HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum.
A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatography (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52 +/- 12%, while analytical recovery was 81 +/- 10%. For the eight samples analyzed, the matrix effect was found to be only -14 +/- 6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addition method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%).
Callipo
Luciano
L
Department of Chemistry, Sapienza University of Rome, Box n masculine 34, Roma 62, Piazzale Aldo Moro 5, 00185, Rome, Italy.
Foglia
Patrizia
P
Gubbiotti
Riccardo
R
Samperi
Roberto
R
Laganà
Aldo
A
D000327:Adsorption; D000328:Adult; D002138:Calibration; D024402:Carbonic Anhydrase II / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D012016:Reference Values; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013499:Surface Properties; D013997:Time Factors; D055815:Young Adult
D010455:Peptides; D024402:Carbonic Anhydrase II