifications [11]. Liquid chromatography was performed on a Shimadzu Nexera system by reversed-phase HPLC (EZ-faast AAA-MS column 250 x 2mm 3µm, Phenomenex) equilibrated at 40°C.…”