CPSA Digest 2001

Proceedings -Thursday, October 11, 2001

ThOC2

Emerging LC/MS/MS Technologies for Accelerating Drug Discovery and Development

Background
Three emerging technologies for accelerating drug discovery and development are discussed and researchers at Schering provide applications for each. These technologies are: 1. High resolution LC/MS/MS using Thermo Finnigan TSQ Quantum 2. Fast serial LC/MS/MS using PE Sciex API 4000 3. Parallel LC/MS/MS using Micromass Ultima with MUX interface.

High resolution LC/MS/MS using Thermo Finnigan TSQ Quantum
The TSQ QUANTUM system was introduced in July 2001 and utilizes HyperQuad technology, i.e., true hyperbolic quadrupole structures that enable high-resolution scans without signal loss. Rugged orthogonal ESI and APCI sources include Ion Sweep technology to reduce source contamination for longer run times. The advanced source technology combines with square quadrupole ion guides to ensure superior ion transmission between analytical quadrupoles. A unique 90-degree square quadrupole collision cell prevents the transmission of unwanted neutral species to the detector and greatly reduces the footprint of the instrument. The TSQ QUANTUM's ultra-high sensitivity, low noise detection system includes a 15kV off-axis conversion dynode and electron multiplier providing wide dynamic range performance. The TSQ QUANTUM can provide the core of an integrated LC/MS/MS system for pre-clinical and clinical studies, drug metabolite and impurity analysis and proteomics applications.

Some of the advantages of the TSQ Quantum, as noted by Schering, are higher sensitivity and high-resolution capability. It has been applied for resolving target compounds in the presence of interferences, and for quantitation under high resolution. As an example, a high throughput LC/MS/MS assay for SCH 29851 and SCH 34117 (standard curve range, 0.025 to 25 ng/mL; isocratic; run time 3.5 min) was validated overnight at both unit resolution and high resolution, simultaneously. Excellent MRM response was obtained with 10 femtograms injected on-column at both unit and high resolution. Absolute signal obtained at high resolution was about 1/3 that of unit resolution. Signal to noise ratios were comparable at unit and high resolution. Quantitation under high resolution could resolve target compounds in the presence of interference.

Fast serial LC/MS/MS using PE Sciex API 4000
pplied Biosystems Group, an Applera Corporation business, through its MDS SCIEX joint venture, began shipping in 2001 the API 4000 LC/MS/MS, a new system for high performance triple quadrupole LC/MS/MS quantitation. Advantages of the API 4000 are higher sensitivity, lower limits of detection, higher flow rate capability, as well as increased ruggedness and higher throughput than have been previously available. The completely redesigned system, including an innovative ion source, is reported to provide up to a ten-fold increase in sensitivity.

The redesigned ion source includes the unique ability to switch ionization modes while using a single ion source housing. Users can switch from electrospray ionization to APCI (atmospheric pressure chemical ionization) by merely switching a single probe. System throughput is enhanced by the high degree of automation and data processing capabilities of Analyst^TM software. Advanced software applications such as Information Dependent Acquisition (IDA) and Automaton increase throughput by maximizing information obtained from a single LC/MS run, and automating methods development and quantitation.

An application for the API 4000 presented by researchers at Schering involves high flow rate "fast chromatography" in combination with serial analysis. This assay for SCH 29851 and SCH 34117 (standard curve range, 0.025 to 25 ng/mL; isocratic) used automated 96-well C18 plates for sample preparation. The system was configured for two-stream serial LC/MS/MS with 2 PE-200 pumps and autosamplers. Mobile phase was delivered at 1.35 mL/min using 20 x 2 mm C8 columns. The run-time was 0.3 min using this technique, as illustrated below.

Their conclusions are reported as follows: … Higher sensitivity (5- to 50-times increase compared with API 3000) … Higher flow rate capability (up to 8 mL/min) … Mass sensitive characteristics … Multi-stream serial LC/MS/MS in combination with fast chromatography provides higher throughput.

Parallel LC/MS/MS using Micromass Ultima with MUX interface
Micromass' Quattro Ultima^TM with MUX-technology^TM allows the interface of 4 LC columns in parallel to one tandem quadrupole, as illustrated below.

Manufacturer-provided information describing this technology is as follows. "MUX-technology^TM typically allows 4 (2.1-3.0 mm i.d.) LC columns running identical gradients (1.0 mL/min per column; total 4.0 mL/min) in parallel to be multiplexed with one Quattro Ultima^TM detector for on-line LC/MS/MS (MRM). Each eluent is split (10:1) post-column to limit the flow per channel entering the MUX-technology^TM interface to 100 uL/min or less. In the MUX-technology^TM variant the conventional electrospray probe and outer source assembly are replaced with a new source housing containing an array of 4 miniaturized, pneumatically assisted electrosprays. The position of the sampling rotor is monitored in real-time enabling the four liquid inlets to be indexed. Micromass' MassLynx^TM data system tracks the data from each of the four liquid streams separately in a secure Windows NT^® environment."

Schering researchers identified the two main advantages of parallel LC/MS/MS using Micromass Ultima with MUX interface to be parallel analysis and 4-times the throughput. However, disadvantages were reported as (a) cross talk between the sprayers, (b) sensitivity less than that of a single sprayer interface, and (c) total cycle time longer than that of a single sprayer interface.

An application using the MUX interface was reported-- simultaneous method validations in rabbit, rat, mouse and dog plasma for SCH 29851 and SCH 34117. Their conclusions were:

• Maximum throughput increase of 4x
• Best utilized for simultaneous method validations or early discovery studies
• Sensitivity is about 3x lower than the single sprayer interface
• Cross talk between sprayers is negligible at concentrations < 100 ng/mL but could be as high as 0.08% at 1,000 ng/mL
• Requires longer cycle time; not compatible with ultra-fast chromatography

Links

Thermo Finnigan TSQ^® QUANTUM

Brian Howard, Thermo Finnigan: A new name, high aspirations
American Laboratory, 44-46 January 2001
(requires Adobe Acrobat)

MDS SCIEX API 4000 LC/MS/MS

Micromass with MUX technology

Patrick Rudewicz and Liyu Yang, Novel Approaches to High Throughput Quantitative LC-MS/MS in a Regulated Environment, American Pharmaceutical Review (Summer 2001).

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