Analysis of Smoker's and Non-Smoker's Urine Using the Pegasus BT 4D

Transcription

1 Analysis of Smoker's and Non-Smoker's Urine Using the Pegasus BT 4D LEC Corporation; Saint Joseph, Michigan USA Key Words: GC-MS, GC GC-MS, GC GC-TFMS, Pegasus BT 4D, smoker's urine Figure 1. GCxGC-TFMS data for non-smoker's urine (NSU, top) and smoker's urine (SU, bottom). The 3D surface plots and spectra for two analytes detected only in SU. 1. Introduction Urine is a favored biological fluid for medical testing since it is easy to obtain in large quantities and provides a window into an individual's exposure, diet, and general health. In this study, a novel analytical approach based on comprehensive two-dimensional gas chromatography-high performance time-of-flight mass spectrometry was utilized for robust identification of compounds in two urine standard reference materials (smoker's and non-smoker's urine). This rich, comprehensive GCxGC-TFMS data can be used to visually differentiate the two urine standards as shown in Figure 1. The analytical methodology resulted in unparalleled urine-component separation and robust identification.

2 Delivering the Right Results 2. Experimental Urine standard reference materials were purchased from NIST (rganic contaminants in smoker's urine, SRM 3672; organic contaminants in non-smoker's urine, SRM 3673). A 6 µl aliquot of the individual urine standard was treated with urease (37 C, 15 min), vortexed (2 min), and then centrifuged (12, g for 1 min). A 2 µl aliquot of supernatant was transferred to a 2mL GC vial and evaporated to dryness (Speed Vac). The dry material was derivatized using a two-step procedure: 1) Treatment with methoxyamine hydrochloride in anhydrous pyridine and 2) reaction with MSTFA. The derivatized samples were analyzed using both GC-TFMS and GCxGC-TFMS (Table 1). Table 1. GC and GC GC-TFMS (Pegasus BT 4D) Instrument Parameters Gas Chromatograph Agilent 789B with LEC Dual Stage Quad Jet Modulator and L-PAL 3 Auto Sampler Injection 1µL, split 28 C Carrier Gas 1.4 ml/min, Constant Flow Column ne Rxi-5ms, 3 m x.25 mm i.d. x.25 µm coating (Restek, Bellefonte, PA, USA) Column Two Rxi-17lMS,.6 m x.25 mm x.25 µm coating (Restek, Bellefonte, PA, USA) Temperature Program.5 min at 5 C, ramped 5 C/min to 15 C, held 1 min, ramped 2 C/min. to 2 C, ramped 5 C/min to 3 C and held 15 min. Secondary oven maintained +1 C relative to primary oven Modulation 4 s with temperature maintained +15 C relative to 2nd oven Transfer Line 3 C Mass Spectrometer LEC Pegasus BT 4D Ion Source Temperature 25 C Mass Range 45-6 m/z Acquisition Rate 15 spectra/s (1D); 2 spectra/s (2D) 3. Results and Discussion Analyses of urine samples resulted in contour plots or fingerprints (Figure 2) displaying a wide variety of different compounds including acids, diacids, amino acids, bases, fatty acids, a large assortment of monosaccharides and disaccharides, and reduced/oxidized sugars (Table 2). The average spectral similarity value for a representative set of compounds in non-smoker's urine was 883/1. NSU, GCxGC-TFMS Figure 2. Contour plot with peak markers for representative compounds in NSU.

3 Table 2. Representative list of compounds in NSU with retention times and spectral similarity values Name R.T. (s) milarity Lactic Acid, 2TMS 588.7, Glycolic acid, 2TMS 612.9, xalic acid, 2TMS 78.17, Methyl-3-hydroxybutyric acid, 2TMS , Hydroxyisovaleric acid, 2TMS , Guaiacol, TMS , Hydroxybutanoic acid, 2TMS 876.3, Methyloctanoic acid, TMS , Benzoic Acid, TMS , Niacin, TMS 96.37, ,2,3-Butanetriol, 3TMS 98.38, Glyceric acid, 3TMS 14.4, Malonic acid, 3TMS , Aminoisobutyric acid, 3TMS , Anthranilic acid, TMS , Pyroglutamic acid, TMS , Malic acid, 3TMS , Uracil 132.7, Hydroxybenzoic acid, 2TMS , Trigonelline TMS 142.7, Hydroxyphenylacetic acid, 2TMS , Hydroxybenzoic acid, 2TMS 154.8, Hydroxybenzeneacetic acid, 2TMS , Vanillyl alcohol, 2TMS , Furoylglycine, TMS , Vanillylmandelic acid, 3TMS , Levoglucosan, 3TMS , Aconitic acid, (E)-, 3TMS 178.1, Name R.T. (s) milarity Citric acid, 4TMS , Methylcitric acid, 4TMS 24.12, Adenine, 2TMS 22.12, m-coumaric acid, 2TMS , ,5-Anhydrohexitol, 4TMS , D-Fructose, MX, 5TMS , L-Ascorbic acid, 2--methyl-3,5,6-tris--TMS , d-galactose, (1E)-MX, 5TMS , d-galactose, (1Z)-MX, 5TMS , d-glucose, (1Z)-MX, 5TMS , D-Mannitol, 6TMS , H-Indole-2-acetic aciid,2tms , D-Sorbitol, 6TMS , Myo-Inositol, 6TMS , D-Gluconic acid, 6TMS , Palmitic Acid, TMS , Scyllo-Inositol, 6TMS 25.16, Kynurenic Acid, 2TMS , N-Acetyl-D-glucosamine, MX (anti), 4TMS , N-Acetyl-D-glucosamine, MX (syn), 4TMS , Stearic acid, TMS , Xanthurenic acid, 3TMS , D-Lactose, MX, 8TMS (isomer 2) , Maltose, 8TMS (isomer 2) , D-(+)-Cellobiose, MX, 8TMS (isomer 2) 298.2, Tryptophan, 4TMS 298.2, Maltose, 8TMS, isomer , Sucrose, 8TMS , X milarity = 883/1 Compound identification was accomplished through automated peak find and deconvolution, spectral similarity searches of large, well-established databases, mass calculations, and retention index filtering. For example, the spectral similarity values for methylmalonic acid and adipic acid were 778 and 854/1 respectively (Figure 3). Further confidence for the identification of the acids was achieved through the comparison of the absolute value of their mass delta values ( M =.2 Da). Retention index filtering was also applied during Peak Find processing resulting in an average absolute value ( RI =) of.54 for a representative set of diacids in NSU (Table 3). 7e5 6e5 5e5 4e5 3e5 2e5 1e M/Z Library Hit - milarity: Library: replib - Methylmalonic acid, 2TMS derivative, Abundance ave A) Methylmalonic acid Exp. RI = 1224 B) 778/1 M + M.2 Da NIST RI = Figure 3. NSU GCxGC-TFMS Peak True spectra, library mass spectra, RI (Experimental and NIST), and Mass Values for methylmalonic acid (A/B) and adipic acid (C/D). 1.6e6 1.4e6 1.2e6 1.e6.8e6.6e6.4e6.2e M/Z 73.6 C) Adipic acid Library Hit - milarity: Library: mainlib - Adipic acid, (2TMS), Abundance D) 854/1 Exp. RI = 1514 M + M -.2 Da NIST RI = 1514

4 P Table 3. Comparison of experimental and NIST RI values for diacids in NSU Delivering the Right Results Name R.T. (s) milarity Mass (Da) Exp RI NIST RI xalic acid, 2TMS 78.17, N/A Methylmalonic acid, 2TMS , Succinic acid, 2TMS 14.4, Methylsuccinic acid, 2TMS 124.4, N/A Fumaric acid, 2TMS 152.4, N/A Itaconic acid, 2TMS 152.4, N/A Methylmaleic acid, 2TMS 164.5, N/A Methylglutaric acid, 2TMS , N/A Adipic acid, 2TMS , Methyladipic acid, 2TMS , N/A xoglutaric acid, MX, 2TMS 138.7, α-hydroxyglutaric acid, 2TMS , Pimelic acid, 2TMS , N/A Tartaric acid, 4TMS , Suberic acid, 2TMS , N/A Azelaic acid, 2TMS , The increase of confidently identified compounds in urine standards was a direct result of transitioning from GC- TFMS to high performance GCxGC-TFMS, which yields cleaner spectra and thus improved spectral similarity scores as shown in Table 4 (516/1 to 877/1). This is a 17% improvement in average spectral similarity. The trend is evident by comparing the 1D and 2D data for parabinic acid which is not found in the 1D data since it perfectly coelutes with citramalic acid, but is detected and identified in the 2D data with a spectral similarity of 855/1. It is clearly evident from these examples that the enhanced chromatographic resolution of GCxGC improves acquired mass spectral data and increases similarity scores resulting in a transformation of 1D unknowns into known compounds. A) Contour Plot Expansion Peak True - sample "2D NSU 1uL S2", Parabanic acid, 2TMS, at s, s, Area (Cou nts) B) Parabanic acid 1.2 4e5 2e5 N N Parabanic acid, 2TMS Library Hit - milarity: Library: mainlib - Parabanic acid, bis--(trimethylsilyl)-, Abundanc e 1 855/ M/Z Peak True - sample "2D NSU 1uL S2", D-(-)-Citramalic acid, 3TMS, at s,.82 s, Area (Counts) C) D-(-)-Citramalic acid 3e e6 D-(-)-Citramalic acid, 3TMS 1e6 Library Hit - milarity: Library: mainlib - D-(-)-Citramalic acid, 3TMS derivative, Abundan ce 864/ M/Z Figure 4. A) NSU contour plot expansion displaying separated parabanic acid, and D-(-)-citramalic acid. B,C) Improved Peak true and library spectra for the chromatographically resolved acids.

5 Table 4. Comparison of GC and GCxGC-TFMS spectral similarity values for acids in NSU. The separation power of GCxGC takes unknowns in 1D separations and makes them knowns. GC-TFMS GCxGC-TFMS Name R.T. (s) milarity R.T. (s) milarity D-(-)-Citramalic acid, 3TMS , Parabanic acid, 2TMS , Kojic acid, 2TMS , Quinolinic acid, 2TMS , rotic Acid, 3TMS , Homovanillic Acid, 2TMS , Hippuric acid, TMS , Vanillylmandelic acid, 3TMS , Pantothenic acid, 3TMS , Caffeic acid, 3TMS , Ave.= 516 Ave.= 877 Unknowns Knowns The rich comprehensive smoker's urine (SU) data was probed to identify important drug and tobacco-related classes of compounds. The characterized compounds included anti-anxiety medication, and over-the-counter medications such as ibuprofen, acetaminophen, Naproxen, and Benadryl (Figure 5). The sample also contained polyaromatic hydrocarbons, phthalate metabolites, phenols, and tobacco metabolites (Figure 6). SU Drugs, GCxGC-TFMS Figure 5. Contour plot and table listing drugs in SU. Name R.T. (s) milarity Mass (Da) Methyl salicylate, TMS , N/A Nudiflorine , Bamethan, 2TMS 138.6, N/A Gabapentin lactam , GabapentinLactam-TMS 142.7, Ibuprofen, TMS , Paracetamol, 2TMS 152.8, Paracetamol, TMS , Caffeine , Benadryl 26.12, N/A Theophylline, TMS , Naproxen, TMS , Acetaminophen 356.2, Pregabalin,,N-N-tri-TMS , N/A

6 Delivering the Right Results SU Tobacco Related Compounds, GCxGC -TFMS Cotinine N N trans-3 -Hydroxycotinine Name R.T. (s) milarity Mass (Da) 3-Pyridinol, TMS 536.3, Benzene, 1,3-dichloro , Phenol, TMS 564.5, Pyridinecarbonitrile 584.7, Carbazole, 2,4,7-trimethyl , Cresol, TMS , Pyridinol, TMS 74.16, p-cresol, TMS derivative , Naphthalene, 2,6-dimethyl , Ethylphenol, TMS 872.3, Catechol, 2TMS 18.4, Pyrene, 1,9-dimethyl- 18.5, Cyanophenol, TMS 114.5, Methylcatechol, 2TMS 112.5, Hydroquinone, 2TMS , Cotinine , Theobromine , trans-3'-hydroxycotinine, TMS , Theobromine, TMS derivative , Hydroxy-3-methylanthraquinone, -TMS , Hydroxy-a-methylnaphthaleneacetic acid, 2TMS , Nitrophenyl-ß-D-galacturonide, 3TMS 328.2, N/A Figure 6. Contour plot and table listing tobacco related compounds in SU. Not surprisingly, targeted data processing (Figure 7) of NSU and SU data files demonstrated increased quantities of tobacco related compounds, such as cotinine and trans-3'-hydroxycotinine in the latter (Table 5). Figure 7. Target Analyte Find (TAF) processing method for rapid and robust identification of tobacco related compounds in comprehensive data files.

7 Table 5. TAF results for SU and NSU Name R.T. (s) SU Area 3-Pyridinol, TMS 536 s,.868 s Benzene, 1,3-dichloro- 536 s, s Phenol, TMS 564 s,.86 s Pyridinecarbonitrile 584 s, s Carbazole, 2,4,7-trimethyl- 672 s,.59 s o-cresol, TMS 692 s,.95 s p-cresol, TMS 728 s,.948 s Naphthalene, 2,6-dimethyl- 772 s,.72 s Ethylphenol, TMS 872 s,.985 s Catechol, 2TMS 18 s,.921 s Pyrene, 1,9-dimethyl- 18 s,.896 s Cyanophenol, TMS 114 s, s Methylcatechol, 2TMS 112 s,.94 s Hydroquinone, 2TMS 1136 s,.936 s Cotinine 1648 s,.122 s Theobromine 1988 s, s trans-3'-hydroxycotinine, TMS 26 s, 3.35 s Hydroxy-3-methylanthraquinone, TMS 244 s, s Hydroxy- -methylnaphthaleneacetic acid, 2TMS 2728 s, s Nitrophenyl- -D-galacturonide, 4TMS 332 s, 1.28 s NSU Area Not Detected Not Detected Conclusion The Pegasus BT 4D facilitated fast and confident compound identification through enhanced two-dimensional chromatographic resolution and high performance TFMS. GCxGC-TFMS contour plots were highly structured showing clustered classes of compounds and provided high quality spectral data that were searched against large, well-established databases. Library hits were filtered using retention index software tools and findings further supported by calculating mass delta values for molecular and fragment ions. Comprehensive data was processed via non-targeted and targeted methods. Comparison of smoker's and non-smoker's results demonstrated increased quantities of tobacco related compounds such as cotinine and trans-3'-hydroxycotinine, but also phenols, and additional nitrogen-containing compounds. LEC, Pegasus are registered trademarks of LEC Corporation. LEC Corporation 3 Lakeview Avenue St. Joseph, MI 4985 Phone: info@leco.com IS-91:28 HQ-Q-994 LEC is a registered trademark of LEC Corporation. Form No /18-REV 218 LEC Corporation