REPEATED PRESSURE-RELATED CHROMATOGRAPHIC FAILURE DURING SALIVARY CORTISOL ANALYSIS BY LC-HRMS/MS: A LABORATORY CASE REPORT AND HYPOTHESIS-DRIVEN ROOT CAUSE ASSESSMENT

SR UMRAO; S ELANGOVAN; MS THARAMMAL; MR QURESHI; SHS MOHAMMED; MM ALAM; F ASHRAF; FU AMIN; MM KHAN

doi:10.64013/bbasr.v2026i1.141

Authors

SR UMRAO Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates
S ELANGOVAN Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates
MS THARAMMAL Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates
MR QURESHI Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates
SHS MOHAMMED Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates
MM ALAM Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates
F ASHRAF Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates
FU AMIN Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates
MM KHAN Life Core Private Clinic LLC, Royal Group Headquarters (7th Floor), Al Salam Street, PO Box 63811, Abu Dhabi, United Arab Emirates

DOI:

https://doi.org/10.64013/bbasr.v2026i1.141

Keywords:

LC-HRMS/MS, Orbitrap, PRM, salivary cortisol, column fouling, back-pressure, matrix effects, method robustness, sample preparation, root-cause analysis

Abstract

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the reference standard method for quantitatively measuring salivary cortisol, largely because of its high analytical specificity and sensitivity. However, saliva is a complex biological matrix that may compromise chromatographic robustness during routine testing. Here, we report a laboratory case of system back-pressure exceedance, chromatographic deterioration, and analytical column collapse during routine LC-HRMS/MS analysis of salivary cortisol using a commercially available sample preparation and analysis kit. Two columns used in assay analysis failed consecutively. Both columns were subjected to the same working conditions. Factors such as how the samples were injected, chromatograms, and the behaviour of the system were analyzed. Possible causes of failures were also evaluated based on the pressure and behaviour of the columns. The team also evaluated possible causes based on column pressure and behaviour. The review indicated that the matrix caused the failure. Poorly removed salivary mucins and other particles clogged the columns. Flow restriction caused retention time to vary. The columns became worn out because the phase failed to remain permanently fixed to the column. Failures occurred in two columns and suggested a limitation of the method regarding matrix interaction with LC-MS/MS. This occurred mainly when high-viscosity saliva samples were analyzed using UHPLC columns. Restrictions were noted particularly at the column inlet. It was recommended that columns be well protected and that preventive maintenance be performed to reduce column failures and loss of phase integrity. This is especially true in laboratories that analyze salivary cortisol using LC-MS/MS. The report presents a case study illustrating the types of column failures caused by matrix limitations in LC-MS/MS analysis.

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