Journal: European Journal of Clinical Pharmacology

Supplements:

Article title:N1-methylnicotinamide as an endogenous probe for drug interactions by renal cation transporters: studies on the metformin-interaction

Authors:Fabian Müller1, Constanza A. Pontones1, Bertold Renner1, Maren Mieth1, Eva Hoier1, Daniel Auge1, Renke Maas1, Oliver Zolk2 and Martin F. Fromm1

Affiliations:1Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

2Institute of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, Ulm, Germany

Adress for correspondence:

Prof. Martin F. Fromm, Director, Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany.Tel: +49 9131 85 22772; Fax: +49 9131 85 22773; E-mail:

Details on analytical methods.

To 200µl precipitating agent (0.1% [v/v] formic acid in acetonitrile) 50µl internal standard solution (metformin-d6 [@rtMolecule, Poitiers, France] / trimethoprim-d6 [Toronto Research Chemicals, North York, Ontario, Canada] 10µg/ml in acetonitrile) was added and mixed with a 100µl sample. The mixture was centrifuged and the supernatant diluted with mobile phase (8mM ammonium formate in acetonitrile-water [80:20] adjusted to pH 4 with formic acid). Chromatographic separation was achieved using an EC 250/2 Nucleodur 100-3 HILIC (Machery Nagel, Düren, Germany) with guard column. The analytical method was fully validated. The lower limits of quantification for metformin were 7.5ng/ml in plasma and 0.25µg/ml in urine and 100ng/ml for trimethoprim in plasma. For metformin the intraday accuracies ranged from 88.3% to 101.0% in urine. In plasma the accuracies ranged from 91.5% to 100.4% for metformin and from 86. 7% to 103.2% for trimethoprim. The intraday coefficients of variation were for both analytes and matrices below 8%. The interday accuracies for metformin ranged from 91.3% to 107.2% in urine and from 94.5% to 102.8% in plasma. For trimethoprim the interday accuracies ranged from 92.9% to 102.9%. The interday coefficients of variation were for both analytes and in all matrices below 13%.

NMN concentrations in plasma, urine and cell lysate were measured by means of HPLC-MS-MS (Agilent 1100 HPLC System, Agilent Technologies, Waldbronn, Germany; API 4000, Applied Biosystems, Darmstadt, Germany) as previously described with minor modifications [1]. In brief, a 100 µl sample was mixed with 100 µl internal standard solution (NMN-d3 [BDG-Synthesis, Wellington, New Zealand] 10µg/ml in acetonitrile) and 200µl acetonitrile. The mixture was centrifuged and the supernatant diluted with mobile phase. Chromatography was carried out isocratically using a mixture of 8mM ammonium formate in acetonitrile-water (80:20) adjusted to pH 4 with formic acid. The analytical method was fully validated. The limits of quantification of NMN were 50ng/ml in urine and 2.5ng/ml in plasma and lysate. Intraday accuracies ranged from 98.7% to 112.4% in urine, from 97.6% to 114.6% in plasma and from 93.9% to 111.6% in lysate. The intraday coefficients of variation were in all matrices below 6%. The interday accuracies ranged from 97.3% to 105.6% in urine, from 89.5% to 111.9% in plasma and from 88.4% to 114.2% in lysate. The interday coefficients of variation were in all matrices below 7%.

Reference:

1. Lang R, Wahl A, Skurk T, Yagar EF, Schmiech L, Eggers R, Hauner H, Hofmann T (2010) Development of a hydrophilic liquid interaction chromatography-high-performance liquid chromatography-tandem mass spectrometry based stable isotope dilution analysis and pharmacokinetic studies on bioactive pyridines in human plasma and urine after coffee consumption. Anal Chem 82:1486-1497