Bioanalysis Zone

An author’s perspective: Ragu Ramanathan on an LC–MS/HRMS assay for quantifying coproporphyrins as organic anion-transporting polypeptide biomarkers

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To help provide insight into the recent article published in Bioanalysis: A highly selective and sensitive LC–MS/HRMS assay for quantifying coproporphyrins as organic anion-transporting polypeptide biomarkers, we spoke with author Ragu Ramanathan, associate research fellow and group leader in the Global Bioanalytical Laboratories at Pfizer Inc. (CT, USA). Ragu explains why he felt this was an important area for bioanalysis and worthy of publication.

Untitled design(1)I am an associate research fellow and group leader in the Global Bioanalytical Laboratories at Pfizer Inc. (CT, USA). I earned my BS degree in Chemistry from the University of Southern Mississippi (MS, USA) (1988) and a PhD degree in Analytical/Physical Chemistry from the University of Florida (FL, USA) (1994). My postdoctoral research was performed at Washington University (MO, USA) (1995-1997). Through a research grant from the NIH, at Washington University Mass Spectrometry resources, I expanded my training in mass spectrometry through application to breast cancer research and protein characterization.  From this training, my interest ventured into application of mass spectrometry at several pharmaceutical research and contract research organizations, including Analytical Bio-Chemistry Laboratories (MO, USA), Schering-Plough Research Institute (now Merck; NJ, USA), Warner-Lambert (now Pfizer; MI, USA), Bristol-Myers Squibb (NJ, USA), QPS (DE, USA), and Pfizer, Inc. (CT, USA). Through collaborations and direct contributions, I am fortunate to have published over 60 peer-reviewed research papers and over 12 book chapters in pharmaceutical bioanalysis, drug metabolism, metabolite identification, ion-molecule reactions, high resolution mass spectrometry and clinical biomarkers. To educate the next generation of bioanalytical/mass spectrometry scientists, I have edited a book entitled; “Mass Spectrometry in Drug Metabolism and Pharmacokinetics” (Wiley, Hoboken, NJ, 2008) and I continue to serve as an instructor for short courses offered by the American Society for Mass Spectrometry (ASMS), Eastern Analytical Symposium (EAS), American Chemical Society (ACS), and Applied Pharmaceutical Analysis (APA). Other contributions, to the scientific community include podium presentation session chairperson at EAS, ACS, ASMS, APA-India, and APA-USA; I am a reviewer for Xenobiotica, Journal of Mass Spectrometry, Bioanalysis, Drug Metabolism and Disposition, Drug Metabolism Letters, Journal of the American Society for Mass Spectrometry, Rapid Communications in Mass Spectrometry, Journal of Pharmaceutical and Biomedical Applications, and Chemical Research in Toxicology.

1. What inspired you to work in this field of bioanalysis?

My inspiration for working in the field of bioanalysis comes from bringing safer and efficacious medicine for patients with optimum timing and investments in the pharmaceutical research and development. I personally witnessed both immediate and close family members battle with diabetes, high blood pressure, and cholesterol-related health issues. Over the past 20 years, I have been involved in applying bioanalytical assays based on liquid chromatography and mass spectrometry (LC–MS) in an effort to bring new drugs, which are proven to possess some combination of improved efficacy and safety as compared to existing drugs. My team and I are routinely involved in developing and using sensitive and selective LC–MS-based assays for detection and quantification of drugs and metabolites in plasma and other biological fluids. Using LC–MS techniques, I also contribute to the characterization of pharmacologically active metabolites and potentially toxic metabolites during the early stages of drug discovery and development. Daily, I am motivated by being at work and bringing new medicines to patients.

2. What impact would you like to see/expect to see as a result of your publication?

In vitro-based transporter drug-drug interaction (DDI) evaluations result in a high number of false positive rates (~30%) and categorize new drugs as potential candidates for dedicated clinical DDI evaluations. Rapidly advancing drug transporter biomarker sciences suggest that selected endogenous compounds, detectable in human plasma and urine, may serve as biomarkers for specific transporters. Among them, coproporphyrin isomers, coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III), bile acids, and N1-methylnicotinamide (NMN) have been evaluated as possible candidate biomarkers for organic anion-transporting polypeptides (OATP1B1 and OATP1B3), organic cation transporters (OCT1 and OCT2), and multidrug and toxin extrusion proteins (MATEs), respectively. The originality of this publication demonstrates the potential application of integrated quantitative/qualitative LC–HRMS for quantitative purposes, especially for endogenous clinical biomarkers. The development of a highly selective and sensitive (CP-I/CP-III) assay affords options for earlier characterization and clinical safety projections for OATP1B1/3-mediated DDIs along with pharmacokinetic analyses of a drug as part of first-in-human clinical studies. By integrating biomarker measurements with drug and metabolite exposure measurements, I would like to see pharmaceutical companies and contract research organizations (CROs) minimize costly clinical DDI studies, thereby conserving patient samples, optimizing cost, improving safety and bringing new treatments to the bedside of the patients within optimum timing. I, also, would like to see the regulatory agencies in the USA and other countries support, accommodate, and promote assays, which are capable of multiplexing in an effort to get maximum information on drugs, metabolites, and biomarkers as part of first-in-human clinical studies.

3. What are the next steps for your research and this field of bioanalysis?

The next steps in clinical transporter biomarker research would involve demonstrating the correlation between in vitro DDI projections and clinical modulations of coproporphyrin-I and coproporphyrin-III. This would be achieved through application of the assay as part of multiple first-in-human clinical studies across multiple pharmaceutical companies. Additionally, I would like to see regulatory agencies adopt and support the data obtained using endogenous biomarkers and stage gate dedicated clinical studies. On the bioanalysis front, I would like to improve the LC–MS/HRMS assay detection limit for CP-III, which is currently around 100pg/mL.

4. Are there any researchers/projects/technologies that you are watching at the moment, and any you think we should be keeping an eye on?

The area of focus of which I am keeping an eye on is the combined use of microflow liquid chromatography and high resolution mass spectrometry; I would like to project the use and augment the combined technology in the pharmaceutical and CRO bioanalytical laboratories.

5. Do you have any advice for anyone who may be interested in working in this field?

Transforming health care by bringing new medicines, while running a profitable business, is not always easy. I want scientists who are interested in this field to start to think differently and openly to adopting new technologies. Adopting new technologies and industrializing can only materialize through partnership between pharmaceutical companies and academia, collaborations between pharmaceutical companies and instrument manufacturers and appropriate trainings. A new technology or a new idea is only as good as the scientist operating, using or driving it. Therefore, maintaining adequate training and experience in today’s constantly changing industry is very important.

Reference: Ramanathan R, King-Ahmad AJ, Holliman CL and Rodrigues AD. A highly selective and sensitive LC–MS/HRMS assay for quantifying coproporphyrins as organic anion-transporting peptide biomarkers. Bioanalysis 9(22): [Epub ahead of print] (2017)

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