Supplements:
Journal:
European Journal of Clinical Pharmacology
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:
1
Institute of Experimental and Clinical Pharmacology and Toxicology,
Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
2
Institute 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 ErlangenNürnberg, Fahrstr. 17, 91054 Erlangen, Germany. Tel: +49 9131 85 22772;
Fax: +49 9131 85 22773; E-mail: martin.fromm@fau.de
Details on analytical methods.
To 200 µl precipitating agent (0.1 % [v/v] formic acid in acetonitrile) 50 µl internal standard
solution (metformin-d6 [@rtMolecule, Poitiers, France] / trimethoprim-d6 [Toronto Research
Chemicals, North York, Ontario, Canada] 10 µg/ml in acetonitrile) was added and mixed with
a 100 µl sample. The mixture was centrifuged and the supernatant diluted with mobile phase
(8 mM 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 7.5 ng/ml in plasma and
0.25 µg/ml in urine and 100 ng/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-MSMS (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] 10 µg/ml in acetonitrile) and 200 µl acetonitrile.
The mixture was centrifuged and the supernatant diluted with mobile phase. Chromatography
was carried out isocratically using a mixture of 8 mM ammonium formate in acetonitrilewater (80:20) adjusted to pH 4 with formic acid. The analytical method was fully validated.
The limits of quantification of NMN were 50 ng/ml in urine and 2.5 ng/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