Common Pitfalls to Avoid LC/MS/MS

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1 Wednesday April 5, :45pm Common Pitfalls to Avoid LC/MS/MS Kathy Nucifora, MPH, MT(ASCP) COLA, Accreditation Division Director Columbia, MD DESCRIPTION: Kathy Nucifora, COLA Accreditation Division Director, with the assistance of COLA Surveyor Gillian Terracina, will present issues seen in laboratories performing LC/MS/MS and discuss how to avoid common pitfalls. A question and answer period with all of the speakers will follow to conclude the workshop. CRI and COLA do not endorse, directly or indirectly, the presentations given at this conference or the products or services provided by the exhibiting vendors. Presentations are intended to be free of bias. The use of any particular product is for demonstration purposes only, and does not imply an endorsement of the product by the presenter or the sponsors of the symposium CRI

2 Specimen integrity Common pitfalls to avoid -LCMSMS APRIL 2017 LDT must establish specimen integrity criteria Specimen age Transport temperature Storage temperature Refrigerated Frozen Freeze/thaw cycles Specimen integrity Conclusions Approval Keep the data! Make sure you stay within your established specimen integrity criteria Method validation procedures Written procedure that describes how the method validation studies were done Criteria for acceptability Store the raw data, the conclusions, and the approval Method validation procedures Accuracy Precision Reportable range Sensitivity Specificity, including evaluation of interfering substances Carryover studies Matrix effects Carryover Test for accuracy of samples that follow ULOQ, 2 x ULOQ, 3 x ULOQ Establish procedures for identifying and correcting carryover

3 Method comparisons As part of the method validation, include, at a minimum: Blank (target analyte absent) Two with target analyte present One in the lower half of the reportable range One in the upper half of the reportable range Method comparisons For rarely detected analytes, spiked samples are acceptable. For some analytes, finding another lab to do comparisons with may be a challenge Review and approval Proficiency Testing Enroll in the best fit module! Be sure to review the analytes that are included with your PT module For analytes not included in the PT module you must do split specimen analyses Twice per year 5 samples Personnel qualifications Specimen preparation is part of the high complexity testing process personnel must be qualified COLA requires that the Technical Supervisor have a minimum of one year documented experience with mass spec General Supervisor provides day to day oversight QC Plan QC Plan needs to include details regarding: Number, type, frequency of QC Acceptable limits Actions to take when QC results are not within acceptable limits Can results be reported if, for example, 1 of 4 controls is out of range and if so can all patients on that run be reported? COLA requires, at a minimum, with each run: Blank Two levels of QC Test procedures Specimen integrity requirements Step by step sample preparation Hydrolysis How are calibrators and controls prepared Detailed identification criteria Ion ratios of the transition ions S/N ratios Elution times How the analytes are reported How long are the specimens retained

4 Internal standard selection Similar to the target analyte Must elute from the column at nearly the same time as the target analyte Usually an isotope labeled analog Corrects for Sample anomalies Matrix effect Ion suppression Ion enhancement Reports Report out must take place at a CLIA certified laboratory Indicate expected or unexpected Include all information necessary for the provider to interpret the results The role of the Clinical Consultant Column Selection The right length The right diameter Test different lots during method development Future availability - Use a reputable manufacturer - Avoid unusual or special (10 Ways to break your LCMS) LC/GC s CHROMacademy powered by Crawford Scientific 1. Not using a Diverter Valve -Diverter valves allow portions of the mobile phase to be sent to waste. This limits the amount going to the source unnecessarily (limits potential for contamination) 2. Using Involatile Buffers -Some manufacturers say this is possible, but in reality it is not practical. Involatile buffers clog the sample orifice and limit ionization 3. Too High a Flow Rate -Higher flow rates lead to larger volume of mobile phase and higher rates of contamination 4. Sample Concentration Too High -Sometimes want to increase sample concentration to increase sensitivity of smaller molecules. This quickly contaminates the source. Try to limit the number on injections. 5. Frequent Venting -MS systems work best when they are running. Frequent venting can damage the turbo pumps, especially the vanes and the bearings. 6. Check the Pump Oil - Rotary pumps use small amounts of oil during normal operation. The oil must be topped off and changed as recommended by the manufacturer.

5 7. Leaks - Check routinely for leaks. If the system alerts you to a leak fix it immediately. Leaks can lead to unnecessary wear and tear on the vacuum system and the detector. 8. Incorrect Assembly - Use extreme caution if you must dismantle any part for cleaning or repair. Parts are easily broken and reassembly is tedious. Always refer to the manufacturer s directions. 9. Source Temperature Too Low - A low source temperature will cause incomplete evaporation. The spray will end up on the source components and lead to contamination. 10. Run a Full Scan During Development - SIM/SRM or MRM runs might show sharp peaks, but you might be missing a big problem. Run in full scan mode to see the big picture. Avoid contamination before it happens. If this is discovered during development, changes to the sample preparation may help. Contact information info@cola.org