HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum
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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%).
KeywordsLiquid chromatography-tandem mass spectrometry Chip Triple quadrupole Peptides Protein absolute quantification
The authors are grateful to Dr. Fiorella Guadagni (Scientific Institute for Research, Hospitalization and Health Care (SIRHHC or IRCCS in the Italian acronym) San Raffaele-Roma) for supplying the serum samples.
A special acknowledgment is given to Agilent Technologies and Agilent staff members, for their invaluable help and technical assistance. In particular, the authors wish to thank Alberto Stocco (Agilent Technologies) for his technical assistance and his infinite helpfulness.
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