Validation of an HPLC method for the determination of urinary and plasma levels of N1-methylnicotinamide, an endogenous marker of renal cationic transport and plasma flow

doi:10.1016/S0731-7085(00)00425-8

Journal of Pharmaceutical and Biomedical Analysis

Volume 24, Issue 3, January 2001, Pages 391-404

https://doi.org/10.1016/S0731-7085(00)00425-8 Get rights and content

Abstract

N¹-Methylnicotinamide (NMN) is an endogenous cationic metabolite of nicotinamide (niacine, vitamine PP) whose renal clearance reflects both the capacity of the renal tubular transport system to secrete organic cations and renal plasma flow. NMN is present in human plasma and urine at the 1–117-ng ml⁻¹ and 0.5–25-μg ml⁻¹ concentration range, respectively, and its level depends notably on pathophysiological (age, renal or hepatic diseases) conditions. We report the optimization and validation of an HPLC method for the measurement of endogenous NMN in biological fluids after derivatization into a fluorescent compound. Plasma is first deproteinized with TCA 20% and the urine diluted 1:10 with HCl 10⁻⁴ M prior to the derivatization procedure, which includes a condensation reaction of NMN with acetophenone in NaOH at 0°C, followed by dehydration in formic acid and subsequent formation of the fluorescent 1,6-naphthyridine derivatives after heating samples in a boiling water bath. The synthetic homologous derivative N¹-ethylnicotinamide (NEN) reacts similarly and is added as internal standard into the biological fluid. The reaction mixture is subjected to reverse phase high performance liquid chromatography on a Nucleosil 100-C18 column using a mobile phase (acetonitrile 22%, triethylamine 0.5%, 0.01 M sodium heptanesulfonate adjusted to pH 3.2), delivered isocratically at a flow rate of 1 ml min⁻¹. NMN and NEN are detected at 7.8 and 10 min by spectrofluorimetry with excitation and emission wavelengths set at 366 and 418 nm, respectively. The addition–calibration method is used with plasma and urine pools. Calibration curves (using the internal standard method) are linear (r²>0.997) at concentrations up to 109 ng ml⁻¹ and 15.7 μg ml⁻¹ in plasma and urine, respectively. Both intra- and inter-assay precision of plasma control samples at 10, 50 and 90 ng ml⁻¹ were lower than 3.3% and concentrations not deviating more than 2.7% from their nominal values. In urine intra- and inter-assay CVs of control samples at 1, 5 and 9 μg ml⁻¹ are lower than 8.3%, with concentrations not deviating more than −9.0 to +11.8% from their nominal values. This analytical method has therefore the required sensitivity and selectivity to measure NMN in plasma and urine, enabling the non-invasive determination of the tubular secretory capacity of the kidney and the renal plasma flow.

Introduction

N¹-Methylnicotinamide (NMN) (Fig. 1) is an endogenous cationic metabolite of nicotinamide (Vitamin PP, niacin). It is filtered at the glomerulus and highly extracted (secreted into the tubules) during its passage through the kidney, without being reabsorbed to a significant extent, and its renal handling reflects therefore both the capacity of the renal tubular transport system to secrete organic cations and the renal plasma flow (RPF). Clearance of endogenous NMN could substitute to p-aminohippuric acid (PAH) clearance for the determination of RPF [1], with the advantages of not being an exogenous product and not having to be infused, eliminating therefore not only technical and financial burden but also adverse effects, though admittedly rare, of PAH administration. Furthermore, though considered the gold standard, PAH has the drawback of being cleared in part (10–15%) by extra-renal mechanisms [2].

To the best of our knowledge, NMN has not been evaluated as a marker of RPF yet in humans. The use of NMN for renal plasma flow determination and clinical tubular function studies has presumably been hampered by the difficulty of routinely analyzing NMN in complex biological fluids. NMN has a very low UV extinction coefficient precluding its direct quantitation at the low nanomolar concentrations found in human plasma samples.

In a different context, the measurement of NMN urinary excretion has been used as a surrogate of niacin nutritional status [3]. Since it is present in urine in the μg ml⁻¹ range, NMN could be directly analyzed in this milieu after ion-exchange chromatography and UV detection [4], [5]. However, UV detection may not provide the necessary selectivity in complex urine matrices found in treated patients. NMN has therefore been commonly analyzed in urine after derivatization procedures without [6], [7] or with [8] subsequent separation of reaction mixtures by high performance liquid chromatography (HPLC). Such an approach has also been applied for measuring subnanomolar levels of endogenous NMN in plasma. The derivatization of NMN requires a preliminary off-line step including a condensation of N¹-alkylnicotinamides with acetophenone in NaOH at 0°C. This is followed by dehydration of the condensed products in formic acid, and a final heating in boiling water, yielding the fluorescent 1,6-naphthyridine derivatives (Fig. 1) [9] subsequently quantitated by reverse phase HPLC with spectrofluorimetric detection.

This approach has been notably evaluated by Somogyi et al. [10] who proposed an optimization of the derivatization method for its application to endogenous NMN in plasma, where sensitivity is a major issue. This important contribution did not made clear whether the very early NMN peak elution could always be resolved from the relatively high baseline noise at the beginning of the relatively short HPLC run. Moreover, the reported direct derivatization of whole plasma samples [10] resulted in our hands in erratic reaction mixtures, producing unstable analytical samples (with clouding and even precipitation in vials over time) not suitable for subsequent HPLC analysis.

We report an adaptation and optimization of the method of Somogyi et al. [10] leading to improved chromatographic profiles, notably for plasma samples, with satisfactory sensitivity and enhanced selectivity, enabling the baseline separation and quantitation of NMN from its synthetic homologous N¹-ethylnicotinamide (NEN)-added as internal standard — and from minor nearby interfering peaks. The detailed analytical method validation has been based on the recommendations published as a conference report of the Washington Conference on Analytical Methods Validation: Bioavailability, Bioequivalence and Pharmacokinetic Studies [11].

The method has been applied to the determination of endogenous NMN in plasma and urine of subjects included in a study assessing the adaptation of renal function during high altitude hypoxia and the development of acute mountain sickness (AMS).

Section snippets

Chemicals and reagents

1-Methylnicotinamide chloride salt (NMN) and trichloroacetic acid (TCA) were purchased from Sigma (Buchs, Switzerland). Formic acid 98–100% and hydrochloric acid 25% were from E. Merck (Darmstadt, Germany). Sodium hydroxide pellets, acetophenone, sodium 1-heptanesulfonate monohydrate, triethylamine and ortho-phosphoric acid 85% were purchased from Fluka (Buchs, Switzerland). HPLC grade acetonitrile was from Romil (Cambridge, UK). All chemicals were of analytical grade and used as received.

Samples preparation

The sample treatment proposed by Somogyi et al. [10] was found satisfactory and could be directly applied to diluted urine samples. Urine samples had to be diluted (1:10) with HCl 10⁻⁴ M prior to derivatization because the native NMN concentrations yielded levels of naphthyridine derivative whose fluorescence signals exceeded the detector response limits. All standard solutions for urine (calibration and control) were diluted similarly (1:10) before the derivatization procedure.

For plasma, the

Conclusion

An optimization and validation of the assay of NMN in biological fluids is presented, using a derivatization reaction first proposed by Huff and Perlzweig [6] and further improved by Clark et al. [7]. Efforts have been made not only for improving the method but also for enabling a fully automatable HPLC analysis of derivatize samples, especially in plasma, where stability problems had to be solved. Indeed analysis of large number of samples over a prolonged period of time (overnight), makes it

Acknowledgements

We are indebted to Thierry Buclin for the statistical analysis and to Ali Maghraoui for the data management and the graphics.

References (16)

L.A. Decosterd et al.
J. Chromatogr. B Biomed. Appl.
(1997)
E.G.A. Carter
Am. J. Clin. Nutr.
(1982)
J.W. Huff et al.
J. Biol. Chem.
(1947)
B.R. Clark et al.
Anal. Biochem.
(1975)
T. Hirayama et al.
Anal. Biochem.
(1985)
B.R. Clark
Methods Enzymol.
(1980)
A. Somogyi et al.
Anal. Biochem.
(1990)
J.L. Magnin et al.
Pharm. Acta Helv.
(1996)

There are more references available in the full text version of this article.

Cited by (45)

The development of a new electrochemical sensor based on Cu<inf>2</inf>O@C core–shell and polyalizarin/nafion polymers for determination of bisphenol A at two area potentials
2021, Surfaces and Interfaces
In this paper, employing a hydrothermal method, we synthesized Cu₂O nanoparticles and Cu₂O@C core–shell. The scanning electron microscope (SEM), X -Ray diffraction (XRD), dynamic light scattering (DLS) and zeta potential techniques were used for characterized of the Cu₂O nanoparticles and Cu₂O@C core–shell. The zeta potential values were obtained to be -11.16 mV and -0.88 mV for Cu₂O and Cu₂O@C, respectively. The average hydrodynamic size of diameter for Cu₂O and Cu₂O@C was obtained as 48-62 nm and 200-250 nm by DLS method. Polyalizarin yellowe R (PAYR) and Nafion (Nf) conductive polymers with Cu₂O@C core–shell immobilizing on glassy carbon electrode (PAYR/Cu2O@C /Nf/GCE), they were applyed for fabrication of an original electrochemical sensor to detect and determine bisphenol A. Cyclic voltammetry (CV) and impedimetric techniques were employed to confirm the proposed sensor construction. Furthermore, the electrocatalytic-based oxidation behavior which electrode showed was studyed by cyclic voltammetry and differential puls voltammetry (DPV) methods. The results showed that, PAYR/Cu₂O@C/Nf composite has a great electrocatalytic features for detecting and determining bisphenol A at 0.12V(OX1) and 0.63V(OX2). For OX1 peak, linear range (1 – 1400 µM), limit of detection (260 nM) and sensitivity (0.0009 µA/µM) were obtained. Moreover, considering the peak of OX2, linear range (1 – 1400 µM), limit of detection (240 nM) and sensitivity (0.0009 µA/µM) were calculated. PAYR/Cu₂O@C/Nf/GCE presented considerable advantages for instance high sensitivity, used for real samples, simple preparation, low detection of limit, and specially the oxidation of bisphenol A at two applied potentials that it reported for the first time.
Highly sensitive paper-based electrochemical sensor for reagent free detection of bisphenol A
2020, Talanta
Bisphenol A is one the most relevant endocrine disruptors for its toxicity and ubiquity in the environment, being largely employed as raw material for manufacturing processes of a wide number of compounds. Furthermore, bisphenol A is released in the drinking water when plastic-based bottles are incorrectly transported under sunlight, delivering contaminated drinking water. For the health of human beings and the environment, rapid and on site detection of bisphenol A in drinking water is an important issue. Herein, we report a novel and cost-effective printed electrochemical sensor for an enzymatic-free bisphenol A detection. This sensor encompasses the entire electrochemical cell printed on filter paper and the reagents for the measurement loaded in the cellulose fiber network, for delivering a reagent-free analytical tool. The working electrode was printed using ink modified with carbon black, a cost effective nanomaterial for sensitive and sustainable bisphenol A determination. Several parameters including pH, frequency, and amplitude were optimized allowing for a detection limit of 0.03 μM with two linear ranges 0.1–0.9 μM and 1 μM–50 μM, using square wave voltammetry as electrochemical technique. The satisfactory recovery values found in river and drinking water samples demonstrated the suitability of this sensor for screening analyses in water samples. These results revealed the attractiveness of this paper-based device thanks to the synergic combination of paper and carbon black as cost-effective materials.
A simple hydroxylated multi-walled carbon nanotubes modified glassy carbon electrode for rapid amperometric detection of bisphenol A
2017, Sensors and Actuators, B: Chemical
Citation Excerpt :
Therefore, the development of new and rapid methods with cheap instrument and real time detection for the determination of BPA has become one attractive approach of research in analytical chemistry because of the practical applications. Various analytical methods, have been employed such as high performance liquid chromatography coupled to UV [14,15], fluorescence [16] and electrochemical detection [17,18], gas chromatography [19], liquid chromatography-mass spectrometry [20,21] and gas chromatography-mass spectrometry [22]. These methods can offer accurate and precise results, but they often require skilled operators and expensive equipment.
A novel amperometric sensor for the determination of bisphenol A (BPA) was fabricated using a glassy carbon electrode easily modified with multi-walled carbon nanotubes functionalized with hydroxyl groups. The catalytic activity of the modifier toward the oxidation of BPA was demonstrated by cyclic voltammetry, giving a well-defined peak at 0.55 V in sodium glycine buffer solution (pH 8.0). The flow injection analysis (FIA) system of BPA exhibited a linear response in the 1–24 μg L⁻¹ concentration range, with a detection limit of 0.81 μg L⁻¹. The current reached the steady-state value with a very fast response time (less than 10 s). The proposed method was successfully applied for the determination of BPA in real samples (water contained in plastic and baby bottles) with satisfactory results, in optimum agreement with those obtained by an independent HPLC method. In the light of our results and previous reports, the proposed system combines good analytical performance with simplicity of sensor fabrication, being ideal for routine sensing applications.
Metabolomic characterization of renal ischemia and reperfusion in a swine model
2016, Life Sciences
Citation Excerpt :
We showed an increase of urinary 1-methylnicotinamide, an intermediate metabolite of the synthesis of reactive species, able to active peroxisome receptors [9], and NAD synthesis [7]. This metabolite is filtered at the glomerulus and highly secreted into the tubules, which reflects the capacity of the renal tubular transport system to secrete organic cations [46] and it was related to tubular injury [38]. We observed, though indirectly, a decrease in glutathione reduced, by the increase in glutamate and 2-aminobutyrate in serum, a precursor of ophthalmic acid, that increased significantly in conjunction with glutathione consumption [58,61].
Acute kidney injury (AKI) is a serious complication in hospitalized and transplanted patients, and is mainly caused by ischemia/reperfusion (I/R). However, the current diagnosis of AKI based on acute alterations in serum creatinine or urine output is late and unspecific. To identify new systemic biomarkers for AKI, we performed serum and urine metabolomic profile analyses during percutaneous unilateral renal I/R in a well-controlled swine model. For this, serial serum and urine samples obtained during the pre-ischemia, ischemia and reperfusion periods were analyzed by ¹H nuclear magnetic resonance at 600 MHz. Through the metabolic profiles over I/R, we identified eight serum metabolites that increased with ischemia and recovered to basal values after reperfusion, delineating the ischemic period. In addition, we identified 13 urinary metabolites that changed during the early reperfusion reflecting the ischemic kidney, being able to differentiate between pre-ischemia and post I/R periods. All selected metabolites are described in terms of disease pathophysiology (change of energetic pathway and oxidative stress), which suggest that these serum and urinary metabolites are candidate AKI biomarkers. Interestingly, the selected metabolites allowed us to identify, well described NFκB, leptin, INF-γ and insulin pathways, and a new pathway (Huntingtin) that had not been previously implicated in renal I/R. Huntingtin showed different fragment patterns in ischemic versus non-ischemic kidneys. Therefore, the metabolomic profile found in renal I/R led to the identification of candidate disease biomarkers and a new pathway associated with renal injury.
Simultaneous quantification of niacin and its three main metabolites in human plasma by LC-MS/MS
2012, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
A sensitive and specific LC–MS/MS method for the simultaneous quantification of niacin (NA) and its three main metabolites nicotinamide (NAM), nicotinuric acid (NUA) and N-methyl-2-pyridone-5-carboxamide (2-Pyr) in human plasma has been developed and validated. Plasma samples (200 μL) were prepared by deproteinization with acetonitrile (500 μL), then the supernatant after centrifugation was evaporated and reconstituted. Chromatography was performed on a phenomenex synergi hydro-RP column with an isocratic elution of methanol-0.1% formic acid (5:95, v/v). The full separation of all analytes was achieved within 9 min. Multiple-reaction monitoring (MRM) using the fragmentation transitions of m/z 124.1 → 80.1, 123.1 → 80.0, 181.0 → 79.0 and 153.1 → 110.2 in positive electrospray ionization (ESI) mode was performed to quantify NA, NAM, NUA and 2-Pyr, respectively. The calibration curves were linear over the concentration range of 2.0–3000 ng/mL for NA and NUA, 10.0–1600 ng/mL for NAM and 50.0–5000 ng/mL for 2-Pyr. This method has been validated in accordance with the US FDA guidelines for bioanalytical method development and applied to the determination of NA and its three main metabolites in Chinese subjects following a single oral dose of niacin extended-release and simvastatin 1000 mg/20 mg. In particular, because of the endogenous NAM and 2-Pyr in human plasma, the concentrations of NAM and 2-Pyr in human plasma after dosing were determined by subtracting blank values of them.
Effect of selected NAD<sup>+</sup> analogues on mitochondria activity and proliferation of endothelial EA.hy926 cells
2010, European Journal of Pharmacology
The aim of the study was to examine the effect of 1-methylnicotinamide (MNA) and 1-methyl-3-nitropyridine (MNP) on mitochondria activity and proliferation of endothelial EA.hy926 cells. The activity of MNA was also referred to nicotinamide (NAM) being MNA metabolic precursor. NAM and MNA used at high concentrations (up to 1 mM) had no effect on mitochondria metabolism and proliferation of EA.hy926 cells. It could be related to the fact that these compounds hardly cross the cell membrane. It supports the results of our previous study suggesting that anti-inflammatory and anti-thrombotic effects of MNA could be associated with its ability to bind to glycosaminoglycans, especially heparins, located on the endothelium membrane without entering into target cells. In contrast, MNP caused substantial changes in mitochondria activity and proliferation of EA.hy926 cells. This compound used at low concentrations (below 100 µM) blocked the cell cycle of EA.hy926 cells in G1 phase and was very effective in inhibiting cell growth (IC₅₀ = 13.8 ± 2.4 µM). At higher concentrations (0.1–1 mM) MNP caused a significant reduction of cell survival. The observed effects of MNP could be related, at least in part, to its ability to influence the ATP and NAD⁺ intracellular levels. MNP caused also important changes in Ca²⁺ intracellular concentration, significant decrease in inner mitochondrial membrane potential and high increase in mitochondrial respiration of EA.hy926 cells. The observed effects of MNP may be related in part to its cellular metabolites detected after 45 min incubation with 250 µM MNP.

View all citing articles on Scopus

View full text

Validation of an HPLC method for the determination of urinary and plasma levels of N1-methylnicotinamide, an endogenous marker of renal cationic transport and plasma flow

Abstract

Introduction

Section snippets

Chemicals and reagents

Samples preparation

Conclusion

Acknowledgements

J. Chromatogr. B Biomed. Appl.

Am. J. Clin. Nutr.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

Methods Enzymol.

Anal. Biochem.

Pharm. Acta Helv.

Validation of an HPLC method for the determination of urinary and plasma levels of N¹-methylnicotinamide, an endogenous marker of renal cationic transport and plasma flow