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High-Throughput Screening of Abused Steroids in Urine Using Direct TOF-MS and LC–High-Resolution TOF-MS with Comprehensive Ion Fragmentation

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High-Throughput Screening of Abused Steroids in Urine Using Direct TOF-MS and LC–High-Resolution TOF-MS with Comprehensive Ion Fragmentation 2 Current Trends in Mass Spectrometry March 2012 www.spectroscopyonline.com High-Throughput Screening of Abused Steroids in Urine Using Direct TOF-MS and LC–High-Resolution TOF-MS with Comprehensive Ion Fragmentation A high-throughput screening and confirmation workflow for the analysis of steroids and metab- olites in urine was tested. Two urine extracts were analyzed per minute by flow injection electro- spray-ionization ultrahigh-resolution time-of-flight mass spectrometry (TOF-MS). Confirmation by liquid chromatography (LC) with high-resolution TOF-MS with comprehensive ion fragmen- tation achieved sub-nanogram-per-milliliter sensitivity and yielded rich, repeatable fragment spectra that allowed confident analyte verification. Ultrahigh-resolution TOF-MS was chosen for screening, as this technique can easily resolve well over 10,000 chemically attributable signals in a single measurement of 1 s or less. LC with high-resolution TOF-MS was chosen for confirma- tion because it provides chromatographic and structural selectivity orthogonal to the screening technique. Kevin Siek, Jeffrey S. Patrick, Erica A. Guice, Stephanie Amaya, and Joe Binkley orkflows for monitoring abused chemical substances abused steroids in urine samples collected from an antidop- often use a high-throughput screening technique ing surveillance program. Urine samples were extracted and Wfollowed by an orthogonal technique to confirm screened using direct flow injection electrospray ionization positive results. This screening-confirmation strategy maxi- (ESI)ultrahigh-resolution time-of-flight mass spectrometry mizes sample throughput while minimizing the chance of false (TOF-MS). Two analyses per minute were recorded without positive results. These workflows typically use two independent advanced programming of the autosampler. technologies: immunoassay, planar chromatography, or a simi- Using exactly the same equipment, positive results were larly low-cost option for screening; and gas chromatography– confirmed by liquid chromatography–high resolution time- mass spectrometry (GC–MS) or liquid chromatography–mass of-flight mass spectrometry (LC–high resolution TOF-MS). spectrometry (LC–MS) for confirmation. A short LC column took the place of a union between the However, if a hyphenated chromatography–mass spectrom- autosampler and the ESI source, and the electrospray sol- etry system that is necessary for confirmation is purchased, vent became the mobile phase. Spectra deconvolved from maximum return on this investment could be achieved by using comprehensive collision-induced dissociation allowed ana- the same instrument for the initial high-throughput screening. lyte confirmation and enabled post hoc analyte-specific and Here, we describe a proof-of-concept application for detecting class-specific searches of the data. www.spectroscopyonline.com March 2012 Current Trends in Mass Spectrometry 3 Theory Screening with Flow-Injection 1.25 1.25 Ultrahigh-Resolution MS (a) Expected profile at R = 52000 (FWHM) (a) Expected profile at R = 88000 (FWHM) Scaled 1st derivative Scaled 1st derivative 1.00 1.00 High resolution MS can most likely re- 14 N 14 N 13 CH 13 CH solve signals from more analytes in a 0.75 0.75 single measurement than any other rapid 0.50 0.50 direct analysis technique. Nevertheless, 0.25 0.25 m/z m/z using this technique alone to screen for 0.00 0.00 499.9000 499.9850 499.9900 499.9950 500.0000 500.0050 500.0100 500.0150 500.0200 500.0250 500.0300 target formulas demands high perfor- 025 499.9800 499.9850 499.9900 499.9950 500.0050 500.0050 500.0100 500.0150 500.0200 500.0250 500.0300 025 mance of the mass spectrometer. Iso- baric interferences present risks for false Figure 1: Resolution of isobars modeled near m/z 500: (a) An instrument operating at 52,000 positive and also false negative results. resolving power would be unable to resolve the illustrated isobars present in a 1:1 ratio; (b) Ultrahigh resolving power, approaching an instrument operating at 88,000 resolving power would be able to resolve the same isobars 100,000 measured at 50% peak height present in a 1:10 ratio. (FWHM), is the minimum requirement for practical implementation. Isobaric interference is not limited (a) to monoisotopic peaks with the same 1000 5 4 nominal mass-to-charge ratio (m/z), but an isotope of one formula may obscure 800 the monoisotopic peak of another for- mula. Consider the formula exchange N 1 vs. 13CH. Such isobaric signals would dif- 600 fer by about 8 mDa. An instrument with 2 400 50,000 resolving power could not distin- 3 guish these signals if they were present in a 1:1 ratio near m/z 500. However, an 200 instrument with 90,000 resolving power is expected to resolve both of these signals 0 275 300 325 350 375 400 425 450 475 500 even if one is 10 times more abundant Time(s) than the other. Expected profile spectra (b) 1.6e51.6e5 generated using a Gaussian peak model Stanozolol and hydrhydroxystanozololoxystanozolol are shown in Figure 1. 1.4e51.4e5 1.2e5 Confirmation by LC–MS with THG and hydroxylated THG Comprehensive Collision-Induced 1.0e5 Dissociation Unambiguous formula assignment 8.0e4 achieved by high-resolution MS is not suffi- 6.0e4 cient for confident identification; orthogo- 4.0e4 nal information is required to discriminate amongst isomers and unresolved isobars. 2.0e4 Chromatographic separation and colli- 0.0e0 275 300 325 350 375 400 425 450 475 500 sion-induced dissociation (CID) provide Time(s) complementary structural selectivity to enable such discrimination. However, iso- Figure 2: A segment of the ultrahigh resolution screening analysis: (a) extracted ion traces: 1 lation of a single nominal m/z before CID = THG, 2 = hydroxylated THG, 3 stanozolol, 4 = hydroxylated stanozolol, 5 = testosterone;. diminishes two of the touted advantages (b) multiple signal correlation traces: greater than zero only if signals for an unmetabolized associated with high resolution MS: the steroid and its hydroxylated metabolite correlate within a flow-injection peak. ability to mine data post hoc for analytes of interest and the ability to leverage accurate Comprehensive CID must be accom- Post Hoc Signal-of-Interest isotope abundance in formula generation. panied by accurate spectral deconvolu- Extraction Comprehensive CID, in which all coeluted tion that correctly reports product and Selected reaction monitoring experi- ions are simultaneously fragmented and precursor signals to be useful. Deconvo- ments performed with essentially 100% analyzed, maintains the advantage of ret- lution algorithms have been developed duty cycle are the benchmark for sensi- rospective analysis and takes full advantage that are capable of this; examples are tive target analysis, but have no capability of a high-resolution mass analyzer. shown with the results. for retrospective data analysis. Further- 4 Current Trends in Mass Spectrometry March 2012 www.spectroscopyonline.com (Morristown, New Jersey). [AUTHOR- correct location for Burdick & Jackson?] 319.22660 313.19842 329.21095 LC–MS-grade formic acid and ammonia 329.22208 329.22984 1.4e4 were obtained from Sigma-Aldrich (St. 328.19452 Louis, Missouri). Polyethylene glycol was 313.21577 313.23615 1.2e4 313.19085 obtained from Alfa Aesar (Ward Hill, 1.0e4 Massachusetts). Urine samples were ob- 313.2 313.22 313 329.20 329.21 329.22 329.23 329.24 tained from an antidoping surveillance 8.0e3 program. The samples were blinded; Intensity (counts) no sample history was received, and no 314.17522 6.0e3 329.00500 327.00786 identification other than a sample num- 323.25796 331.20921 4.0e3 321.24214 320.23004 ber was given. 312.15696 2.0e3 Sample Preparation 0.0e0 Samples were prepared using conven- 332 332 332 332 332 332 332 332 332 332 m/z tional procedures of hydrolysis followed by solid phase extraction. Details have Figure 3: Ultrahigh resolution spectrum of a flow injection peak showing signals for THG (m/z been given by Guice (2). 313.216) and hydroxylated THG (m/z 329.211). Flow-Injection Ultrahigh-Resolution MS An Agilent 1290 series pump and autos- ampler were used for flow injection sam- (a) 323.17677 (b) 329.25774 2.0e4 pling (Agilent Technologies, Santa Clara, 1.0e4 1.8e4 8.0e3 1.5e4 1.3e4 California). 6.0e3 1.0e4 4.0e3 7.5e3 5.0e3 The injection volume was 3 μL and 2.0e3 2.5e3 0.0e0 0.0e0 the flow rate was 300 μL/min; the injec- 100 150 200 250 300 100 150 200 250 300 350 Peak True (Cid) - sample “3002KS2011w43-049 150 ng_mL”, Peak 49, at 262.183 s Peak True (Cid) - sample “3002KS2011w43-069 150 ng_mL”, 19 Stanozolol, at 278.75 s tion solvent consisted of a volume of 10% 143.02559 329.25776 Intensity (counts) Intensity (counts) Intensity (counts) 0 8 mobile phase A and 90% mobile phase B 4.0e3 2 7.0e3 3.5e3 6.0e3 as described in the section discussing LC 4 3.0e3 4 5.0e3 9 9 9 9 0 131.0256 81.10444 81.10444 3 4.0e3 7 323.1768 2.0e3 3.0e3 1.5e3 procedures. 2.0e3 345.1587 345.1587 113.0706 113.0706 95.06012 175.1479 1.0e3 175.1479 157.0412 135.0913 121.0756 121.0756 5.0e2 1.0e3 149.110700 0.0e0 0.0e0 Detection was achieved using a LECO 100 150 200 250 300 100 150 200 250 300 350 m/zm/z m/zm/z Citius LC-HRT high-resolution TOF-MS system with an ESI source operated in Figure 4: Deconvolved spectra: (a) clostebol at collision offsets of 10 V (top) and 60 V (bottom); positive-ion mode (LECO Corporation, (b) stanozolol at collision offsets of 10 V (top) and 75 V (bottom). St. Joseph, Michigan). The MS system was operated in ultrahigh-resolution mode, more, Thurman and Ferrer (1) discussed theoretically improve the signal-to-noise which yielded a mass resolving power the concept of a crossover point between ratio (S/N) as long as the second m/z has of about 100,000 measured at FWHM.
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