MSACL 2025 Abstract

Start as You Mean to Go On; Controlling Batch Effects in Quantitative LC-SRM Measures

Russell P Grant, Erin M Lewis, Matthew L Crawford, Stacy Dee, Christopher M. Shuford
Labcorp, Burlington, NC, USA

Russell Grant, PhD (Presenter) Labcorp

Presenter Bio: Dr. Grant earned a first-class honors degree in Industrial Chemistry from Cardiff University and a PhD in Chromatographic and Mass Spectrometric technologies from the University of Swansea, Wales, United Kingdom. He continued his scientific training in various industrial settings, which have included senior scientist at GSK, Principal scientist at Cohesive Technologies, Technical director at Eli Lilly, and Director of Mass Spectrometry at Esoterix Endocrinology. Dr Grant is currently the Vice President of Research and Development and co-discipline director for Mass spectrometry at Labcorp. Dr Grant has pioneered the use of direct injection technologies, chromatographic systems multiplexing, microsampling, utility of automation, and other new analytical platforms in direct patient care. His research goals are focused upon improvements in speed, sensitivity, and quality of liquid chromatography with tandem mass spectrometric (LC-MS/MS) analytical systems and assays. Dr Grant has been awarded 100 patents and received both the MSACL Distinguished contribution award and ASMS AL Yergey “Unsung Hero” Award in 2024 for his contributions to Clinical Diagnostics using Mass Spectrometry.

Relevant Financial Disclosures (within past 24 months, reported on Apr 23, 2026) Other Potential Conflicts HepQuant / Consultant LabCorp / Stock LabCorp / Salary Conflict mitigation NOT REQUIRED.

1. Problem
Conventionally, the start of a batch incorporates the fundamental parameters of assay determination, namely, calibrators for accuracy, IS response expectations and ion ratio response for outlier detection. This presentation will highlight issues and solutions we have implemented to overcome errors and drift when operating LC-MS/MS in clinical care.

2. Method Information
• Direct injection of diluted samples (TFC-LC) or SPE extracted samples (Plasma Catecholamines, PCats and Plasma Metanephrines, PMets)
• ARIA TX4 (4 streams).
• SCIEX API5000 and 7500
• MP-A: Load/Elute A: 0.1% Formic Acid
• Load B: 1:1 ACN:MeOH + 0.1% Formic acid (v/v), Elute B: 90:10 MeOH: H2O + 0.1% Formic acid (v/v)
• 3 min gradient LC program, 1-2 mL/min flow rate
• Column 1: TFC Cyclone P, 50 x 0.5mm, Column 2: Agilent XDB C18 50 x 2.1 mm, 5m 50z2mm Polymeric 0 x 3 mm, 2.6μm C18, with guard cartridge
• Injection volume 10-20 μL
• Quantitative SRM acquisition

3. Troubleshooting Steps
Initial runs included calibration curve re-injection at the end of a run, demonstrating unacceptable drift of back-calculated calibrator accuracy, IS response and/or Ion ratio. Repeat preparation and individual bracketing calibrators ruled out preparative errors leading to drift. Either a full plate of calibration curves or a single sample across a plate (prep/re-pool and re-plate) was used to study drift phenomenon as a function of source conditions (cold start) and/or multiplexing (switching between assays). Removal of the bypass valve (initiated pre and post analyte elution) failed to resolve changes in the ion source. Assay specific solutions were as follows.

Clozapine/Norchlozapine: While provisionally we accepted the 50% signal loss for running in parallel with identical chemistry for MPA/MPAG (different composition during elution) due to IS correcting drift and narrow analytical range. Implementation did not run in parallel due to IS recovery inaccuracies and included repeat priming injections (SST’s) prior to batch start (inefficient MS utilization). The assay was moved to another Mass spec to improve MS utilization and remove IS recovery bias.

Fluconazole (antifungals): Repeat injection of calibration curves indicated 2 full curves required for system passivation to ensure calibrator accuracy and Ion ratio drift (used 3rd injection of curve for calibration) for launch of fluconazole assay. Eventually, commercial availability of a better IS (D4 – 3C13 change) ameliorated concentration and ion ratio drift.

Chlorpromazine: D3-Chlorpromazine response was stable but Chlorpromazine responses drifted after a cold start (quantitative error) even using the same neutral losses. Analysis of a single sample across a run and review of two analyte/IS transition pairs (mismatched neutral losses) for response drift correction of Chlorpromazine and D3 IS, indicating low energy CE transitions liable to batch effects that are not conserved between analyte and IS.

Riboflavin: Pre-screened Roboflavin and 13C4,15N2 IS transitions using 3 independent transitions indicated response drift for 1 of 3 transitions for the IS not observed by analyte to rule out batch effects by selecting transitions not liable to changes in response over the start of a run.

Plasma Catecholamines: Bypass in/out did not resolve disparate responses (>50% losses in parallel) for PCats assay, used PCats elution solvent on second pump added after PMets eluted on Pmets assay and introduced into the mass spectrometer to precondition source from PMets to PCats solvent chemistry prior Pcats sample analysis (response loss <10%).

4. Outcome
In each instance, drift in IS response (instrument sensitivity/outlier detection), ion ratio response (outlier detection) or analyte response drift that was not corrected by the IS (concentration error) was systematically resolved through multiple different approaches. This presentation will help the audience understand and resolve issues that we all have in assay design and operational utilization.