Lee Goodwin

Bioanalytical approaches to analyzing peptides and proteins by LC–MS/MS

Peptides and proteins have been utilized as therapeutic agents for over 40 years. Traditional approaches to quantify these molecules in biological matrices have utilized immunoassay approaches that can be time inefficient, lack assay specificity and have limited analytical ranges. The advances in sample preparation technologies, chromatographic systems and their chemistries, mass spectrometers and their software over the last decade have meant that LC-MS/MS approaches to peptide and protein quantification are feasible and can overcome the problems associated with quantification by immunoassay. In this article we present an overview of the challenges and approaches to overcome them when performing quantitative bioanalysis of peptides and proteins by LC-MS/MS.

Recommendations on the interpretation of the new European Medicines Agency Guideline on Bioanalytical Method Validation by Global CRO Council for Bioanalysis (GCC)

) Guideline on Bioanalytical Method Validation (BMV), during the 4th GCC (23 October 2011, Washington DC, USA) and 5th GCC (14 November 2011, Barcelona, Spain) Closed Forums. These North American and European events provided a unique opportunity for CRO leaders to openly share opinions and perspectives and to agree on unified bioanalytical recommendations specifically in relation with the new EMA guideline.The Global CRO Council for Bioanalysis (GCC)

Validation of a bioanalytical method for the quantification of a therapeutic peptide, ramoplanin, in human dried blood spots using LC‐MS/MS

A method has been developed and validated for the quantification of ramoplanin, a 2554 Da peptide antibiotic, in human dried blood spots using high-performance liquid chromatography with tandem mass spectrometric detection. The validation data meet FDA acceptance criteria for bioanalytical assays and cover the quantification of ramoplanin over the range 10-5000 ng/mL. The assay determines ramoplanin at the same lower limit of quantification as conventional liquid sample methods. Dried blood spot analysis provides an approach for quantification of peptide therapeutics and delivers significant benefits for sample collection and handling and also sample cleanup over conventional plasma and serum assays.

Application of the DBS methodology to a toxicokinetic study in rats and transferability of analysis between bioanalytical laboratories.

BACKGROUND There are little published data on either the comparison of liquid blood and dried blood spots (DBS) analyses or the ability to generate comparable DBS data at different analytical laboratories. We assess the comparative results of samples stored as liquid blood and DBS. We also determine the transferability of DBS samples by comparing the analysis at two laboratories. RESULTS Bioanalytical methods for the analysis of pioglitazone in DBS and liquid blood samples were validated to US FDA guidelines. Pharmacokinetic data generated from DBS and liquid blood samples demonstrated area under the time-concentration profile (0-24 h) values within 3% of each other and maximum plasma concentration values within 7% of each other. Comparing DBS sample results at different laboratories showed more than 99% of results agreeing within 20%. CONCLUSIONS The results indicate that comparable concentration results are obtained from DBS and whole blood samples within the same laboratory, indicating that changing between the two matrices is viable. The comparable results of DBS samples analyzed at two laboratories using different analytical methodologies demonstrate that the technique is robust and transferable.

Development of a bioanalytical method for quantification of a 15-mer oligonucleotide at sub-ng/ml concentrations using LC–MS/MS

LC-MS/MS provides a powerful technique for the selective quantification of therapeutic oligonucleotides; however, the LOQ (typically >1 ng/ml) may be higher than desirable for clinical bioanalysis. A method has been developed to allow quantification of a 15-mer unmodified DNA oligonucleotide in human plasma using SPE and UHPLC with MS/MS detection. The LOQ of this assay was 0.05 nM (∼250 pg/ml). This method was then further developed by the inclusion of online SPE to increase loading and apply additional sample cleanup. This allowed for improved assay precision at lower concentrations and increased signal, thus allowing the method to be validated over the range of 10-4000 pM (approximately 50-20,000 pg/ml). The method is accurate, precise and selective and it provides proof-of-concept for sub-ng/ml, high-throughput quantification of oligonucleotides using online SPE coupled to ion-pair, reversed-phase LC-MS/MS.

Quantification of oligonucleotides by LC–MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites

BACKGROUND LC-MS/MS allows quantification of therapeutic oligonucleotides in biological fluids at low ng/ml concentrations. Achieving selectivity between metabolites and parent molecules in a single assay is one of the biggest challenges when developing a method. We present a strategy that allows quantification of an 18-mer antisense therapeutic, trabedersen, and six metabolites in human plasma. RESULTS/METHODOLOGY: The method utilizes phenol-chloroform and SPE with UHPLC-MS/MS to independently quantify trabedersen and the 5´n-1, 5´n-2, 5´n-3, 3´n-1, 3´n-2 and 3´n-3 metabolites in a single assay. The qualification data indicate that if the method was validated it would meet regulatory expectations for precision, accuracy and selectivity. CONCLUSION We show that quantification of an oligonucleotide and multiple metabolites, including isobaric 3´ and 5´ metabolites, is achievable in a single assay through good sample clean-up and careful optimization of the LC-MS/MS parameters. The strategy presented here can be applied elsewhere and may be useful for other oligonucleotides and their metabolites.

Alternative strategies for mass spectrometer-based sample dilution of bioanalytical samples, with particular reference to DBS and plasma analysis.

BACKGROUND The analysis of bioanalytical samples has required a physical dilution of high-concentration samples to bring concentrations into the validated calibration range of an assay. RESULTS A reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry method for the quantitative analysis of pioglitazone in dried blood spots has been used to partially validate two novel techniques to analyze sample concentrations that lie above a particular calibration range. The first of the two techniques is mass spectrometer signal dilution, which consists of lowering the signal that reaches the detector. The second technique designated isotope signal ratio monitoring looks at [M+2]+1 ions (caused by naturally occurring isotopes) for samples above the upper limit of quantification. CONCLUSIONS The newly developed methods have the potential to simplify the analysis of bioanalytical samples for which previously a physical dilution of the sample was required to bring analytes within the calibration range of an assay.