Preparation of Isotopically Labelled Standards of Creatinine Via H/D Exchange and Their Application in Quantitative Analysis by LC-MS.

Remigiusz Bąchor,Andrzej Konieczny,Zbigniew Szewczuk

doi:10.3390/molecules25071514

Remigiusz Bąchor, Andrzej Konieczny + Show 1 more

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https://doi.org/10.3390/molecules25071514

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Abstract

Kidneys play a crucial role in maintaining metabolic homeostasis in a body. Serum creatinine concentration is a simple test used as an indicator of renal function. One of the known ways of quantifying creatinine concentration is the liquid chromatography-mass spectrometry (LC-MS) method, using an isotopically labeled analog of creatinine as an internal standard. Unfortunately, such isotope-labeled analogs are expensive and their synthesis is complex. Here we demonstrate a facile preparation of deuterated analogues of creatinine, via the H/D exchange of hydrogens located at the α-carbon (α-C) of the N-methylated amino acid part, under basic conditions. The stability of retrieved isotopologues was analyzed under both neutral or acidic conditions, and the results revealed that the introduced deuterons do not undergo back-exchange. In addition, the coelution of deuterated and non-deuterated forms under acidic and neutral conditions was observed. The prepared isotopologues were successfully applied in the quantitative LC-MS analysis of urine samples, and the results demonstrated that the presented strategy is novel and inexpensive, and that the quantification correlates with the commonly used Jaffe test method.

Highlights

Creatinine, 2-amino-1-methyl-1,5-dihydro-4H-imidazol-4-on, is a breakdown product of creatine phosphate in muscle
The main goal of this work was to synthesize the deuterated standards of creatinine, at α-C of the
According to our previous study [14,17], we tested the influence of the 1% TEA/D2 O solution on the hydrogen/deuterium exchange (HDX) at the α-C of the other N-methylated amino acid part as milder reaction conditions

Summary

Introduction

Creatinine, 2-amino-1-methyl-1,5-dihydro-4H-imidazol-4-on, is a breakdown product of creatine phosphate in muscle. The loss of a water molecule from creatine results in the formation of creatinine as a heterocyclic compound. Creatinine is transferred to the kidneys by blood plasma, whereupon it is eliminated from the body by glomerular filtration and partial tubular excretion. Creatinine is usually produced at a fairly constant rate by the body. Measuring serum creatinine is a simple test, and it is the most commonly used indicator of renal function. A rise in blood creatinine levels is observed only with marked damage to functioning nephrons. The detection of renal hypofunction is highly desired. The serum concentrations of creatine (sCr) and creatinine are commonly used for the determination of the renal function. Urinary creatinine (uCre) analysis may be used to calculate creatinine clearance, confirm the completeness of 24-h collections, or serve as a reference quantity for other analyses, such as in the calculation of the albumin or protein/creatinine ratio

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