PK and Metabolite Identification
The ability to gather both quantitative and qualitative data in a single run using high-resolution accurate mass spectrometry coupled with UPLC for high-throughput data acquisition can substantially improve efficiency in pharma sample analysis, maintained Suma Ramagiri, Ph.D., application scientist at AB Sciex.
“For example, it’s not necessary to reanalyze the same sample two or three times by separate departments within the same company such as the bioanalytical group for quantitative information and the biotransformation group for qualitative information.”
At “Pittcon”, Dr. Ramagiri presented work demonstrating the ability to gather a buspirone PK quantitation profile in rat plasma along with identification of major metabolites over an eight-hour time period, all in single injection per time point.
The accurate mass high-resolution LC/MS assay was developed on the AB Sciex TripleTOF™ 5600 system using on-the-fly multiple mass defect filter acquisition method. Calibration curve ranging from 0.1 ng/mL–1,000 ng/mL was prepared in rat plasma and evaluated for its linearity, accuracy, and precision. Buspirone-d8 was used as an internal standard. A simple sample protein precipitation was used as sample-extraction method.
The current LC/MS/MS assay showed linearity (R>0.99) with accuracy ranging from 80–120%. Absolute quantification of parent and relative quantification of metabolites was processed by MultiQuant™ 2.0.1 software. From the same sample, both phase I and II metabolites were identified by MetabolitePilot™ 1.0 software.
Dr. Ramagiri believes this approach has wide applicability in drug R&D, including early discovery (in vitro metabolic stability and soft-spot analysis), development (in vivo PK analysis in different animal species like mouse, rat, dog, monkey), and preclinical/early clinical stages (definitive metabolite identification with quantitative capability to address metabolite safety and toxicity issues under MIST guidelines).
One challenge, noted Dr. Ramagiri, “is that more data generation with large file size creates data-storage and data-transfer problems. But with advances in computer technology, this can be controlled.”
Complex Sample Analysis
In a similar vein, complex sample analysis has long been challenging. Historically, it required two separate analytical experiments, and dealing with the large number of chromatographic and mass spec peaks is often problematic.
Lester Taylor, Ph.D., LC/MS platforms and programs manager, Agilent Technologies, said it is now possible to routinely acquire and use high-resolution accurate mass data (from unbiased full scan acquisition) for both qualitative and quantitative information. Moreover, it is not necessary to define target analytes prior to analysis.”
Dr. Taylor reviewed various approaches to the task and in particular examined the role of sophisticated software data-mining tools (molecular feature extraction, molecular formula generation, statistical differentiation and MS/MS compound libraries) to minimize time-consuming manual data review.
Working with data from a rat PK study and using the Agilent 6540 QTOF LC/MS/MS system for the quantitation of Clozapine, Norclozapine, and Clozapine-N-oxide, Dr. Taylor’s results indicated:
- LOD in rat plasma is 1 ng/mL and 1 pg on column.
- Calibration curves in rat plasma show excellent linearity (>0.995) over three orders of dynamic range.
- Assay statistics for accuracy, reproducibility, and precision were well within accepted limits.
- The concentrations of Clozapine and metabolites measured in rat plasma PK samples with good reproducibility (%RSD<5%).
- Sophisticated software processing tools aid metabolite identification.
He pointed out that qual-quant approaches such as the one used have a natural application in drug-metabolism studies where they possess the potential to speed and simplify analyses. Vendors recognize this, according to Dr. Taylor, and there is a trend “to provide robust and reliable instruments to generate accurate and precise data—and develop software tools to facilitate automated data mining, processing, and reporting.”
Magnetic sector ICP-MS, both single and multicollector, is being asked to provide lower limits of detection across the periodic table and to do so with high throughput, explained Charles Douthitt, isotope ratio mass spectrometry specialist at Thermo Fisher Scientific.
“It is continuously being asked to improve the precision for measurement of isotope ratios, both at natural abundance and in the nuclear fuel cycle,” he said. “And it is being challenged to provide measurements of radionuclides, which have hitherto been the province of photon-based technologies.”
Major advances, according to Douthitt, include changes to the ICP interface which have yielded a 100-fold increase in sensitivity; improvements to the detection system that contribute to precise measurement of very weak signals; and significant developments to the handling of very small samples.
Although his talk focused on measuring radionuclides in environmental samples, the approach “can apply to any problem where one needs to measure sub ppt levels in uL sample amounts.”
Currently, sector field ICP-MS instruments are about 5–10% of the total global market for ICP-MS, according to Douthitt, and are used primarily in research settings. However adoption is expanding as researchers extend SF machine capabilities by adding chromatography (GC, LC, CE, FFF) for on-line automated sample preparation, on line isotope dilution, and laser-sampling technology.”