Human C-peptide Quantitation by LC-MS Isotope-Dilution Assay in Serum or Urine Samples

Abstract

Introduction C-peptide is a 31-amino acid central part of the pro-insulin molecule. Equal amounts of insulin and C-peptide are released from cleavage of proinsulin by specific beta cell endopeptidases within the pancreatic islets of Langerhans. The exact biological role of C-peptide is not completely understood. C-peptide has generally been considered a byproduct of insulin biosynthesis, but recent data suggest that it may have biological significance [1-3]. In contrast to serum insulin, which is cleared by the liver, C-peptide is cleared at a much slower rate by the kidney and thus represents a more useful indicator of intrinsic insulin secretion (due to 1:1 stoichiometric ratio with insulin) [4-6]. Low C-peptide concentrations in plasma can indicate early insulin secretory failure in the preclinical stages of diabetes. Additionally, for diabetes patients who take insulin, C-peptide measurement allows indirect assessment of endogenous insulin production. The reference interval for human C-peptide concentration in plasma is 0.5-10 ng/mL (0.15-3 nmol/L) [7,8]; its concentration in urine is approximately one order of magnitude higher [9, 10]. Mass-spectrometric (MS) analysis of C-peptide in processed blood (plasma samples) was first performed in 1996 [7] and was one of the first successful applications of the MS isotope dilution assay (IDA) for quantitative analysis of endogenous peptides. The authors used reversed phase chromatography in LC-MS quantitation, and employed solid phase extraction for sample preparation. The same general principle was utilized by other investigators who contributed to further method development [10-16], mainly aimed at increasing analysis throughput. To overcome the negative effects of poor ionization efficiency for C-peptide in MS analysis, a method that utilized two-dimensional reversed phase-reversed phase chromatography was introduced in 2006 [14,15]. A relatively small peak heart-cut fraction, less than 0.3mL containing the C-peptide peak was eluted using a shallow linear gradient during the first dimension separation; this fraction was then transferred to a second dimension reversed phase column prior to MS analysis. Two-dimensional chromatography was thus realized without complete sample mapping [17]. Although, in each dimension the same separation mechanism based on hydrophobic interaction was used, the signal to noise ratio was greatly improved, presumably due to the fact that different hydrophobic interaction stationary phases were employed Stage Procedure Conditions 1 precipitation with methanol v/v (4:6-8:2) followed by centrifugation at 12 g, 7 min 2 SepPak C18 in presence of methanol >70% 3 HiTrap SP HP Methanol<20%, pH 2.95 4 HiTrap Q HP Methanol<20%,sample application pH 7.0, elution Table 1: C-peptide purification scheme. Methanol precipitation was followed by centrifugation; the supernatant was then loaded onto C18 SepPak cartridge. The effluent was then applied to the ion exchanger after dilution of the methanol concentration and pH adjustment. Added organic solvent content, S, v/v Approximate final dilution factor 0.2 (20%) 1.25 (80%) 0.3 (30%) 2.15 (47%) 0.4 (40%) 3.3 (30%) 0.5 (50%) 5 (20%) 0.6 (60%) 7.5 (13%) 0.7 (70%) 12 (8.6%) 0.8 (80%) 20 (5%) 0.9 (90%) 45 (2.2%) Table 2: Final sample dilution factor as a result of two step purification. After methanol precipitation, additional sample dilution is caused by the necessity of reducing the content of organic solvent to the desired concentration (20% in this particular case, s=0.2). This "double dilution" results in very fast analyte content decline per sample volume, as given by the relationship: f=(1-S)(s/S). The values are reported without taking into account a correction for volumes non-additivity. Abstract In this communication we report a simple and efficient approach to C-peptide quantitation using isotope dilution mass-spectrometry analysis. The method facilitates quantitation of C-peptide levels at least one order of magnitude lower compared to concentration levels achieved with an IDA method reported previously. The improvement was due to more intensive sample preparation procedure that, in turn, makes it possible to increase the sample load without a corresponding increase in matrix effects. We also show the results of a comparison study with a second laboratory using a similar previously reported method for C-peptide quantitation. sequentially using different ion-pairing agents [15], which is resulted in different column selectivities. Importantly, the most abundant fragment ion of C-peptide is Y1 [m/z 147.1], which represents a yield of only 1.5% using collision induced fragmentation (the standard fragmentation 0.4% of formic acid (pH~2)