= Emerging. More than 5 years before clinical availability. (16.60%, 2024)
= Expected to be clinically available in 1 to 4 years. (37.02%, 2024)
= Clinically available now. (46.38%, 2024)
MSACL 2024 : Chegwidden

MSACL 2024 Abstract

Self-Classified Topic Area(s): Troubleshooting > Troubleshooting > none

Poster Presentation
Poster #3a
Attended on Wednesday at 18:45

You Don’t Know What You Don’t Know : How Automating Data Review Helped Uncover Chromatography Variations and Issues

Emily Chegwidden, Caitlyn Flannery, Drew Payto, Jessica Colón-Franco PhD
Cleveland Clinic, Cleveland, OH

Emily Chegwidden, MPH (Presenter)
Cleveland Clinic


Through the implementation of automated chromatography review in our clinical laboratory, we identified notable differences in instrument performance affecting chromatography, particularly peak shape (tailing). Prior to this, acceptable tailing criteria were not established for manually reviewed results from the instrument software, and visual assessment of peak tailing was purely subjective. Upon automating review with defined peak tailing criteria, chromatography from one instrument consistently met the assay’s established limits while the other did not for 25-50% of samples per batch. We hypothesized that the differences are the result of an ongoing LC problem that was not identified by manual review of chromatograms. Troubleshooting and service support is ongoing to resolve the underlying issue. Here, we highlight the value of using an automated software for chromatography data review as a tool for early identification of assay performance issues and to guide troubleshooting.

Manual chromatography review is time-intensive and subjective, and it relies heavily on the experience and expertise of the reviewers. Advancements in analytical software have improved the review process by making it easier for clinical laboratories to apply rules or flags to various aspects of chromatographic results. Automation of chromatography review achieves standardization, reduces subjectivity, decreases manual time and labor, and improves data quality and consistency. Standardization of chromatography review is essential in ensuring high quality and timely and accurate reporting of clinical laboratory results.

Automated chromatography review was implemented in our clinical laboratory for the analysis of methylmalonic acid (MMA) in plasma and serum by LC-MS/MS. The MMA assay is validated on two LC-MS/MS instruments. During the development and implementation of automated chromatography review, decision criteria for chromatography flags were initially determined using recommended values and laboratory data collected by the primary LC-MS/MS instrument. While reviewing data and applying preliminary and recommended criteria, we noticed that a significant number of patient samples displayed asymmetrical peak shape (on average 25 – 50% of samples per batch of ~60 samples) and were flagged by the automated review software for chromatography issues (peak tailing). The recommended acceptable ratio for peak tailing is generally ≤ 2.00 (measured at 20% of peak max). Even with a more liberal setting (> 2.30), we found that a number of patient samples (5 or fewer per batch) still flagged for peak tailing. MMA data were then collected and reviewed from the other LC-MS/MS instrument, for which we observed a noticeable improvement of peak symmetry and did not observe flags for peak tailing. Final settings for peak tailing were established based on data review from this other LC-MS/MS instrument (<1.70), and when all MMA results were analyzed using this criteria, we found evidence that one LC-MS/MS instrument consistently produces better chromatography than the other. Under manual review conditions (instrument software and subjective judgment), these differences in chromatography went undetected.

2.Method Information
Analytical Method:
MMA is measured by LC-MS/MS. MMA is extracted from serum or plasma with a deuterated internal standard using solid phase extraction. The eluent is dried down, reconstituted, and injected onto the LC-MS/MS for separation and quantitative SRM acquisition.
a. Instrument(s): Thermo LX-2 Transcend II HPLC and TSQ Quantis MS/MS
b. Column: Restek Force C18, 100 mm x 3.0 mm, 3 µM
c. Mobile Phase A: 0.5% formic acid in water
d. Mobile Phase B: 0.5% formic acid in methanol
e. Flow rate: 0.7 mL/min (95% A: 5% B)
f. Injection volume: 25 µL

Automated Chromatography Review Software System:
Raw data collected by the LC-MS/MS software is transferred to the automated chromatography review software system and undergoes data analysis, quantification, and quality control assessment using custom-built and laboratory defined parameters.
a. Software System: Indigo BioAutomation Ascent v 4.2.0
b. Select rules (flags) with established laboratory criteria for MMA:
i. RT Shift X min > 0.05 min
ii. Peak Tailing at 20% X > 1.70
iii. ISTD Response Recovery X% > 170% or <50%

3.Troubleshooting Steps
We verified that the instrument methods and parameters were identical on both instruments and matched validated settings. We performed duplicate runs on both instruments using the same extracted samples, mobile phases, and analytical column to rule out variation from sample preparation, reagents, or consumables. Manual review of chromatographic results showed slightly more tailing from one instrument; automated chromatography review of these same results clearly demonstrated peak tailing for the same instrument with multiple samples flagged. We used the software to evaluate select historical batches (collected at least 1 month prior or earlier) to determine when chromatography quality began to decline.

Additional on-site troubleshooting has been provided by field service engineers. Troubleshooting measures have focused on the LC and have included, but are not limited to, verification of all connections and fittings and replacement of various consumables (i.e.: inject valve and stator and rotor seal on bypass valve). Using automated chromatography review to evaluate the results obtained throughout the troubleshooting process has demonstrated that there are still chromatography issues with one of the two LC-MS/MS instruments.

The laboratory is still investigating the source of peak tailing in the MMA assay, including determining when the chromatography quality between the two LC-MS/MS instruments started to diverge. Additional (escalated) analytical troubleshooting is in progress with the technical support team. Currently, all MMA runs are being performed on one single LC-MS/MS.

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