{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T02:43:05Z","timestamp":1792204985253,"version":"4.4.0"},"reference-count":32,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2022,6,15]],"date-time":"2022-06-15T00:00:00Z","timestamp":1655251200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the Tomi-Hopf-Stiftung, Switzerland"},{"name":"the Lorenzo-Piaggio Foundation, Switzerland"},{"name":"the Neuromuscular Research Association Basel, Switzerland"},{"name":"the University of Basel Children\u2019s Hospital"},{"name":"the Department of Neurology, University Hospital Basel"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Metformin (N,N-dimethylguanylguanidine) is one of the most prescribed drugs with pleiotropic, exerted in part by not fully elucidated mechanisms of action. We developed and validated a gas chromatography\u2013mass spectrometry (GC\u2013MS) method for the quantitative analysis of metformin (metformin-d0) in 10-\u00b5L aliquots of human serum and urine using N,N-[dimethylo-2H6]guanylguanidine (metformin-d6) as the internal standard. The method involves evaporation of the samples to dryness, derivatization with pentafluoropropionic (PFP) anhydride in ethyl acetate (30 min, 65 \u00b0C), and extraction into toluene. The negative-ion chemical ionization GC\u2013MS spectra of the PFP derivatives contain a single intense ion with mass-to-charge (m\/z) ratios of m\/z 383 for metformin-d0 and m\/z 389 for metformin-d6. Our results suggest that all amine\/imine groups of metformin-d0 and metformin-d6 are converted to their N,N,N-tripentafluoropropionyl derivatives, which cyclize to form a symmetric triazine derivative, of which the non-ring amine group is amidated. Quantification was performed by selected-ion monitoring (SIM) of m\/z 383 and m\/z 389. Upon validation, the method was applied to determine serum and urine metformin concentrations in 19 patients with Becker muscular dystrophy (BMD). Serum and urine samples were collected at baseline (Visit I), after six weeks of supplementation (Visit II) with metformin (3 \u00d7 500 mg\/d; metformin group; n = 10) or l-citrulline (3 \u00d7 1500 mg\/d; citrulline group; n = 9) followed by a six-week supplementation with 3 \u00d7 500 mg\/d of metformin plus 3 \u00d7 1500 mg\/d l-citrulline. At Visit I, the metformin concentration in the serum and urine was very low in both groups. The metformin concentrations in the serum and urine of the patients who first took metformin (MET group) were higher at Visit II and Visit III. The metformin concentration in the serum and urine samples of the patients who first took l-citrulline (CITR group) were higher at Visit III. The serum and urine concentrations of metformin were insignificantly lower in the CITR group at Visit III. The mean fractional excretion (FE) rate of metformin was 307% (Visit II) and 322% (Visit III) in the MET group, and 290% in the CITR group (Visit III). This observation suggests the accumulation of metformin in the kidney and its secretion in the urine. The GC\u2013MS is suitable to measure reliably circulating and excretory metformin in clinical settings.<\/jats:p>","DOI":"10.3390\/molecules27123850","type":"journal-article","created":{"date-parts":[[2022,6,16]],"date-time":"2022-06-16T03:01:22Z","timestamp":1655348482000},"page":"3850","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Stable-Isotope Dilution GC\u2013MS Measurement of Metformin in Human Serum and Urine after Derivatization with Pentafluoropropionic Anhydride and Its Application in Becker Muscular Dystrophy Patients Administered with Metformin, l-Citrulline, or Their Combination"],"prefix":"10.3390","volume":"27","author":[{"given":"Svetlana","family":"Baskal","sequence":"first","affiliation":[{"name":"Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30623 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Bollenbach","sequence":"additional","affiliation":[{"name":"Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30623 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bettina","family":"Henzi","sequence":"additional","affiliation":[{"name":"Division of Paediatric Neurology, University of Basel Children\u2019s Hospital, 4056 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia","family":"Hafner","sequence":"additional","affiliation":[{"name":"Division of Paediatric Neurology, University of Basel Children\u2019s Hospital, 4056 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0707-9201","authenticated-orcid":false,"given":"Dirk","family":"Fischer","sequence":"additional","affiliation":[{"name":"Division of Paediatric Neurology, University of Basel Children\u2019s Hospital, 4056 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6320-0956","authenticated-orcid":false,"given":"Dimitrios","family":"Tsikas","sequence":"additional","affiliation":[{"name":"Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30623 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/S0021-9673(00)86962-9","article-title":"Gas chromatography of amino acids","volume":"113","author":"Macek","year":"1975","journal-title":"J. Chromatogr. A"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.chroma.2016.03.019","article-title":"Profiling of urinary amino-carboxylic metabolites by in-situ heptafluorobutyl chloroformate mediated sample preparation and gas-mass spectrometry","volume":"1443","year":"2016","journal-title":"J. Chromatogr. A"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"121819","DOI":"10.1016\/j.jchromb.2019.121819","article-title":"Analytical strategies for the determination of amino acids: Past, present and future trends","volume":"1132","author":"Guillarme","year":"2019","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1071","DOI":"10.1007\/s00726-020-02884-7","article-title":"Analytical methods for amino acid determination in organisms","volume":"52","author":"Xu","year":"2020","journal-title":"Amino Acids"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1226","DOI":"10.1016\/j.jchromb.2010.11.013","article-title":"Derivatization of carbohydrates for GC and GC\u2013MS analyses","volume":"879","author":"Sanz","year":"2011","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1016\/j.jchromb.2014.08.040","article-title":"Improved stability of TMS derivatives for the robust quantification of plant polar metabolites by gas chromatography-mass spectrometry","volume":"970","author":"Jousse","year":"2014","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5565","DOI":"10.1021\/acs.analchem.7b00660","article-title":"High Throughput and Quantitative Measurement of Microbial Metabolome by Gas Chromatography\/Mass Spectrometry Using Automated Alkyl Chloroformate Derivatization","volume":"89","author":"Zhao","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1007\/s00726-018-02688-w","article-title":"Development and validation of GC\u2013MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: Evidence of altered methylation, transamidination, and arginase activity","volume":"51","author":"Hanff","year":"2019","journal-title":"Amino Acids"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1021\/ac60353a067","article-title":"Simple elcetron capture gas chromatographic method for the determination of oral hypoglycemic biguanidis in biological fluids","volume":"47","author":"Matin","year":"1975","journal-title":"Anal. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1002\/cpt1978246683","article-title":"Disposition of metformin (N,N-dimethylbiguanide) in man","volume":"24","author":"Sirtori","year":"1978","journal-title":"Clin. Pharmacol. Ther."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1007\/BF00562061","article-title":"Pharmacokinetics of metformin after intravenous and oral administration to man","volume":"16","author":"Neuvonen","year":"1979","journal-title":"Eur. J. Clin. Pharmacol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1111\/j.1365-2125.1981.tb01206.x","article-title":"Metformin kinetics in healthy subjects and in patients with diabetes mellitus","volume":"12","author":"Tucker","year":"1981","journal-title":"Br. J. Clin. Pharmacol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1391","DOI":"10.1007\/s00726-018-2614-7","article-title":"Effects of single and combined metformin and l-citrulline supplementation on l-arginine-related pathways in Becker muscular dystrophy patients: Possible biochemical and clinical implications","volume":"50","author":"Hanff","year":"2018","journal-title":"Amino Acids"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Hafner, P., Bonati, U., Erne, B., Schmid, M., Rubino, D., Pohlman, U., Peters, T., Rutz, E., Frank, S., and Neuhaus, C. (2016). Improved muscle function in Duchenne Muscular Dystrophy through l-arginine and metformin: An investigator-initiated, open-label, single-center, proof-of-concept-study. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0147634"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1186\/s13063-016-1503-1","article-title":"Treatment with l-citrulline and metformin in Duchenne muscular dystrophy: Study protocol for a single-centre, randomised, placebo-controlled trial","volume":"17","author":"Hafner","year":"2016","journal-title":"Trials"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1111\/j.1365-2125.1978.tb00852.x","article-title":"Determination of metformin in biological samples","volume":"6","author":"Lennard","year":"1978","journal-title":"Br. J. Clin. Pharmacol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"81","DOI":"10.2165\/11534750-000000000-00000","article-title":"Clinical pharmacokinetics of metformin","volume":"50","author":"Graham","year":"2011","journal-title":"Clin. Pharmacokinet."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1920","DOI":"10.2967\/jnumed.116.177774","article-title":"In vivo imaging of human 11C-metformin in peripheral organs: Dosimetry, biodistribution, and kinetic analyses","volume":"57","author":"Gormsen","year":"2016","journal-title":"J. Nucl. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1080\/10408347.2017.1304200","article-title":"Analytical Methods for Metformin Estimation","volume":"47","author":"Patel","year":"2017","journal-title":"Crit. Rev. Anal. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2015","DOI":"10.4155\/bio-2017-0182","article-title":"Bioanalytical methods for the detection of antidiabetic drugs: A review","volume":"9","author":"Fachi","year":"2017","journal-title":"Bioanalysis"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1080\/10408347.2017.1374165","article-title":"Metformin: A Review of Characteristics, Properties, Analytical Methods and Impact in the Green Chemistry","volume":"48","author":"Kogawa","year":"2018","journal-title":"Crit. Rev. Anal. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e4789","DOI":"10.1002\/jms.4789","article-title":"Design of experiment-based LC\u2013MS\/MS method development for simultaneous estimation of nateglinide and metformin hydrochloride in rat plasma","volume":"56","author":"Patel","year":"2021","journal-title":"J. Mass Spectrom."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Chaudhari, K., Wang, J., Xu, Y., Winters, A., Wang, L., Dong, X., Cheng, E.Y., Liu, R., and Yang, S.H. (2020). Determination of metformin bio-distribution by LC\u2013MS\/MS in mice treated with a clinically relevant paradigm. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0234571"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1380","DOI":"10.1002\/dta.2866","article-title":"Development of a new GC\u2013MS\/MS method for the determination of metformin in human hair","volume":"12","author":"Arbouche","year":"2020","journal-title":"Drug Test Anal."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4723","DOI":"10.1039\/c3ay40507a","article-title":"The development and validation of a gas chromatography-mass spectrometry method for the determination of metformin in human plasma","volume":"5","year":"2013","journal-title":"Anal. Methods"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"10935","DOI":"10.1021\/acs.analchem.5b03003","article-title":"Thermal Degradation of Small Molecules: A Global Metabolomic Investigation","volume":"87","author":"Fang","year":"2015","journal-title":"Anal. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"122902","DOI":"10.1016\/j.jchromb.2021.122902","article-title":"Thermal degradation of metabolites in urine using multiple isotope-labelled internal standards for off-line GC metabolomics\u2014Effects of injector and oven temperatures","volume":"1181","author":"Tsochatzis","year":"2021","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Baskal, S., Bollenbach, A., and Tsikas, D. (2021). Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC\u2013MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts. Molecules, 26.","DOI":"10.3390\/molecules26061726"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/j.jchromb.2016.03.034","article-title":"Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings","volume":"1047","author":"Hanff","year":"2017","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2582","DOI":"10.1016\/j.jchromb.2010.04.025","article-title":"GC\u2013MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaff\u00e9, HPLC and enzymatic assays","volume":"878","author":"Tsikas","year":"2010","journal-title":"J. Chromatogr. B Anal. Technol. Biomed. Life Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2074","DOI":"10.2337\/diabetes.51.7.2074","article-title":"Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes","volume":"51","author":"Musi","year":"2002","journal-title":"Diabetes"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"379","DOI":"10.2133\/dmpk.20.379","article-title":"Metformin is a superior substrate for renal organic cation transporter OCT2 rather than hepatic OCT1","volume":"20","author":"Kimura","year":"2005","journal-title":"Drug Metab. Pharmacokinet."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/27\/12\/3850\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:32:21Z","timestamp":1760139141000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/27\/12\/3850"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,15]]},"references-count":32,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["molecules27123850"],"URL":"https:\/\/doi.org\/10.3390\/molecules27123850","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,15]]}}}