title: Enzymatic tissue digestion as an alternative sample preparation approach for quantitative analysis using liquid chromatography-tandem mass spectrometry.
abstract: Compound extraction from biological tissue often presents a challenge for the bioanalytical chemist. Labor-intensive homogenization or sonication of whole or powdered tissue is performed before compounds can be extracted and analyzed. Enzymatic digestion is commonly used for tissue dissociation and cell harvesting and offers the advantages of unattended sample preparation, potential automation, and low cost. The feasibility of enzymatic digestion as an alternate tissue preparation technique was evaluated for bioanalysis of drugs in conjunction with LC/MS/MS. Two different enzymes (collagenase and proteinase K) that are known to degrade connective tissues to allow tissue dissolution were chosen for evaluation, employing well-known antidepressants desipramine and fluoxetine as test compounds in dog and rat brain tissue. Comparison between enzymatic digestion and conventional homogenization tissue preparation was performed, including investigation of matrix ionization suppression of both methods using a postcolumn infusion system. Results showed that enzymatic digestion has extraction efficiency comparable to homogenization. Matrix ionization suppression was not observed for either the test compounds evaluated or the sample extraction method. Test compound levels of incurred tissue samples prepared by enzymatic digestion were in good agreement with the values obtained by the conventional homogenization tissue preparation, indicating that enzymatic digestion is an appropriate tissue sample preparation method.
authors.affiliation: Pfizer Global Research and Development, Bioanalytical Research, Department of Pharmacokinetics, Dynamics & Metabolism, 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA.
authors.forename: Chongwoo
authors.initials: C
authors.lastname: Yu
authors.forename: Lara D
authors.initials: LD
authors.lastname: Penn
authors.forename: John
authors.initials: J
authors.lastname: Hollembaek
authors.forename: Wenlin
authors.initials: W
authors.lastname: Li
authors.forename: Lucinda H
authors.initials: LH
authors.lastname: Cohen
mesh_terms: D000818:Animals; D000928:Antidepressive Agents / Q000032:analysis; D001921:Brain / Q000201:enzymology* / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D017364:Collagenases / Q000378:metabolism; D003891:Desipramine / Q000008:administration & dosage / Q000032:analysis* / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D019286:Endopeptidase K / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D005473:Fluoxetine / Q000008:administration & dosage / Q000032:analysis* / Q000378:metabolism; D006220:Haloperidol / Q000008:administration & dosage / Q000032:analysis* / Q000378:metabolism; D066298:In Vitro Techniques; D013058:Mass Spectrometry / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D000928:Antidepressive Agents; D005473:Fluoxetine; D004950:Esterases; D019286:Endopeptidase K; D017364:Collagenases; D006220:Haloperidol; D003891:Desipramine