This observation is linked to the fact that them/zposition of a monoclonal light chain peak (S) relative to the polyclonal background (N) impacts the final S/N calculation. EXENT, the sample was then subjected to analysis by LC-MS. For the LC-MS analysis, the sample eluate, obtained after the MALDI-TOF MS spotting step, was collected and transferred to an autosampler tray for subsequent analysis using LC-MS. == Conclusion == LC-MS has the capability to detect monoclonal immunoglobulins that are no longer detected by EXENT. Reflexing samples to LC-MS for analysis does not involve additional sample handling, allowing for a faster time-to-result compared to current methods, such as Next-Generation Sequencing, Next-Generation Flow, and clonotypic peptide methods. Notably, LC-MS offers equivalent sensitivity in detecting these specific monoclonal immunoglobulins. == Introduction == For over two decades LC-MS has been used extensively in the development of therapeutic monoclonal antibodies (t-mAbs)[1]. The technology is usually ideally suited for the characterization of t-mAbs since monoclonality is usually defined as polypeptides with identical amino acid sequences, resulting in a single molecular mass. Recent studies have shown the power of mass spectrometry in confirming correct t-mAb expression by analyzing intact immunoglobulin molecular mass, reduced light chain and heavy chain molecular masses, and tryptic peptide molecular masses[2],[3]. Initial investigations describing the use of LC-MS to characterize endogenous immunoglobulins in blood circulation were conducted in parallel with t-mAb characterization work, focusing on immunoglobulins with an IgG isotype. These studies explored aspects such as heavy chain Fc region oxidation, allelic variants, and post-translational modifications, such as glycosylation[4],[5]. Research into the analysis of endogenous monoclonal immunoglobulins in serum began with methods that combined enzymatic digestion and LC-MS/MS to monitor constant region peptides common to specific isotypes[6]and, later, variable region clonotypic peptides[7],[8]. Subsequently, LC-MS methods with higher throughput were developed to monitor endogenous monoclonal immunoglobulins. In the beginning, Chlorpheniramine maleate these methods involved either no purification or enrichment using Melon Gel prior to LC-MS. These methods yielded significant findings including, the ability to isotype kappa and lambda light chains using top-down MS/MS[9], direct detection of monoclonal light chains from urine[10], insights into the origins of light chain polyclonal molecular mass distributions[11],[12], methods designed to quantify a t-mAb while monitoring oligoclonal immunoglobulin patterns[13], and comparison of oligoclonal immunoglobulins in cerebrospinal fluid (CSF) and serum[14]. The studies pointed out previously highlight the usefulness of LC-MS to identify and quantify endogenous monoclonal immunoglobulin. However, incorporating an LC-MS system in a laboratory that primarily employs gel-based analytical methods was challenging due to the associated Chlorpheniramine maleate costs of instrumentation and infrastructure. As an alternative, researchers explored the use of MALDI-TOF MS due to its lower cost, smaller footprint, reduced infrastructure requirements, and the availability of devices bearing a Class I medical device rating, a prerequisite for in vitro diagnostic (IVD) applications. A proof-of-concept study exhibited that MALDI-TOF MS could be utilized to identify endogenous monoclonal immunoglobulins, potentially replacing LC-MS, despite it being typically less sensitive[15]. Chlorpheniramine maleate Following this, option methods for isotype-specific immunoprecipitation were developed along with he optimization of MALDI-TOF MS sample preparation and acquisition parameters. This resulted in a viable methodology for detecting monoclonal immunoglobulin in a clinical laboratory establishing[16],[17],[18]. EXENT is usually a comprehensive clinical analyzer that consists of three main components: an automated liquid handler, EXENT-iP 500, a MALDI-TOF mass spectrometer, EXENT-iX 500, and instrument control and data analysis software, EXENT-iQ. Its main purpose is usually to detect and quantify monoclonal immunoglobulins in serum, making it a valuable device for clinical laboratories seeking IVD compliance. One notable advantage of using the MALDI-TOF MS technology employed by EXENT is usually its requirement for only a small sample volume for analysis. This ensures that a sufficient amount of the sample remains available for further analysis by LC-MS in instances where EXENT TNC yields a negative result. In this study, we present the results obtained from serum samples that were in the beginning analyzed using EXENT and subsequently subjected to reflex analysis by LC-MS. Our findings demonstrate that this eluates purified by EXENT can be directly analyzed by LC-MS without the need for any additional sample manipulation. This approach provides complementary data to that obtained from EXENT, enhancing comprehensive sample characterization. == 2. Materials and methods == Monoclonal Immunoglobulins and Serum: Serum samples from patients with a confirmed disease diagnosis of multiple.