Jason Lai (Presenter)
Thermo Fisher Scientific
Bio: Jason Lai, Ph.D., MBA, is the Senior Product Marketing Manager in Clinical & Toxicology Marketing, LSMS Applied Product Marketing of Chromatography and Mass Spectrometry Division at Thermo Fisher Scientific. His industrial experiences include clinical diagnostics, medical device, toxicology (clinical & forensic), newborn screening, diabetes monitoring, point-of-care, life sciences and biotechnology. He has served various roles in marketing, product development, product management, market and business development, and R&D. In addition to 20 peer-reviewed journal articles, he has made more than 50 presentations internationally in various topics of clinical diagnostics and related technologies.
Authorship: Jason Lai, Jia Wang, Ilham Birkan, Brad Hart
Thermo Fisher Scientific, CA, USA
| Short Abstract The recent growing interest of LC-tandem MS in clinical laboratories is mainly because this analytical technique can provide definitive identification and accurate quantitation of target compounds. Today’s clinical laboratories are constantly challenged to increase sample throughput with method flexibility and reduce sample turnaround time. Multichannel HPLC in couple with a tandem-MS allows effective utilization of a single mass spectrometer with method flexibility. Two approaches of multichannel HPLC designs are presented to address different clinical labs’ needs for speed, efficiency and flexibility; One design with TurboFlow and laminar flow for both sample cleanup and separation, while the other design with laminar flow for separating compounds only. For in vitro diagnostic use. Not available in all countries. |
Long Abstract
Introduction
The recent growing interest of LC-tandem MS in clinical laboratories is mainly because this analytical technique can provide definitive identification and accurate quantitation of target compounds. Today’s clinical laboratories are constantly challenged to increase sample throughput with method flexibility and reduce sample turnaround time. Multichannel HPLC in couple with a tandem-MS allows effective utilization of a single mass spectrometer with method flexibility. Two approaches of multichannel HPLC designs are presented to address different clinical labs’ needs for speed, efficiency and flexibility; One design with TurboFlow and laminar flow for both sample cleanup and separation, while the other design with laminar flow for separating compounds only.
When analytes elute for only a portion of an HPLC run, mass spectrometers configured in a single channel LC-MS workflow may only be in use in a fraction of the method time. The design of multichannel optimization brings the productivity of multichannel HPLC to a single mass spectrometer, with staggered sample injections, the idle time of the mass spectrometer can be removed.
Methods
Two-Channel HPLC
The first design is a two channel HPLC, which includes a TurboFlow flow column and a laminar flow column for each channel. The TurboFlow column provides online sample cleanup, and the laminar flow column provides analytical separations. This HPLC can run identical or two different methods.
This HPLC instrument has two HPLC channels, and four injection ports.
(a) Two injection ports (one each for the two channels) are placed before the beginning of TurboFlow column, where online sample cleanup occurs. After sample cleanup, the analytes are transferred to the analytical compound for separation. This separation mode uses both TurboFlow and laminar flow, and is designated as TLX mode.
(b) The other two injection ports (one each for the two channels) are placed directly before the laminar flow column (by passing TurboFlow column), which is suitable for samples that have already been pre-processed or of less complex matrices that can be dilute-and shoot. This separation mode uses laminar flow only, and is designated as LX mode.
Four-Channel HPLC
The second design is a four channel HPLC, which includes a laminar flow for each channel for providing analytical separation of compounds. This HPLC instrument has four LC channels, and operates in LX mode only. The channels have separate injectors, tubing, heaters, pumps, columns, and solvents. The channels are synchronized to a single mass spectrometer. Each channel operates independently, allowing four identical or different tests to run simultaneously. In addition, identical or different analytical columns and mobile phases can be used with each channel, providing method flexibility. Multichannel optimization ensures the maximum performance of the mass spectrometer with little to no idle time. When four channels run simultaneously, the data collection times are scheduled with staggered starts so that the data collections do not occur at the same time.
Samples
In this study, calibration Standards, stable isotopically labeled Internal Standards (IS), QC sample and test sample were spiked in synthetic urine, or synthetic serum.
Mass Spectrometer
Tandem MS was operated in selected reaction monitor (SRM) mode with heated electrospray ionization (HESI), or APCI, in either positive ion mode or negative ion mode, and specific SRM transitions of precursor ion to product ion were selected for identification and quantitation of each compound.
Results and Conlcusion
Two-Channel HPLC
A total of 2000 crashed synthetic serum samples spiked with Alprazolam and isotopically-labeled internal standard were analyzed continuously for 100 hours, with an additional 44 QC samples inserted at intervals during the same 100 hours. Cross-channel RSD’s (n=2000) of retention time, concentration and ion ratio (m/z 274 to m/z 281) of Alprazolam were observed at <2%. The precision studies of between-instruments (n=120, 5 replicates, 4 runs, 2 channels, 3 units) of different ionization modes on HESI probe and APCI probe were conducted using four example compounds in polarity switching mode (+/-) in either HPLC TLX workflow, or HPLC LX workflow. HESI (LX workflow with synthetic urine sample matrix): Reserpine(+); Chloramphenicol (-); APCI (TLX workflow with synthetic serum sample matrix): Testosterone (+); Estradiol (-). The corresponding between-instruments RSD’s of concentrations were < 4% for Reserpine and Testosterone, and <10% for Chloramphenicol and Estradiol.
Four-Channel HPLC
For identical method, a test mixture of four example compounds (Atenolol, Warfarin, Lidocaine, Imipramine) in synthetic urine were analyzed (dilute and shoot) in four HPLC channels. Each HPLC channel used the same liquid chromatographic elution method, the same composition of binary mobile phases from its own solvent bottles, and the same type of analytical column. A total of 1976 samples were run unattended and continuously for about 60 hours. QC samples were inserted in every 30 samples for each channel. RSD’s of all test compounds were less than 3% in retention time, and less than 2% in concentration measurements.
For different methods, a precision study was conducted with four different methods using this four channel HPLC (n=40 for each channel; 10 replicates per run, 4 runs per channel). In this study, identical mobile phases and identical analytical columns were used for each channel. Each HPLC channel ran with a different HPLC elution method and a different set of example compounds (Channel 1: Atenolol, Warfarin, Lidocaine, Imipramine; Channel 2: Amphetamine, Methamphetamine; Channel 3: Alprazolam, α-hydroxyalprazolam; Channel 4: Oxycodone, Noroxycodone).
Within-instrument precision (n=40), all test compounds showed the RSD’s of less than 5% in both concentration and retention time; Between-instrument precision (n=40 X 3 units), showed RSD’s of less than 10%. The test results demonstrated that this four channel HPLC can continuously run tests unattended for about 60 hours. In addition to pre-processed samples, samples of less complex matrices can be injected directly with a dilute-and-shoot process.
For in vitro diagnostic use. Not available in all countries.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Thermo Fisher Scientific |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |