Introduction

Osteoporosis is a skeletal disease characterized by low bone mass and loss of bone microarchitecture, which represents a major global health issue [1–3]. It accounts for more than 4% of the annual EU health expenditure[2–4]. The disease is caused by an increased rate of bone resorption, which involves osteoclasts releasing acids and proteases like cathepsin K to digest type I collagen.[5] This process results in the release of collagen-derived peptides into the bloodstream. Due to the importance of finding biomarkers for osteoporosis and other bone metabolism disorders, these peptides have been extensively studied and investigated as potential biomarkers[6–11]. During the course of these investigations, the primary emphasis was put on the analysis of pyridinoline/deoxypyridinoline crosslinks, highly specific to collagen molecules, and peptides bound to these crosslinks. This particular focus was intentional, as the preference for these markers over linear peptides stemmed from the fact that pyridinoline crosslinks present in biofluids are primarily derived from bones due to bone resorption [12]. This specificity makes them indicative of bone metabolism, whereas linear peptides could originate from various tissues rich in type I collagen. Furthermore, the inclusion of deoxypyridinoline crosslinks in the study was driven by their specificity to bones and dentin.[13,14] The outcomes of these investigations led to the identification and characterization of trivalently crosslinked C-telopeptide of the α1-chain of type I collagen (ICTP) and free pyridinoline[15–24]. These characterizations relied on preparative chromatography coupled with UV detection, MALDI TOF spectrometry, and N-sequencing analysis[25–27]. Subsequently, immunoassays were developed for the quantitative analysis of ICTP[28,29]. While the trivalently crosslinked C-Terminal telopeptide (CTX), trivalently crosslinked N-Terminal telopeptide (NTX), and amino-terminal propeptide of type I procollagen (PINP) can presently be quantified through immunoassays, their comprehensive characterization remains incomplete. Only specific portions of these molecules, such as the sequence EKAHDGGR in CTX, are recognized and employed as epitopes in immunoassays[28,30,31]. CTX and NTX are known to be made up of three peptides trivalently crosslinked together by a pyridinoline crosslink[32]. CTX and ICTP both originate from the same type I collagen region and share a common origin. However, they are released under different conditions and are cleaved by distinct enzymes. Specifically, CTX is released through the activity of cathepsin K during physiological bone resorption, while ICTP is likely cleaved by matrix metalloproteases during pathological bone resorption, as observed in bone metastasis[33]. To this day, CTX and PINP serve as the recommended markers for bone resorption and bone formation, respectively, according to the guidelines set forth by the International Osteoporosis Foundation and the International Federation of Clinical Chemistry and Laboratory Medicine for monitoring the therapeutic progress of osteoporotic patients[34,35]. Despite CTX endorsement, the absence of a reference method and variability in immunoassay results have diminished clinician confidence, leading to underutilization. The lack of complete marker characterization hampers the development of a definitive reference method, like liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), for precise quantitation. HR-MS analysis. Consequently, our research is dedicated to the comprehensive characterization of CTX found in human plasma and serum. Developing a LC-MS/MS method necessitates precise knowledge of the exact structure of the targeted molecule. This is crucial to achieve the highest level of specificity attainable by triple quadrupole mass spectrometry. In contrast to the development of immunoassays, where specific regions of a molecule can be targeted, LC-MS/MS methods require the comprehensive targeting of the entire molecule. This is because the method relies on the measurement of both the mass and charge of the molecule, demanding a comprehensive understanding of its structure. Therefore, we conducted an analysis of the various CTX species present in plasma and serum using a comprehensive workflow involving preparative chromatography, affinity chromatography, and HR-MS analysis.