Skip to main content

Advertisement

SpringerLink
Log in

Development and validation of an LC-ESI-MS/MS method for the triple reuptake inhibitor indatraline enabling its quantification in MS Binding Assays

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

We herein present the first LC-MS/MS quantification method for indatraline, a highly potent nonselective inhibitor of the three monoamine transporters (for dopamine, DAT; norepinephrine, NET; serotonin, SERT), and its application to MS Binding Assays. For HPLC, an R18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L-1, pH 10.0) in a ratio of 90:10 (v/v) at a flow rate of 600 μL min-1 was used. Recording indatraline at m/z 292.2/261.0 and (2H7)-indatraline, employed as internal standard, at m/z 299.2/268.0 allowed reliable quantification from 5 pmol L−1 (LLOQ) to 5 nmol L−1 in biological matrices without additional sample preparation. Validation of the developed quantification method showed that selectivity, calibration standard curve, accuracy, as well as precision meet the criteria of the CDER guideline. Applying this method to mass spectrometry (MS) Binding Assays, a label-free MS-based alternative to conventional radioligand binding assays, binding of indatraline’s eutomer, (1R,3S)-indatraline, towards NET could be characterized directly for the first time, revealing an equilibrium dissociation constant (K d) of 805 pmol L−1. Additionally, it could be shown that the established MS Binding Assays enable characterization of test compounds in competition experiments. As the established setup is based on a 96-well format and an LC MS/MS method with a short chromatographic cycle time (1.5 min), the developed MS Binding Assays enable considerable throughput and are therefore well suited as substitute for corresponding radioligand binding assays.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Schildkraut JJ, Kety SS (1967) Biogenic amines and emotion. Science 156(3771):21–37

    Article  CAS  Google Scholar 

  2. Lapin IP, Oxenkrug GF (1969) Intensification of the central serotoninergic processes as a possible determinant of the thymoleptic effect. Lancet 1(7586):132–136

    Article  CAS  Google Scholar 

  3. Skolnick P, Popik P, Janowsky A, Beer B, Lippa AS (2003) “Broad spectrum” antidepressants: is more better for the treatment of depression? Life Sci 73(25):3175–3179

    Article  CAS  Google Scholar 

  4. Marks DM, Pae CU, Patkar AA (2008) Triple reuptake inhibitors: the next generation of antidepressants. Curr Neuropharmacol 6(4):338–343. doi:10.2174/157015908787386078

    Article  CAS  Google Scholar 

  5. Schermann SM, Simmons DA, Konermann L (2005) Mass spectrometry-based approaches to protein-ligand interactions. Expert Review Proteomics 2(4):475–485. doi:10.1586/14789450.2.4.475

    Article  CAS  Google Scholar 

  6. Jonker N, Kool J, Irth H, Niessen WM (2011) Recent developments in protein-ligand affinity mass spectrometry. Anal Bioanal Chem 399(8):2669–2681. doi:10.1007/s00216-010-4350-z

    Article  CAS  Google Scholar 

  7. Holdgate GA, Anderson M, Edfeldt F, Geschwindner S (2010) Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: matching high information content with high throughput. J Struct Biol 172(1):142–157. doi:10.1016/j.jsb.2010.06.024

    Article  CAS  Google Scholar 

  8. Geoghegan KF, Kelly MA (2005) Biochemical applications of mass spectrometry in pharmaceutical drug discovery. Mass Spectrom Rev 24(3):347–366. doi:10.1002/mas.20019

    Article  CAS  Google Scholar 

  9. Annis DA, Nickbarg E, Yang X, Ziebell MR, Whitehurst CE (2007) Affinity selection-mass spectrometry screening techniques for small molecule drug discovery. Curr Opin Chem Biol 11(5):518–526. doi:10.1016/j.cbpa.2007.07.011

    Article  CAS  Google Scholar 

  10. Siegel MM (2005) Mass-spectrometry based drug screening assays for early phases in drug discovery. In: Lee MS (ed) Integrated strategies for drug discovery using mass spectrometry. Wiley, New York, pp 27–70

    Chapter  Google Scholar 

  11. Hess M, Höfner G, Wanner KT (2011) Development and validation of a rapid LC-ESI-MS/MS method for quantification of fluoxetine and its application to MS binding assays. Anal Bioanal Chem 400(10):3505–3515. doi:10.1007/s00216-011-4997-0

    Article  CAS  Google Scholar 

  12. Zepperitz C, Höfner G, Wanner KT (2006) MS-binding assays: kinetic, saturation, and competitive experiments based on quantitation of bound marker as exemplified by the GABA transporter mGAT1. ChemMedChem 1(2):208–217. doi:10.1002/cmdc.200500038

    Article  CAS  Google Scholar 

  13. Bogeso KP, Christensen AV, Hyttel J, Liljefors T (1985) 3-Phenyl-1-indanamines. Potential antidepressant activity and potent inhibition of dopamine, norepinephrine, and serotonin uptake. J Med Chem 28(12):1817–1828

    Article  CAS  Google Scholar 

  14. Rothman RB, Cadet JL, Akunne HC, Silverthorn ML, Baumann MH, Carroll FI, Rice KC, de Costa BR, Partilla JS, Wang JB et al (1994) Studies of the biogenic amine transporters. IV. Demonstration of a multiplicity of binding sites in rat caudate membranes for the cocaine analog [125I]RTI-55. J Pharmacol Exp Ther 270(1):296–309

    CAS  Google Scholar 

  15. Lengyel K, Pieschl R, Strong T, Molski T, Mattson G, Lodge NJ, Li YW (2008) Ex vivo assessment of binding site occupancy of monoamine reuptake inhibitors: methodology and biological significance. Neuropharmacology 55(1):63–70. doi:10.1016/j.neuropharm.2008.04.014

    Article  CAS  Google Scholar 

  16. Froimowitz M, Wu KM, Moussa A, Haidar RM, Jurayj J, George C, Gardner EL (2000) Slow-onset, long-duration 3-(3′,4′-dichlorophenyl)-1-indanamine monoamine reuptake blockers as potential medications to treat cocaine abuse. J Med Chem 43(26):4981–4992

    Article  CAS  Google Scholar 

  17. Xu C, Coffey LL, Reith ME (1995) Translocation of dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane (WIN 35,428) measured under identical conditions in rat striatal synaptosomal preparations. Inhibition by various blockers. Biochem Pharmacol 49(3):339–350

    Article  CAS  Google Scholar 

  18. Deutsch HM, Schweri MM (1994) Can stimulant binding and dopamine transport be differentiated? Studies with GBR 12783 derivatives. Life Sci 55(6):PL115–PL120

    Article  CAS  Google Scholar 

  19. Valchar M, Hanbauer I (1993) Comparison of [3H]WIN 35,428 binding, a marker for dopamine transporter, in embryonic mesencephalic neuronal cultures with striatal membranes of adult rats. J Neurochem 60(2):469–476

    Article  CAS  Google Scholar 

  20. Cheetham SC, Viggers JA, Butler SA, Prow MR, Heal DJ (1996) [3H]nisoxetine—a radioligand for noradrenaline reuptake sites: correlation with inhibition of [3H]noradrenaline uptake and effect of DSP-4 lesioning and antidepressant treatments. Neuropharmacology 35(1):63–70

    Article  CAS  Google Scholar 

  21. Rothman RB, Silverthorn ML, Glowa JR, Matecka D, Rice KC, Carroll FI, Partilla JS, Uhl GR, Vandenbergh DJ, Dersch CM (1998) Studies of the biogenic amine transporters. VII. Characterization of a novel cocaine binding site identified with [125I]RTI-55 in membranes prepared from human, monkey and guinea pig caudate. Synapse 28(4):322–338. doi:10.1002/(SICI)1098-2396(199804)28:4<322::AID-SYN8>3.0.CO;2-B

    Article  CAS  Google Scholar 

  22. Martin RS, Henningsen RA, Suen A, Apparsundaram S, Leung B, Jia Z, Kondru RK, Milla ME (2008) Kinetic and thermodynamic assessment of binding of serotonin transporter inhibitors. J Pharmacol Exp Ther 327(3):991–1000. doi:10.1124/jpet.108.142307

    Article  CAS  Google Scholar 

  23. Apparsundaram S, Stockdale DJ, Henningsen RA, Milla ME, Martin RS (2008) Antidepressants targeting the serotonin reuptake transporter act via a competitive mechanism. J Pharmacol Exp Ther 327(3):982–990. doi:10.1124/jpet.108.142315

    Article  CAS  Google Scholar 

  24. Navarro HA, Xu H, Zhong D, Blough BE, Ross WP, Kuhar MJ, Carroll FI (2001) [(125)I]3beta-(4-ethyl-3-iodophenyl)nortropane-2beta-carboxylic acid methyl ester ([(125)I]EINT): a potent and selective radioligand for the brain serotonin transporter. Synapse 41(3):241–247. doi:10.1002/syn.1081

    Article  CAS  Google Scholar 

  25. Cheetham SC, Viggers JA, Slater NA, Heal DJ, Buckett WR (1993) [3H]paroxetine binding in rat frontal cortex strongly correlates with [3H]5-HT uptake: effect of administration of various antidepressant treatments. Neuropharmacology 32(8):737–743

    Article  CAS  Google Scholar 

  26. Hulme EC (1992) Receptor ligand interactions—a practical approach. Oxford University Press, New York

    Google Scholar 

  27. Grimm SH, Allmendinger L, Höfner G, Wanner KT (2013) Enantiopurity determination of the enantiomers of the triple reuptake inhibitor indatraline. Chirality 25:923–933. doi:10.1002/chir.22235

    Article  CAS  Google Scholar 

  28. Allmendinger L, Wanner KT (2014) Synthesis of [2H7]-Indatraline. J Labelled Compd Radiopharm. doi:10.1002/jlcr.3245

  29. Hess M, Höfner G, Wanner KT (2011) (S)- and (R)-fluoxetine as native markers in mass spectrometry (MS) binding assays addressing the serotonin transporter. ChemMedChem 6(10):1900–1908. doi:10.1002/cmdc.201100251

    Article  CAS  Google Scholar 

  30. Unger KK, Weber E (1999) Handbuch der HPLC Teil 1, 2nd edn. Git Verlag, Darmstadt

    Google Scholar 

  31. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  32. Weissberg A, Dagan S (2011) Interpretation of ESI(+)-MS-MS spectra—towards the identification of “unknowns”. Int J Mass Spectrom 299:158–168

    Article  CAS  Google Scholar 

  33. Zhang M, Gao F, Cui X, Zhang Y, Sun Y, Gu J (2011) Development and validation of an improved method for the quantification of sertraline in human plasma using LC-MS-MS and its application to bioequivalence studies. J Chromatogr Sci 49:89–93

    Article  Google Scholar 

  34. Watson JT, Sparkman OD (2007) Introduction to mass spectrometry—instrumentation, applications and strategies for data interpretation, 4th edn. Wiley, New York

    Google Scholar 

  35. Nierenberg DW, Lester DC (1985) Determination of vitamins A and E in serum and plasma using a simplified clarification method and high-performance liquid chromatography. J Chromatogr 345(2):275–284

    Article  CAS  Google Scholar 

  36. Haas R, Rosenberry TL (1995) Protein denaturation by addition and removal of acetonitrile: application to tryptic digestion of acetylcholinesterase. Anal Biochem 224(1):425–427. doi:10.1006/abio.1995.1061

    Article  CAS  Google Scholar 

  37. Sindelar M, Wanner KT (2012) Library screening by means of mass spectrometry (MS) binding assays-exemplarily demonstrated for a pseudostatic library addressing gamma-aminobutyric acid (GABA) transporter 1 (GAT1). ChemMedChem 7(9):1678–1690. doi:10.1002/cmdc.201200201

    Article  CAS  Google Scholar 

  38. FDA US (2001) Guidance for industry, bioanalytical method validation. Available at: http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf

  39. Davenport AP, Russel FD (1996) Radioligand binding assays: theory and practice. In: Mather S (ed) Current directions in radiopharmaceutical research and development. Springer, New York, pp 169–179

    Chapter  Google Scholar 

  40. Lammertsma AA, Leysen JE, Heylen L, Langlois X (2012) Receptors: binding assays. Springer, SpringerReference. Available at: http://www.springerreference.com/docs/html/chapterdbid/169194.html. Accessed 01 July 2014

  41. Eshleman AJ, Carmolli M, Cumbay M, Martens CR, Neve KA, Janowsky A (1999) Characteristics of drug interactions with recombinant biogenic amine transporters expressed in the same cell type. J Pharmacol Exp Ther 289(2):877–885

    CAS  Google Scholar 

Download references

Acknowledgments

We thank Prof. Harald H. Sitte for providing the pRc/CMV vector containing the cDNA coding for hNET. For synthesizing indatraline and (2H7)-indatraline, we are very thankful to Lars Allmendinger and Gerd Bauschke (LMU München, Department of Pharmacy).

Author information

Authors and Affiliations

  1. Department Pharmazie—Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 7, 81377, Munich, Germany

    Stefanie H. Grimm, Georg Höfner & Klaus T. Wanner

Authors
  1. Stefanie H. Grimm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Georg Höfner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Klaus T. Wanner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Klaus T. Wanner.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 587 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Grimm, S.H., Höfner, G. & Wanner, K.T. Development and validation of an LC-ESI-MS/MS method for the triple reuptake inhibitor indatraline enabling its quantification in MS Binding Assays. Anal Bioanal Chem 407, 471–485 (2015). https://doi.org/10.1007/s00216-014-8312-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-8312-8

Keywords

Search

Navigation