Russell Grant (Presenter)
Laboratory Corporation of America
Authorship: Dr Russell Grant, Yvonne Wright, Matthew Crawford, Patricia Holland, Dr Christopher Shuford
Laboratory Corporation of America, Burlington, NC, USA, 27215
| Short Abstract Development of a Multi-analyte psychostimulant panel comprising Fluphenazine, Chlorpromazine, Risperidone, 9-OH Risperidone, Methylphenidate and Haloperidol was undertaken using TFC-LC-MS/MS. The assay development posed an initial challenge related to assay LLOQ (0.1ng/mL for Fluphenazine and 10ng/mL for Chlorpromazine) with a common measurement range (250 fold). Sequential observations were resolved including phospholipid removal (TFC loop injection and LC gradient modulation), transition detuning (and selection for equivalent response ranges), transition summing (least sensitive ionization/transmission efficiency analyte focus), improved imprecision/accuracy (echo transition summing, scheduled MRM and differential transition selection between IS and analyte) and determination of LC eluent stream multiplexing (staggered parallel LC compatibility). |
Long Abstract
1. Problem
Unanticipated issues in method development of a multiplexed psychostimulants panel (100-fold different LLOQ's for analytes, identical measurement range) requiring systematic and sequential correction through informed experimentation.
2. Method Information
- 50uL sample diluted 10-fold with 0.1% aqueous Formic acid containing internal standards
- Aria Transcend TX4 multiplexed LC
- Turbulent flow (Cyclone P, 50 x 0.5mm ID) and Agilent XDB C18 (50 x 2, 5uM particle) columns (TFC-LC)
- Quantitative SRM acquisition using duplicate transitions for analyte and IS
3. Troubleshooting Steps
Multiple solutions were applied sequentially to resolve observations. Step 1 involved differential recovery of analytes and phospholipids using reduced TFC loop composition (5-10% extraction efficiency) and LC gradient shaping.
Step 2 involved determination of appropriate injection volume and influence on IS response through calibration (IS suppression), together with transitions detuning (or selecting) to reduce curve quadratic nature due to deferential molar on column amounts measured.
Step 3 involved use of echo transition summing to improve S:N for least "sensitive" analyte (fluphenazine, sum of LLOQ and MS/MS transmission).
Step 4 involved the use of Scheduled MRM to increase the dwell time per transition and enable >12 points across each pair of transitions).
Step 5 Involved determination of drift observed for analyte:IS response ratio for Chlorpromazine, resolved through the use of differential transition pairs (not identical neutral losses) for analyte and equivalent stable labelled IS.
Step 6 involved the determination of the influence of alternate assays assayed in staggered parallel mode (bias) to establish compatibility with established assays using the same instrument.
4. Outcome
Through critical observation and systematic modification, an assay was developed enabling validation of an assay that met all applicable regulatory standards. Experimental designs, data review and subsequent next steps will be described, together with analytical validation results confirming success.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Laboratory Corporation of America |
| Board Member | yes | Scientific advisory board for Vanderbilt University (unpaid) |
| Stock | yes | Laboratory Corporation of America |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |