Small Molecule Science: Experimental designs for achieving ultra trace analysis Michael P. Balogh Principal scientist Waters Corporation 211 Waters Corporation 1
www.cosmoscience.org The Society for Small Molecule Science ~51c (3) non-profit~ 212 Providence, RI (Joint meeting with SMASH NMR 211 Chapel Hill, NC 21 Portland, OR (Joint meeting with SMASH NMR) 29 Boston, MA 28 San Jose, CA 27 Chapel Hill, NC 26 San Diego, CA 25 Bristol, RI 24 Bristol, RI 211 Waters Corporation 2
Improving the design of your experiment Separation LC or GC? Column choice UPLC Ionization ESI (8) APCI, APPI Thermal desorption/ ionization (DART, ASAP) MS TOF (accurate mass) Tandem (nominal mass) Defining the workflow predicts practical definitions of 'peak capacity' and 'sensitivity' 211 Waters Corporation 3
Changing nature: MS applications Fenn ESI publications 1984 ASMS formed 1969 [1952 as ASTM committee E- 14] QTOF Fenn Nobel awarded 22 6 ave since 22 27 ave since 22 Gas Phase interests dominant until mid198s Today 1/3 abstracts contain the word peptide 51 st ASMS Conference on Mass Spectrometry and Allied Topics report 211 Waters Corporation 4
2 Monitors Multiple Integrators & CPUs A Wellequipped LC & MS Lab ca. 1988 (2-pen chart 2 Keyboards recorder) 211 Waters Corporation 5
Speed and Resolution MS E & rapid chromatography Fastnet_431_water68 F1 243.64.1Da 1 9.56e4 28 datapoints across spiroxamine peak Fragments = 14 data points Fastnet_431_water68 F2 1.4 144.139.1Da 1 2.46e4 MS E = All the masses in ONE injection m/z = 144.1388 ΔM =. ppm 1.25 1.5 1.75 Fastnet_431_water68 1.4 1 F1 298.275.1Da 9.4e4 Time.5 1. 1.5 Hundreds of Pesticides, <85 seconds m/z = 298.2746 ΔM = 1.3 ppm Time 1.25 1.5 1.75 Precursors = 14 data points 211 Waters Corporation 6
In Spectrum Dynamic range - Matrix Tolerance 1 XIC 1.6 1 99.82 315.67 389.1552 m/z 2 4 6 8 1.5 1. 1.5 1 97.771 38.166 1 1.34 TIC m/z 2 4 6 8 1 1 326.2351 Time.5 1. 1.5 Malaoxon in sewage effluent,.2 µg/l Theoretical mass = 337.487 328.17 331.1977 337.485 341.139 m/z 325 33 335 34 211 Waters Corporation 7
Atmospheric Pressure Ionization LC methods 8 of LCMS work today is done using ESI assumptions: Mid- to highly polar analytes more basic than the solvent Deal with ion suppression (1 of 3 abstracts at ASMS) Solubilize (prep and handle) samples APCI or APPI address some of the remainder Requires method change in many cases MP Balogh 21 211 Waters Corporation 8
Addressing Chemical Diversity: ASAP - Solvent in the gas phase limits ionization to molecules more basic than the solvent (exception photoionization - not acid/base ionization but still mediated by solvent) - Removing solvent and water vapor from ionization region increases types of compounds that can be ionized at atmospheric pressure 211 Waters Corporation 9
Evaluating the tools With today s technology the first step in some solutions may require intensive sample prep and column-based separation to achieve whereas other samples would be better suited to methods adapted to intact final products. How do we tell them apart? Realities of separations technology in practice including SFC William Farrell Senior Principal Scientist Pfizer Global Research & Development, La Jolla, CA Development of chromatographic materials spurred by MS demands Diane Diehl, PhD Director, Waters Corporation, Milford, MA 211 Waters Corporation 1
Creatively Applying the Tools Application of APGC in extractable testing Baiba Cabovska, PhD Senior Scientist, Mannkind Corporation, Danbury, CT Containers for packaging pharmaceutical products are required to be tested for extractables to verify the absence of toxic impurities that could transfer to the drug. Often the resin manufacturer does not provide all the necessary compound information and/or additional compounds are formed in the molding process. Therefore, a considerable need exists for identification of substances in the polymer which can potentially contaminate the drug product. Typically, LC-MS and GC-MS are used for extractable analysis. However, for some analytes GC-MS does not provide sufficient sensitivity for positive identification. 211 Waters Corporation 11
Creatively Applying the Tools Identification and Quantitation of Bioactives using UPLC-QuanTOF- MS, HPLC-MS/MS and IMS-TOF-MS Dr. Timo Stark, Chair, Food Chemistry & Molecular Sensory Science Technical University Munich Stable isotope dilution analysis (SIDA) was developed for the quantitative analysis of the health-promoting phytoalexin trans-resveratrol in red wines using UPLC-QuanTOF- MS. Recovery measured at 96.2±.8 RSD demonstrating the robustness and accuracy of the SIDA-UPLC-QuanTOF-MS method. Cross validation against a SIDA- HPLC-MS/MS analysis using a triple quadrupole mass spectrometer revealed comparable data, but the SIDA-UPLC-QuanTOF-MS was four times faster making it preferential for an accurate high-throughput analysis of wine samples. Urine samples from coffee drinkers compared with non-coffee drinkers using HILIC- UPLC/TOF-MS-based metabolite profiling. Found two molecules contributing most to the dissimilarities between both groups: N-methylpyridinium (NMP) and trigonelline. Their suitability as coffee-specific biomarkers was validated by means of a pilot coffee intervention study. Thermozeaxanthins are the end products of the carotenoid biosynthesis of the thermophilic eubacterium Thermus thermophilus. These compounds are zeaxanthin glucoside esters carrying fatty acid moieties of different chain lengths. Zeaxanthin, β- cryptoxanthin and β-carotene as well as these highly hydrophobic zeaxanthin glucoside fatty acid esters were separated and identified using direct infusion coupled to ion mobility spectrometry-time-of-flight mass spectrometry (IMS/TOF-MS). 211 Waters Corporation 12