Development of liquid chromatography with tandem mass spectrometry method for measurement of sepsis biomarkers

Background: Sepsis is a life-threatening condition that is caused by organ dysfunction due to dysregulated host-response to systemic pathogen infection. The most common sepsis diagnostic methods such as blood culture and molecular assays are often costly, time-consuming, and lack sensitivity. There is a need to develop alternative diagnostic methods which can produce faster results and detection of biomarker using mass spectrometry (MS) technique emerges as an appealing sepsis diagnostic tool. MS-based assay enables accurate measurement of biomarkers in serum and offers multiplexing capability. Measurements of biomarkers aid clinicians in their decision making and helps them to assess the efficacy of treatment. Hence, leading to improvement in the patient’s health and quality of life.

Objective: The aim of this study was to develop and validate a tandem mass spectrometry (MS/MS)-based approach that could be used for the simultaneous measurement of multiple biomarkers in septic patients. Surrogate peptides were identified to represent sepsis protein biomarkers C-reactive protein (CRP) and procalcitonin (PCT) for quantitation. Two different tandem MS modes: multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) were developed using triple quadrupole (QqQ) and Orbitrap mass spectrometer, respectively, for measurement of fragment ions of the chosen surrogate peptides. The detection performance such as limit of detection (LOD), limit of quantification (LOQ), and specificity of these two methods were compared.

Results: The result highlighted superiority of PRM method developed using the Orbitrap compared to MRM assay using QqQ. MRM method developed using an API 3000 QqQ is limited to monitor predefined fragment ions and less than three transition ions from CRP and PCT surrogate peptides could be monitored, which compromises the selectivity of the assay. In contrast, using the Orbitrap in PRM mode improves specificity by producing full-scan MS/MS spectra for the precursor ions of surrogate peptides of CRP and PCT.

The LOD of CRP and PCT in buffer using the MRM method developed using an API 3000 QqQ mass spectrometer was found to be 500 ng/mL and 1000 ng/mL respectively. Using a more modern QqQ LC-MS 8060 with capillary flow, LOD for both CRP and PCT in buffer was found to be 150 and 100 ng/mL, respectively. This was not deemed to be a significant improvement. On the other hand, using the Orbitrap in PRM mode CRP and PCT in buffer could be detected at 10 and 12.5 ng/mL, respectively.

Detection of biomarkers in serum sample using MS remains a challenge due to the presence of high-abundance proteins. Immunocapture technique using magnetic beads adopted in this study greatly improved detection of sepsis biomarker in human serum. The LOD and LOQ of CRP in serum using this method was found to be 0.4 and 1.33 μg/mL, respectively. The LOQ is below the CRP level found in non-septic patients of <6 μg/mL. On the other hand, The LOD and LOQ of PCT in serum using this method was found to be 0.5 and 1.8 μg/mL, respectively. The LOQ is still higher than the PCT level found in non-septic patients of <0.1 ng/mL.

Conclusions: This study has demonstrated that MS/MS coupled with upstream immunocapture using magnetic beads method can be used in detection of biomarker of sepsis in human serum and has a potential to be adopted in clinical laboratory. Nevertheless, the assay still has to be improved by performing more thorough optimization and validation, particularly to allow for the detection and quantification of PCT at a low level in serum for the diagnosis of sepsis. Additionally, the assay needs to be validated and examined on actual clinical samples before it can be implemented as a routine diagnostic tool for sepsis.