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.

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.

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.

Aim: Osimertinib (Tagrisso, AZD9291) has been approved for the treatment of patients with metastatic EGFRm T790M non-small-cell lung cancer. Results: Rapid and sensitive LC–MC/MS methods were developed for osimertinib and its metabolites, AZ13597550 and AZ13575104, in human plasma (low- and high-range), urine and cerebrospinal fluid. We discuss the challenges of these multi-analyte and multiple matrix assays. The methods have been successfully validated and used for the analysis of over 20,000 clinical samples, with successful incurred sample reproducibility. Conclusion: The assays have been shown to be selective, accurate and robust, providing high-throughput analysis during the clinical development of osimertinib.

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.