Bioanalysis Zone

2016 New Investigator: Sheelendra Pratap Singh


sheelandra pratap singhNominee: Sheelendra Pratap Singh – Analytical Chemistry Laboratory, CSIR-Indian Institute of Toxicology Research (Uttar Pradesh, India)

Nominated By: Professor Alok Dhawan – Director, CSIR-Indian Institute of Toxicology Research (Uttar Pradesh, India)


Supporting Comments: I am pleased to recommend Dr Sheelendra Pratap Singh for the Bioanalysis New Investigator Award 2016. He is a bright, dynamic and highly motivated bioanalytical scientist. He has vast experience in the field of bioanalysis using hyphenated mass spectrometric techniques.

Prior to joining this organization he was working in the area of drug discovery and published several bioanalytical papers in reputable journals. Within a short span of time, after joining CSIR-IITR, he has done excellent bioanalytical work in the field of environmental science. He is trying to promote the dry blood spot (DBS) micro-volume technique in environmental science community to facilitate biomonitoring and toxicokinetics studies for environmental toxicants.

He has recently published a DBS paper in Scientific Reports 10;6:22447(2016) on the same topic. He has recently taken up a problem of co-occurrence of pesticides (e.g. synthetic pyrethroids) and developed the bioanalytical method for simultaneous quantification of seven pyrethroids, in order to study the effect of cumulative exposure of pyrethroids on human health. He is playing a key role in the institute for bioanalysis and has clear road map to advance in the field of bioanalysis especially for protein analysis to support toxicity biomarkers research. He has received Young Mass Spectrometrist Award-2014 from the Indian Society of Mass Spectrometry (ISMAS), India for his excellent contribution in the field of Mass spectrometry.

What made you choose a career in bioanalysis?

I graduated with my masters in Pharmacology. During my post-graduation, I was taught that the therapeutic and toxicologic outcome of a drug is determined by its pharmacokinetic disposition in biological system which primarily involves quantitative determination of the drug in various body fluids. From there onwards, I got motivated towards the field of drug metabolism and pharmacokinetics (DMPK). After my graduation, I joined DMPK department in a pharmaceutical industry and realized the importance and challenges associated with bioanalysis for DMPK studies. I learnt about bioanalysis and took this field as my core areas of expertise to do DMPK studies.

Describe the main highlights of your bioanalytical work, and its importance to the bioanalytical community.

My research focus has been the application of leading edge high throughput mass spectrometry techniques for various in vitro and in vivo assays contributing to the overall drug discovery process. I have worked as a bioanalytical and pharmacokinetic scientist in various drug discovery teams for malaria, cancer and osteoporosis projects.

Additional focus has been to evaluate interaction potential of principal components of dietary supplements with marketed drugs for which I have validated several high throughputs, LC–MS/MS methods for marketed drugs, such as centchroman, lumefantrine, raloxifene, tamoxifen and their metabolites and for components (e.g. biochanin A, formononetin, genistein) of herbal neutraceuticals/dietary supplements.

Recently, I have been involved in the quantitative estimation and ADME studies of environmental toxicants along with their metabolites using mass spectrometry. Determination of highly polar low molecular weight metabolites in body fluids, to facilitate xenobiotic metabolism studies and biomarker research, has been a frequent challenge in bioanalytical laboratories. Therefore, currently, I am exploring various derivatization techniques along with micro-extraction (e.g. DLLME) techniques to manage matrix effects and achieve reproducibility and sensitivity required for this application.

Further, I am also exploring the utility of dry blood spot (DBS) technique in conjunction with LC–MS/MS for quantitative bioanalysis to support the pharmacokinetic/toxicokinetic and biomonitoring studies of environmental toxicants.

What is the impact of your work beyond your home laboratory?

I have developed & validated several bioanalytical methods for pharmaceutical drugs, NCEs and compounds of natural origin to support pharmacokinetics (preclinical and clinical)& herb-drug interaction studies and findings from some of herb-drug interaction studies have been included in the NIEHS-funded Comparative Toxicogenomics Database. One of my methods for simultaneous quantification of 9 permeability markers is of significant importance for a bioanalyst/researcher who is trying to set up an intestinal perfusion model for absorption assessment of chemicals. This paper was awarded the Dr P.D. Sethi award, India for best paper in pharmaceutical analysis category. Recently, i have developed a micro-volume DBS method as a proof of concept for quantification of environmental toxicants in rat and human blood to facilitate/promote the biomonitoring (especially in pediatrics) and toxicokinetic studies. I have investigated the presence of a compound called methylenecyclopropyl-glycine in litchi pulp by LC–MS/MS, which may be responsible for producing hypoglycemia in undernourished children thereby causing death in Muzaffarpur area. This paper appeared on the cover page of Current Science 109, 2195–2197 (2015). Attempts are being made to develop a method for detection of MCPG in biological fluids, which shall be useful for the society.

Describe the most difficult challenge you have encountered in the laboratory and how you overcame it.

For LC–MS/MS analysis one of the major problems I routinely faced is the matrix effect due to endogenous plasma constituents and presence of excipients in plasma coming through formulation administration in animals. Due to differences in the quantity of the formulation excipients in plasma samples collected following oral versus intravenous administration, it causes significant difference in the matrix effect in the two study samples and therefore significant error in the interpretation of the results of the pharmacokinetic study. To overcome these problems, we have started using the transition pair for most abundant plasma constituents (e.g. phospholipids) and excipients (e.g. PEGs) in the bioanalytical method along with analyte’s transition pair. Further, the methods are optimized such that analyte elution time is different from the elution time of phospholipids and excipients (e.g.PEG400) of the formulation. This has helped us to significantly reduce the matrix effect and consequently, the error associated with bioanalysis in interpreting the results of the pharmacokinetic studies.

Presently, i am working on environmental toxicants quantification using GC–MS /MS. The major problem, I have faced while working with GC–MS/MS is the use of time consuming, intensive sample preparation and tedious derivatization technique. To overcome such challenges, we are using simultaneous derivatization and dispersive liquid-liquid micro-extraction (DLLME) technique, and injector port silylation technique so that extracted samples can be directly injected with minimal processing time.

Describe your role in bioanalytical communities/groups.

Presently, I am involved in the development and validation of bioanalytical methods for estimation of environmental pollutants (e.g. pesticides, pharmaceuticals, PAHs etc.) and their metabolites in biological and non-biological matrices. The validated methods are being utilized for studying the metabolism and pharmacokinetics/toxicokinetics of these toxicants to support their risk assessment upon human consumption. My special emphasis is to promote the DBS approach, microvolume technique, in environmental science community in order to facilitate the toxicokinetics and biomonitoring studies of environmental toxicants especially in pediatrics which is the need of the hour for health risk assessment. I am also working in the area of food testing, where currently I am involve in the determination of hazardous compounds such as residues of pesticides, polyaromatic compounds, phthalates and bisphenols in food commodities to meet the demand of risk free food supply. Another primary focus has been the development of bioanalytical methods for simultaneous estimation of co-administered drugs/herbs/neutraceuticals to study their interaction potential and hence safe administration to humans. Such methods have widely utility in both in vitro and in vivo settings.

Please list up to five of your publications in the field of bioanalysis (posters and presentations accepted):
  1. Raju KS, Taneja I, Rashid M, Sonkar AK, Wahajuddin M*, Singh SP. DBS-platform for biomonitoring and toxicokinetics of toxicants: proof of concept using LC–MS/MS analysis of fipronil and its metabolites in blood. Scientific Reports. 10(6), 224–247 (2016).
  2. Singh SP, Dwivedi N, Raju KS, Taneja I, Wahajuddin M. Validation of a Rapid and Sensitive UPLC–MS-MS Method Coupled with Protein Precipitation for the Simultaneous Determination of Seven Pyrethroids in 100 μL of Rat Plasma by Using Ammonium Adduct as Precursor Ion. Journal of Analytical Toxicology. 40(3), 213–21 (2016).
  3. *Wahajuddin M, *Singh SP, Raju KS, Nafis A, Jain GK. Simultaneous determination of nine model compounds in permeability samples using RP-HPLC: Application to prove the cassette administration principle in single pass intestinal perfusion study in rats. Journal of Pharmaceutical and Biomedical Analysis. 67–68, 71–6 (2012). (*Equal contribution)
  4. SP Singh, MM Ali, K Kohli, GK Jain. Liquid chromatography-mass spectrometry method for the quantification of tamoxifen and its metabolite 4-hydroxytamoxifen in rat plasma: Application to interaction study with biochanin A (an isoflavone). Journal of Chromatography B. 879(27), 2845–2851 (2011).
  5. Singh SP; Wahajuddin M, Ali MM, Jain GK. High‐throughput quantification of isoflavones, biochanin A and genistein, and their conjugates in female rat plasma using LC–ESI–MS/MS: Application in pharmacokinetic study. Journal of Separation Science. 33(21), 3326–3334 (2010).
Please select one publication from above that best highlights your career to date in the field of bioanalysis and provide an explanation for your choice.

I will choose the paper entitled “DBS-platform for biomonitoring and toxicokinetics of toxicants: proof of concept using LC–MS/MS analysis of fipronil and its metabolites in blood”. I feel proud of this research work since this work is likely to attract the attention from the scientific community working in the field of environmental research and will facilitate the biomonitoring studies of environmental toxicants especially in pediatrics. Biomonitoring of environmental toxicants is crucial for the risk assessment of chemicals exposure on human health. Further, DBS approach use will facilitate the exposure assessment in toxicity study itself rather than carrying out separate toxicokinetic study.

Find out more about this year’s New Investigator Award, the prize, the judging panel and the rest of our nominees.


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