Sensitive quantitative analysis of C-peptide in human plasma by 2-dimensional liquid chromatography-mass spectrometry isotope-dilution assay.
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.
Department of Medicine and General Clinical Research Center at Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.
Eduard
E
Rogatsky
Beate
B
Balent
Gayotri
G
Goswami
Vlad
V
Tomuta
Harsha
H
Jayatillake
Greg
G
Cruikshank
Louis
L
Vele
Daniel T
DT
Stein
D002096:C-Peptide / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007201:Indicator Dilution Techniques; D013058:Mass Spectrometry; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity
D002096:C-Peptide