Dilute-and-Shoot HPLC-UV Method for Determination of Urinary Creatinine as a Normalization Tool in Mycotoxin Biomonitoring in Pigs.

Agnieszka Tkaczyk,Piotr Jedziniak

doi:10.3390/molecules25102445

Abstract

A simple, rapid, and accurate HPLC-UV method was developed for the determination of creatinine in pig urine. Usually, it is determined in urine in biomonitoring of xenobiotics to correct for variations in dilutions of urine samples. The colorimetric method (based on Jaffe reaction), which was mainly used for this purpose in mycotoxin biomonitoring, is not a reliable approach for pig urine. Therefore, a novel and accurate HPLC method for creatinine determination was developed. The sample preparation was based on the dilute and shoot approach. An HPLC separation was performed with a porous graphitic carbon column with an aqueous mobile phase to achieve satisfactory retention time for creatinine. The method has been successfully validated, applied for the determination of creatinine in pig urine, and compared with other methods commonly used for that purpose—a colorimetric method based on Jaffe reaction and commercial ELISA test. The developed HPLC method shows the highest precision and accuracy for pig urine samples. Finally, the method was applied as a normalization tool in LC-MS/MS mycotoxin biomarkers analysis. The standardization to a constant creatinine level (0.5 mg/mL) enables similar matrix effects for eleven mycotoxin biomarkers for pig urine samples with different creatinine levels.

Highlights

Creatinine (2-amino-1-methyl-2-imidazoline-4-one) is the final metabolism product of creatine in mammals [1], which is excreted exclusively by the kidneys via glomerular filtration and, to a lesser extent, by tubular secretion [2]
Recent studies have shown the advantages of hydrophilic interaction chromatography (HILIC) over the Reversed-phase high-performance liquid chromatography (RP-HPLC) for the separation of highly polar compounds, small biological metabolites [26,27]
An alternative to commonly used in reversed-phase chromatography alkyl-bonded (e.g., C18) silicas sorbents is porous graphitic carbon

Summary

Introduction

Creatinine (2-amino-1-methyl-2-imidazoline-4-one) is the final metabolism product of creatine in mammals [1], which is excreted exclusively by the kidneys via glomerular filtration and, to a lesser extent, by tubular secretion [2]. Its excretion throughout the day is relatively constant, the amount of creatinine produced is proportional to the muscle mass of the individuals [3]. Creatinine concentration in urine can be taken as a measure of the dilution of the urine [4]. Creatinine concentration is commonly used as a standardization tool for quantifying xenobiotics in urine (biomarkers) [6]. The World Health Organization (WHO) recommends if a sample is too diluted (creatinine concentration < 0.3 mg/mL) or too concentrated (creatinine concentration > 3.0 mg/mL), another urine void should be collected (WHO, 1996). Dividing biomarker concentration by the creatinine level measured in the same sample can account

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Conclusion