title: High-sensitivity quantitation of a Nanobody® in plasma by single-cartridge multidimensional SPE and ultra-performance LC-MS/MS.
abstract: BACKGROUND
  A major challenge in protein quantitation based on enzymatic digestion of complex biological samples and subsequent LC-MS/MS analysis of a signature peptide is dealing with the high complexity of the matrix after digestion, which can reduce sensitivity considerably.
  
  
  RESULTS
  Using single cartridge multidimensional SPE, sufficient selectivity was introduced to allow quantitation in 50 µl of plasma down to 10.0 ng/ml (~0.3 nM). An inhouse prepared (18)O-labeled signature peptide was used as the internal standard. The procedure was validated for human and rabbit plasma.
  
  
  CONCLUSIONS
  The developed SPE procedure allowed the sensitive and selective LC-MS/MS quantitation of the Nanobody(®) without the use of antibodies. When appropriate precautions are taken, the (18)O-labeled peptide is a practical and economical alternative to custom synthesis.
authors.affiliation: Bioanalytical Laboratory, PRA Health Sciences, Early Development Services, Westerbrink 3, 9405 BJ, Assen, The Netherlands.
authors.forename: Kees J
authors.initials: KJ
authors.lastname: Bronsema
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Bouche
authors.forename: Kjell
authors.initials: K
authors.lastname: Mortier
authors.forename: Nico C
authors.initials: NC
authors.lastname: van de Merbel
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D061905:Single-Domain Antibodies / Q000097:blood*
chemical_list: D061905:Single-Domain Antibodies
keywords: LC–MS/MS; Nanobody; antibody-free; biopharmaceutical; enzymatic digestion; high sensitivity; multidimensional SPE; oxygen exchange internal standard