Research abstracts

Matrix-assisted laser desorption/ionization mass spectrometry analysis of dimethyl arginine isomers from urine

Isomeric asymmetric and symmetric dimethyl arginine (ADMA and SDMA respectively) residues are excreted in urine and are putative markers of cardiovascular and chronic kidney diseases. In this work, we demonstrated simultaneous and quantitative detection of endogeneous ADMA and SDMA from urine samples of healthy subjects using MALDI-TOF MS without any chromatographic separation. The DMA isomers yielded [M+H]+ ions along with their product ions formed due to MALDI in-source fragmentation. The precursor ions were validated using MALDI-TOF MS/MS  as well as direct injection ESI-Q-TOF MS/MS. ADMA and SDMA generated unique product ions at ~m/z 46 and ~m/z 172 respectively in the MS-mode itself. These were advantageously used for full scan-mode absolute quantification of the isomeric metabolites. The m/z observed for all the ions was within 10 ppm mass accuracy. The calibration method was established by generating internal standard normalized peak area-based concentration response curves using synthetic standards. Good linearities (R2 > 0.95) with acceptable intra-assay, inter-assay variation (within 15% RSD) and excellent recoveries were observed for quality control samples. Finally, endogeneous concentrations of the metabolites were determined in urine from healthy subjects (n=11). ADMA and SDMA were found to be in the range of 1.6-8 µM and 2.9-9.1 µM in urine and were in agreement with previously reported physiological levels.

Reference: N. Bhattacharya, A. Singh, A. Ghanate, G. Phadke, D. Parmar, D. Dhaware, T. Basak, S. Sengupta and V. Panchagnula, “Matrix-assisted laser desorption/ionization mass spectrometry quantitation of dimethyl arginine isomers from urine” Anal. Methods 2014, DOI:10.1039/C4AY00309H

High throughput quantitative analysis of melamine and triazines by MALDI-TOF MS

Quantitative and high throughput MALDI MS analysis of symmetrical triazines (s-triazines), and milk contaminated with melamine has been demonstrated. s-Triazines from a multi component aqueous test mixture, and melamine, a known adulterant in milk, were quantified at low parts-per-million (ppm) level using 2,5-dihydroxybenzoic acid (2,5-DHB) as a matrix. Mass spectral peak intensity ratios of the analyte and a suitable internal standard were used for the quantitation. Limit of detection at 20 ‘parts-per-billion’ (nano molar concentration) has been achieved for the s-triazines from the aqueous mixture. Quantitation from MALDI MS peak intensities was validated using LC-PDA and LC-ESI-MS analysis using both spectral peak intensities and areas under the chromatographic curves. Excellent linearity in low ppm concentrations along with good precision and accuracy, all of which were comparable to LC-ESI-MS data, were obtained with the advantage of rapid analysis.

Reference: Singh, A. and Panchagnula, V. “High throughput quantitative analysis of melamine and triazines by MALDI-TOF MS” Analytical Methods, 2011, 3, 2360-2366. DOI:10.1039/C1AY05262D

Rapid and direct quantitation of pharmaceutical drugs from urine using MALDI MS

Quantitative estimation of drugs from biofluids in pharmacokinetic studies requires high throughput bioanalytical methods. We report a rapid, simple and efficient method for simultaneous quantitative and determination of different pharmaceutical drugs directly from urine samples.  Acetamiophen, griesofluvin, ampicillin and verapamil spiked in human urine were characterized directly by MALDI TOF-MS followed by tandem MS to confirm their identities. Further, quantitative profiling of drugs was carried out directly from spiked urine samples using MALDI TOF-MS without any chromatographic separation. Linearity was observed for the relevant concentration ranges of the metabolites found in urine. The quantitative analysis successfully produced acceptable precision and high accuracy of recoveries of drugs.

Reference: Singh, A.; Bhattacharya N.; Ghanate, A.D.; Panchagnula, V . “Rapid and direct quantitation of pharmaceutical drugs from urine using MALDI MS” Submitted to Current Trends in Mass Spectrometry, LCGC North America

Enhanced protein stability and enzyme activity retention with bicontinuous microemulsions

Bicontinuous microemulsions (BCMEs) have excellent solubulizing properties along with low interfacial tension and aqueous content that can be controlled. In this work, water soluble plant protease inhibitor (PI), well characterized for its activity against insect pests, was incorporated into a BCME system and explored for permeation on hydrophobic leaf surfaces and protease inhibition activity. The bicontinuous nature of the microemulsion containing water:2-propanol:1-butanol (55:35:10 w/w) was characterized using conductivity and self-diffusion coefficient measurements. The PI was soluble in the water-rich bicontinuous domains, stable in the microemulsions, and protease inhibition activity was retained for a prolonged duration. The microemulsions ensured greater wettability and a wider spread of the PI on hydrophobic leaf surfaces as revealed by contact angle measurements. Significantly, trypsin inhibition activity assays of the PI recovered from the leaves after delivery from the microemulsion indicated a significant increase in the PI retention on the leaf. This BCME enabled greater leaf permeation and retention of the PI can be attributed to a temporary disruption of the waxy leaf surface followed by self-repair without causing any long term damage to the plant.

Reference: Tamhane, V.A.; Dhaware, D.G.; Khandelwal, N.; Giri, A.P.; Panchagnula, V. “Enhanced permeation, leaf retention and plant protease inhibitor activity with bicontinuous microemulsions” Journal of Colloid and Interface Science, 2012, 383(1), 177-183. DOI: 10.1016/J.JCIS.2012.06.025