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Chemical Analytics and Synthesis

Staff
Staff
• Monika Seiler
   laboratory technician
  Georg Heinkele
  chemical engineer
Portrait
Main focus of the department is quantitative, mass spectrometric analysis. Analytical methods for quantification have been developed for more than 100 different drugs and metabolites, being used primarily for pharmacokinetic studies and for in vitro investigations of enzyme kinetics and drug transport.

As part of metabolomics projects in the Virtual Liver Network (a major national initiative on systems biology of the liver, funded by the German Federal Ministry for Education and Research) intracellular and extracellular concentrations of endogenous metabolites in hepatocytes and liver tissue are determined. Liver-specific intermediates of glucose metabolism (glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway), lipid metabolism (ketone bodies, bile acids, intermediates in cholesterol synthesis) and amino acid metabolism are quantitatively determined.

The main task of the synthesis section is the chemical synthesis of otherwise not available substances (mostly drugs and their metabolites and stable isotope-labeled compounds). These substances are needed in virtually every area: In the analysis as reference substances and internal standards, for conducting in vitro studies on metabolism and transport of drugs and stable isotope-labeled drugs for clinical trials.

LiSym (Liver Systems Medicine)
Equipment
 

 
 
 
Mass spectrometers

 

 

  • GC-MS
  • GC-MSD 5975C inert XL, Agilent

    LC-MS(-MS)
  • Single Quadrupol 6120, Agilent
  • Triple Quadrupol 6460, Agilent
  • Triple Quadrupol 6495B, Agilent
  • Q-TOF iFunnel 6550 with UHPLC, Agilent
  • Ion Trap HCT plus, Bruker

HPLC Systems

  • 2 gradient HPLC systems (Agilent 1100/1200) with UV-Diode array and fluorescence detector, fraction collector
  • Devices for sample preparation

Chemical synthesis

  • Microwave reactor
  • Preparative flash chromatography system

 

Established quantitative methods for endogenous metabolites, mainly for cells and tissue samples

glycolytic metabolites (glucose, pyruvate, lactate, glucose 6-P, glucose 1-P, fructose 6-P, fructose 1,6-P2, 3-phosphoglycerate, phosphoenolpyruvate); pentose phosphate pathway (6-phosphogluconate, ribulose 5-P, ribose 5-P); TCA cycle (malate, fumarate, citrate, isocitrate, aconitate, 2-oxoglutarate); amino acids and urea (all 20 proteinogenic amino acids, ornithine, urea); co-metabolites for detoxification (UDP-glucuronic acid); cholesterol biosynthesis and metabolites (mevalonate, desmosterol, lathosterol, cholesterol, 4ß-hydroxycholesterol); bile acids (glycocholate, taurocholate, glycochenodeoxycholate, muricholate); nucleotides (energy charge, ATP, ADP, AMP); nucleosides (deoxycytidine, methyldeoxycytidine for determination of total DNA methylation); nucleobases (uracil, thymine and metabolites); lipids (about 25 individual triglycerides, present in liver, phosphatidylcholines, sphingomyelins, fatty acids, phosphatidylethanolamines semiquantitative, phosphatidylglycerols semiquantitative, phosphatidylserines semiquantitative); steroid hormones (cortisol, 6ß-hydroxycortisol, estradiol, estrone, estrone sulfate, DHEA, DHEAS, testosterone, androstenedione, progesterone, epitestosterone)

Established quantitative methods for drugs and drug metabolites (plasma, urine, cells, tissue, in vitro experiments)

5-ASA (acetyl-5-ASA); 5-fluorouracil; amodiaquine (desethyl amodiaquine); antipyrin (all major metabolites); budesonide; bupropion (hydroxybupropion); caffeine (paraxanthine); clomiphene (8 metabolites); cyclophosphamide; dextromethorphan (dextrorphan, hydroxymorphinan, methoxymorphinan, glucuronides); digoxin; doxorubicin (doxorubicinol); fexofenadine; ifosfamide; lansoprazole; letrozole (carbinol metabolite, carbinol glucuronide); MAP kinase inhibitors; mefloquine (mefloquine enantiomers, carboxymefloquine); mephenytoin (nirvanol, 4-hydroxymephenytoin); metformin; metoprolol (α-hydroxymetoprolol); midazolam (1'-hydroxymidazolam, 4-hydroxymidazolam); nabumetone (5 metabolites); omeprazole (5-hydroxyomeprazole, omeprazole sulfone); opiates (codeine, morphine, dihydrocodeine and desmethyl and glucuronide metabolites); phenacetine (acetaminophen); piritramide; pridinol (hydroxylated metabolites); propafenone (5-hydroxypropafenone, propafenone glucuronide, N-desalkylpropafenone); rabeprazol; statins (atorvastatin, lovastatin, simvastatin, pravastatin, rosuvastatin and metabolites); tamoxifen (endoxifen and further 30 metabolites); tenatoprazol; thiopurine drugs (6-thioguanine in plasma, all nucleotide metabolites in RBC); tolbutamide (hydroxytolbutamide); tyrosine kinase inhibitors (dasatinib, imatinib, N-desmethylimatinib, nilotinib); verapamil (all major metabolites)

Precht JC, Ganchev B, Heinkele G, Brauch H, Schwab M, Mürdter TE. Simultaneous quantitative analysis of letrozole, its carbinol metabolite, and carbinol glucuronide in human plasma by LC-MS/MS. Anal Bioanal Chem. 2012; 403(1):301-8.
Hofmann U, Heinkele G, Angelberger S, Schaeffeler E, Lichtenberger C, Jäger SU, Reinisch W, Schwab M. Simultaneous quantification of eleven thiopurine nucleotides by liquid chromatography - tandem mass spectrometry. Anal Chem. 2012;84(3):1294-301
Mürdter TE, Kerb R, Turpeinen M, Schroth W, Ganchev B, Böhmer GM, Igel S, Schaeffeler E, Zanger U, Brauch H, Schwab M. Genetic polymorphism of cytochrome P450 2D6 determines oestrogen receptor activity of the major infertility drug clomiphene via its active metabolites. Hum Mol Genet. 2012;21(5):1145-54
Dengler MA, Staiger AM, Gutekunst M, Hofmann U, Doszczak M, Scheurich P, Schwab M, Aulitzky WE, van der Kuip H. Oncogenic stress induced by acute hyper-activation of Bcr-Abl leads to cell death upon induction of excessive aerobic glycolysis. PLoS One. 2011;6(9):e25139.
Ganchev B, Heinkele G, Kerb R, Schwab M, Mürdter TE. Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography-electrospray ionization-tandem mass spectrometry. Anal Bioanal Chem. 2011;400(10):3429-41.
Mürdter TE, Schroth W, Bacchus-Gerybadze L, Winter S, Heinkele G, Simon W, Fasching PA, Fehm T; German Tamoxifen and AI Clinicians Group, Eichelbaum M, Schwab M, Brauch H. Activity levels of tamoxifen metabolites at the estrogen receptor and the impact of genetic polymorphisms of phase I and II enzymes on their concentration levels in plasma. Clin Pharmacol Ther. 2011;89(5):708-17.
Burk O, Brenner SS, Hofmann U, Tegude H, Igel S, Schwab M, Eichelbaum M, Alscher MD. The impact of thyroid disease on the regulation, expression, and function of ABCB1 (MDR1/P glycoprotein) and consequences for the disposition of digoxin. Clin Pharmacol Ther 2010;88(5):685-94.
Feidt DM, Klein K, Hofmann U, Riedmaier S, Knobeloch D, Thasler WE, Weiss TS, Schwab M, Zanger UM: Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using A New LC-MS/MS Cocktail Assay in Human Hepatocytes. Drug Metab Dispos 2010;38(9):1589-97
Maier K, Hofmann U, Reuss M, Mauch K. Dynamics and control of the central carbon metabolism in hepatoma cells. BMC Systems Biology 2010; 4:54.
Riedmaier S, Klein K, Hofmann U, Keskitalo JE, Neuvonen PJ, Schwab M, Niemi M, Zanger UM. UDP-glucuronosyltransferase (UGT) polymorphisms affect atorvastatin lactonization in vitro and in vivo. Clin Pharmacol Ther. 2010;87(1):65-73.
Birkenfeld AL, Jordan J, Hofmann U, Busjahn A, Franke G, Krüger N, Igel S, Mürdter T, Drescher S, Shi S, Engeli S, Schwab M, Eichelbaum M, Luft FC, Fromm MF. Genetic influences on the pharmacokinetics of orally and intravenously administered digoxin as exhibited by monozygotic twins. Clin Pharmacol Ther. 2009;86(6):605-8.
Maier K, Hofmann U, Bauer A, Niebel A, Vacun G, Reuss M, Mauch K. Quantification of statin effects on hepatic cholesterol synthesis by transient 13C flux analysis. Metab Eng. 2009;11(4-5):292-309.
Maier K, Hofmann U, Reuss M, Mauch K. Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part II. Flux estimation. Biotechnol Bioeng. 2008;100(2):355-70.
Hofmann U, Maier K, Niebel A, Vacun G, Reuss M, Mauch K. Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations. Biotechnol Bioeng. 2008;100(2):344-54.
Heinkele G, Mürdter TE. Synthesis of [2H3]-labelled sulfamethoxazole and its main urinary metabolites. J Labelled Comp Radiopharm 2007;50: 656-9
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