BACKGROUND: Isotope-dilution assays (IDAs) are well established for quantification of metabolites or small drug molecules in biological fluids. Because of their increased specificity, IDAs are an alternative to immunoassays for measuring C-peptide. METHODS: We evaluated a 2-dimensional liquid chromatography-mass spectrometry (2D LC/MS) IDA method. Sample preparation was by off-line solid-phase extraction, and C-peptide separation was performed on an Agilent 1100 2D LC system with a purification method based on high-pressure switching between 2 high-resolution reversed-phase columns. Because of the low fragmentation efficiency of C-peptide, multiple-reaction monitoring analysis was omitted and selective-ion monitoring mode was chosen for quantification. Native and isotope-labeled ([M+18] and [M+30]) C-peptides were monitored in the +3 state at m/z 1007.7, 1013.7, and 1017.7. RESULTS: The assay was linear (r(2) = 0.9995), with a detection limit of 300 amole (1 pg) on column. Inter- and intraday CVs for C-peptide were < or =2%. Comparison with an established polyclonal-based RIA showed high correlation (r = 0.964). Plasma concentrations of total C-peptide measured by RIA were consistently higher than by IDA LC/MS, consistent with the higher specificity of IDAs compared with immunoassays. CONCLUSIONS: The 2D LC/MS IDA approach eliminates matrix effects, enhancing assay performance and reliability, and has a detection limit 100-fold lower than any previously reported LC/MS method. Isotope-labeled C-peptide(s) can be clearly differentiated from endogenous C-peptide by the difference in m/z ratio, so that both peptides can be quantified simultaneously. The method is highly precise, robust, and applicable to pharmacokinetic detection of plasma peptides.
BACKGROUND: Isotope-dilution assays (IDAs) are well established for quantification of metabolites or small drug molecules in biological fluids. Because of their increased specificity, IDAs are an alternative to immunoassays for measuring C-peptide. METHODS: We evaluated a 2-dimensional liquid chromatography-mass spectrometry (2D LC/MS) IDA method. Sample preparation was by off-line solid-phase extraction, and C-peptide separation was performed on an Agilent 1100 2D LC system with a purification method based on high-pressure switching between 2 high-resolution reversed-phase columns. Because of the low fragmentation efficiency of C-peptide, multiple-reaction monitoring analysis was omitted and selective-ion monitoring mode was chosen for quantification. Native and isotope-labeled ([M+18] and [M+30]) C-peptides were monitored in the +3 state at m/z 1007.7, 1013.7, and 1017.7. RESULTS: The assay was linear (r(2) = 0.9995), with a detection limit of 300 amole (1 pg) on column. Inter- and intraday CVs for C-peptide were < or =2%. Comparison with an established polyclonal-based RIA showed high correlation (r = 0.964). Plasma concentrations of total C-peptide measured by RIA were consistently higher than by IDA LC/MS, consistent with the higher specificity of IDAs compared with immunoassays. CONCLUSIONS: The 2D LC/MS IDA approach eliminates matrix effects, enhancing assay performance and reliability, and has a detection limit 100-fold lower than any previously reported LC/MS method. Isotope-labeled C-peptide(s) can be clearly differentiated from endogenous C-peptide by the difference in m/z ratio, so that both peptides can be quantified simultaneously. The method is highly precise, robust, and applicable to pharmacokinetic detection of plasma peptides.
Authors: Weixun Wang; Bernard K Choi; Wenyu Li; Zhege Lao; Anita Y H Lee; Sandra C Souza; Nathan A Yates; Timothy Kowalski; Alessandro Pocai; Lucinda H Cohen Journal: J Am Soc Mass Spectrom Date: 2014-02-06 Impact factor: 3.109
Authors: Alexander V Stoyanov; Curt L Rohlfing; Shawn Connolly; Matthew L Roberts; Christopher L Nauser; Randie R Little Journal: J Chromatogr A Date: 2011-11-02 Impact factor: 4.759
Authors: Alexander V Stoyanov; Eduard Rogatsky; Daniel Stein; Shawn Connolly; Curt L Rohlfing; Randie R Little Journal: Proteomics Clin Appl Date: 2013-10-18 Impact factor: 3.494
Authors: Benjamin Y Owusu; Hannah Pflaum; Russell Garner; North Foulon; Thomas J Laha; Andrew N Hoofnagle Journal: J Mass Spectrom Adv Clin Lab Date: 2020-12-28