Simple and sensitive quantification of bioactive peptides in biological matrices using liquid chromatography/selected reaction monitoring mass spectrometry coupled with trichloroacetic acid clean-up.
A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed for the quantification of bioactive peptides in biological fluids. The method employs protein precipitation with 4% trichloroacetic acid (TCA) and selected reaction monitoring (SRM) using an immonium ion as the product ion. This method was applied to determine the synthetic parathyroid hormone (PTH) analog (MW 1721) in rat plasma and human hepcidin-25 (MW 2789) in human serum. TCA clean-up showed a sufficient recovery for peptides with a MW of less than 3000, and would be useful as a simple and rapid method because of direct injection of the supernatant without evaporation or dilution. In addition, TCA clean-up allowed us not only to reduce sample preparation time, but also to select an immonium ion as a product ion of SRM, which led to detection more sensitive than SRM using other types of product ions. The lower limits of quantitation (LLOQs) of the PTH analog and the human hepcidin-25 were 0.2 ng/mL and 5 ng/mL, respectively. This method was fully validated with acceptable linearity, intra- and inter-assay precisions, and accuracy. Furthermore, this simple and rapid method is applicable to pharmacokinetic studies.
Murao
Naoaki
N
Fuji Gotemba Research Labs, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. muraonoa@chugai-pharm.co.jp
Ishigai
Masaki
M
Yasuno
Hideyuki
H
Shimonaka
Yasushi
Y
Aso
Yoshinori
Y
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000097:blood / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D064451:Hepcidins; D006801:Humans; D010281:Parathyroid Hormone / Q000097:blood / Q000737:chemistry; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry*; D051381:Rats; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014238:Trichloroacetic Acid / Q000737:chemistry*
D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; C578703:Hamp protein, rat; D064451:Hepcidins; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D014238:Trichloroacetic Acid