The potential for Isotope Dilution-LC-MS/MS to improve laboratory measurement of C-peptide: reasons and critical determinants

Abstract Background: Serum C-peptide concentrations reflect pancreatic function in different clinical and diagnostic settings; however, the utility of C-peptide testing is limited by the lack of standardized commercial immunoassays. Standardization can best be done by split-sample comparison with a hierarchically higher reference measurement procedure with a set of native sera. For serum peptides, isotope-dilution liquid chromatography–mass spectrometry (ID-LC/MS) is recommended as a reference measurement procedure. Methods: We evaluated the analytical performance characteristics of an ID-LC/tandem MS procedure for measurement of serum C-peptide after a 2-step solid-phase extraction. To investigate the feasibility of this procedure for use in standardization, we also performed a method comparison with 3 representative commercial assays. Results: The ID-LC/tandem MS procedure showed maximum within-run, between-run, and total CVs on dedicated sera (C-peptide concentrations, 1.6 and 4.0 μg/L) of 2.1%, 2.5%, and 2.9%, respectively; an accuracy of 94.6%–104.1%; a minimum trueness of 98.1% (95% confidence interval, 96.2%–100.0%), and limits of quantification and detection of 0.15 and 0.03 μg/L, respectively. Deming linear regression analysis of the method-comparison data showed that the immunoassays correlated well with ID-MS and were specific, but lacked intercomparability and trueness. We propose that the deficiencies can be resolved by recalibration on the basis of the method comparison. Conclusions: The ID-LC/tandem MS procedure is suitable for specific and accurate measurement of basal and stimulated serum concentrations of proinsulin C-peptide fragment 33–63 and is suitable for use in standardization of C-peptide immunoassays.

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Use of cation exchange chromatography for human C-peptide isotope dilution – Mass spectrometric assay

Journal of Chromatography A ◽

10.1016/j.chroma.2011.10.080 ◽

2011 ◽

Vol 1218 (51) ◽

pp. 9244-9249 ◽

Cited By ~ 8

Author(s):

Alexander V. Stoyanov ◽

Curt L. Rohlfing ◽

Shawn Connolly ◽

Matthew L. Roberts ◽

Christopher L. Nauser ◽

...

Keyword(s):

Cation Exchange ◽

Isotope Dilution ◽

Exchange Chromatography ◽

Mass Spectrometric ◽

Cation Exchange Chromatography ◽

C Peptide

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Sensitive Quantitative Analysis of C-Peptide in Human Plasma by 2-Dimensional Liquid Chromatography–Mass Spectrometry Isotope-Dilution Assay

Clinical Chemistry ◽

10.1373/clinchem.2005.063081 ◽

2006 ◽

Vol 52 (5) ◽

pp. 872-879 ◽

Cited By ~ 35

Author(s):

Eduard Rogatsky ◽

Beate Balent ◽

Gayotri Goswami ◽

Vlad Tomuta ◽

Harsha Jayatillake ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Detection Limit ◽

Isotope Dilution ◽

Matrix Effects ◽

Multiple Reaction Monitoring ◽

Plasma Concentrations ◽

Liquid Chromatography Mass Spectrometry ◽

C Peptide ◽

Chromatography Mass Spectrometry

Abstract 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 (r2 = 0.9995), with a detection limit of 300 amole (1 pg) on column. Inter- and intraday CVs for C-peptide were ≤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.

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