Determination of Creatinine in Human Urine with Flow Injection Tandem Mass Spectrometry

Background/Aims: Excretion of urinary compounds in spot urine is often estimated relative to creatinine. For the growing number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays of urine-excreted molecules, a fast and accurate method for determination of creatinine is needed. Methods: A high-throughput flow injection tandem mass spectrometry method for exact quantitation of creatinine in urine has been developed and validated. Sample preparation used only two-step dilution for protein precipitation and matrix dilution. Flow injection analysis without chromatographic separation allowed for total run times of 1 min per sample. Creatinine concentrations were quantitated using stable isotope dilution tandem mass spectrometry. Selectivity and coelution-free quantitation were assured by qualifier ion monitoring. Results: Method validation revealed excellent injection repeatability of 1.0% coefficient of variation (CV), intraday precision of 1.2% CV and interday precision of 2.4% CV. Accuracy determined from standard addition experiments was 106.1 +/- 3.8%. The linear calibration range was adapted to physiological creatinine concentrations. Comparison of quantitation results with a routinely used method (Jaffe colorimetric assay) proved high agreement (R-2 = 0.9102). Conclusions: The new method is a valuable addition to the toolbox of LC-MS/MS laboratories where excretion of urinary compounds is studied. The `dilute and shoot' approach to isotope dilution tandem mass spectrometry makes the new method highly accurate as well as cost-and time-efficient. Copyright (C) 2012 S. Karger AG, Base

Folate catabolites in spot urine as non-invasive biomarkers of folate status during habitual intake and folic acid supplementation.

Folate status, as reflected by red blood cell (RCF) and plasma folates (PF), is related to health and disease risk. Folate degradation products para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (apABG) in 24 hour urine have recently been shown to correlate with blood folate. Since blood sampling and collection of 24 hour urine are cumbersome, we investigated whether the determination of urinary folate catabolites in fasted spot urine is a suitable non-invasive biomarker for folate status in subjects before and during folic acid supplementation. Immediate effects of oral folic acid bolus intake on urinary folate catabolites were assessed in a short-term pre-study. In the main study we included 53 healthy men. Of these, 29 were selected for a 12 week folic acid supplementation (400 µg). Blood, 24 hour and spot urine were collected at baseline and after 6 and 12 weeks and PF, RCF, urinary apABG and pABG were determined. Intake of a 400 µg folic acid bolus resulted in immediate increase of urinary catabolites. In the main study pABG and apABG concentrations in spot urine correlated well with their excretion in 24 hour urine. In healthy men consuming habitual diet, pABG showed closer correlation with PF (rs = 0.676) and RCF (rs = 0.649) than apABG (rs = 0.264, ns and 0.543). Supplementation led to significantly increased folate in plasma and red cells as well as elevated urinary folate catabolites, while only pABG correlated significantly with PF (rs = 0.574) after 12 weeks. Quantification of folate catabolites in fasted spot urine seems suitable as a non-invasive alternative to blood or 24 hour urine analysis for evaluation of folate status in populations consuming habitual diet. In non-steady-state conditions (folic acid supplementation) correlations between folate marker (RCF, PF, urinary catabolites) decrease due to differing kinetics

Correlation of folate catabolites in spot urine with 24 hour urine and blood folate concentrations.

<p><b><i>Comparison of folate catabolites in spot urine and 24 hour urine.</i></b> Creatinine normalized concentration of para-aminobenzoylglutamate (pABG) (A) and para-acetamidobenzoylglutamate (apABG) (B) in spot urine is correlated with daily excretion in 24 hour urine. <b><i>Correlations of urinary pABG with plasma and red blood cell folate, a comparison between baseline and week 12.</i></b> Scatterplots for para-aminobenzoylglutamate (pABG)/creatinine in spot urine related to plasma folate and red blood cell folate concentrations, at baseline (C, E, n = 51) and week 12 (D, F, n = 25). For ease of comparison, dashed lines in C, D, E and F indicate the lowest value of the respective biomarker at week 12.</p

Description of participants and biochemical parameters during habitual diet (baseline).

<p>Values are given as median, IQR in parentheses. Results of the two baseline measurements were averaged, if valid data were available from both examinations. Relations between variables were analyzed by Spearman’s rank correlation. Significance level:</p>*<p>p<0.05,</p>**<p>p<0.001. pABG: para-aminobenzoylglutamate, apABG: para-acetamidobenzoylglutamate, MMA: methylmalonic acid, ratio_p/ap: ratio of pABG to apABG.</p>†<p>One extreme outlier was excluded.</p

Time course of plasma, red blood cell folate, urinary pABG and apABG during folic acid supplementation.

<p>Time course of the median concentrations (error bars = inter quartile range (IQR)) of plasma folate (nmol/L) (A), red blood cell folate (nmol/L) (B), pABG (nmol/mmol creatinine) (C) and apABG (nmol/mmol creatinine) (D) during folic acid supplementation. Significant changes from baseline: *: p = 0.005 and **: p≤0.001 (Wilcoxon’s test).</p

RCF, PF, urinary folate catabolites during folic acid supplementation (week 6, 12).

<p>Values are given as median and IQR in parentheses, relations between variables were analyzed by Spearman’s rank correlation. pABG: para-aminobenzoylglutamate, apABG: para-acetamidobenzoylglutamate, RCF: red blood cell folate, PF: plasma folate. Spearman’s rank correlation significance level:</p>*<p>p<0.05. Significance of changes from baseline was calculated using Wilcoxon’s test and given as p-value.</p

Time course of urinary apABG and pABG after folic acid intake (short-term study).

<p>Time course (medians, error bars = inter quartile range (IQR)) of urinary apABG (nmol/mmol creatinine) (A) and pABG (nmol/mmol creatinine) (B), in fasted state (black line) and after oral intake of 400 µg folic acid (dashed line) during the first three hours. (C) Time course of the ratio between pABG and apABG (ratio_p/ap) in spot urine without (black) and with (dashed line) intake of folic acid at time 0. Significant differences from time point 0 are marked with an asterisk (*: p≤0.05, Wilcoxon’s test). (D) Histograms of observed frequencies of ratio_p/ap in spot urine in non-dosed state (light grey bars) and 2 h after oral folate intake (dark grey bars), displayed with continuous kernel density function estimates (black lines).</p

Flow chart of study selection process.

<p>Flow chart of study selection process.</p