The Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) by LC/MS/MS Using an Atmospheric Pressure Photoionization Source
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1 Application Note Mass Spectrometry The Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) by LC/MS/MS Using an Atmospheric Pressure Photoionization Source The atmospheric pressure photoionization source, PhotoSpray ion source (Robb, Covey and Bruins 1 ) uses photons to ionize large quantities of a dopant molecule added along with the vaporized mobile phase. Analyte molecules are efficiently ionized through secondary reactions initiated by the charged dopant. This new technique was used to develop an analytical method for polycyclic aromatic hydrocarbons (PAHs), comparing the difference under reverse phase and normal phase chromatographic conditions. A total of 16 PAHs were analysed including the determination of unknown quantities in atmospheric samples from both rural and urban areas. Introduction Polycyclic aromatic hydrocarbons (PAHs) are a group of over 100 different chemicals, typically formed during incomplete combustion, which are present in a variety of environmental matrices. Airborne PAHs, usually found in aerosol particles, are implicated in heterogeneous atmospheric processing and as important indicators of gas-phase atmospheric processes. Certain carcinogenic PAHs may be carried by ultrafine particles in ambient air into the lungs, and are often the targets of rigorous environmental monitoring. Currently, environmental PAHs are most commonly analysed by HPLC or GC/EI-MS; techniques which require long, high-resolution Figure 1. PAH structures. separations, and complicated MS methods. Atmospheric pressure photoionization is a new technique that uses the energy of photons to generate ions from the vaporized eluent of an LC. This study compares the analysis of PAHs by LC/MS/MS using both reverse phase and normal phase chromatographic conditions. Materials and methods A 20 ppm stock standard solution containing 16 common PAHs (see Figure 1). Under optimized LC/MS/MS conditions for both reversed phase and normal phase chromatographic conditions, the performance, linearity and LOD levels were determined. The most abundant single positive transition for each PAH compound was used to construct an +MRM method. Concentrations from ambient air for several PAHs were determined from samples collected on high volume filters at both urban and rural sites. Standard solutions were prepared by serial dilution in acetonitrile/water and iso-octane for reversed phase and normal phase conditions, respectively. Spectrometer: API 3000 LC/MS/MS System with Analyst Software PhotoSpray Source (see Figure 2 and 3) HPLC: Perkin-Elmer 200 Series Autosampler and Micro pumps
2 Figure 3. PhotoSpray source. LC conditions: Acetonitrile/water (gradient mode) flow at 1.0 ml/min Figure 2. Schematic diagram of the PhotoSpray source Column: Hypersil PAH, 100 x 4.6 mm; 5 µm, 50:50 post column split Normal phase conditions: 100% iso-octane (isocratic mode) flow at 0.5 ml/min Column: Spherisorb Silica, 250 x 4.6 mm; 5 µm Dopant: Toluene at a flowrate of 100 µl/min. Results Figure 4. Reversed phase chromatography. The source uses a modified housing of a conventional APCI source. The corona discharge needle has been removed and replaced with a connection for the lamp gas (nitrogen) which protects the Magnesium Fluoride optical window from the vaporised mobile phase. Reversed phase PAH separation was accomplished by gradient flow (ACN/H2O) through a Hypersil PAH column, 100 x 4.6 mm; 5 µm. Individual MRM transitions were monitored simultaneously for 16 common PAHs (see Figure 1 for corresponding PAH). Figure 4 is a 20 µl injection of a 5 ng/µl solution (100 ng on column). Figure 5. Normal phase chromatography.
3 Normal phase PAH separation was accomplished with 100% iso-octane through a Spherisorb Silica column, 250 x 4.6 mm; 5 µm. Individual MRM transitions were monitored for 16 common PAHs (see Figure 1 for corresponding PAH). Figure 5 is a 20 µl injection of a 5 ng/µl solution (100 ng on column). The observed response for PAHs under normal phase conditions show an increase on the order of 10 to 70 times of that observed under reversed phase conditions (Figure 6). Figure 6. Response comparison for normal phase and reversed phase conditions. The post-column addition of dopant flow can have an effect on the overall response. This will vary from compound to compound. As shown in Figure 7, the response of naphthalene is increased by a factor of 7, while the response of benzo(a)pyrene is relatively unchanged. Under normal phase conditions, 5000 pg/µl fell out of the calibration range for most PAHs, except Indeno(1,2,3,c,d) pyrene and Dibenz(a,h)anthracene. Reversed phase conditions could be calibrated as high as 5000 pg/µl (Figure 8). Figure 7. Effect of dopant on response. Ambient air samples were collected on high volume filters(subsequently solvent extracted, dried to 0.2 ml) from three locations, rural, urban and a traffic tunnel. Sample chromatogramsare shown in Figure 9, and calculated ambient concentrations are shown in Table 2. Figure 8. PAH calibrations under normal phase conditions.
4 Conclusions The new PhotoSpray source provides a sensitive ionization technique for analysis of polycyclic aromatic hydrocarbons by LC/MS/MS. Normal phase conditions provide an increase in sensitivity by a factor of at least 10 and as much as 70 over reversed phase conditions. The APPI LC/MS/MS normal phase method provides adequate sensitivity to be useful in the analysis of atmospheric aerosol samples for PAHs. Table 1. Limits of detection for 16 common PAHs under normal phase and reversed phase conditions. References 1 Robb, D. B., Covey, T. R., Bruins, A. P., Anal. Chem. 2000, 72, Authors Gary Impey, Byron Kieser, Jean-François Alary, Applied Biosystems/MDS SCIEX Table 2. Ambient concentrations at three sites: rural, urban, and traffic tunnel. *Not shown in Figure 9. Acknowledgements The authors wish to thank Satoshi Irei (York University, Toronto, Canada) for supplying PAH standards and the ambient air samples for analysis.
5 iscience. To better understand the complex interaction of biological systems, life scientists are developing revolutionary approaches to discovery that unite technology, informatics, and traditional laboratory research. In partnership with our customers, Applied Biosystems provides the innovative products, services, and knowledge resources that make this new, Integrated Science possible. Worldwide Sales Offices Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For international office locations, please call the division headquarters or refer to our Web site at Applera is committed to providing the world s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Applied Biosystems/MDS SCIEX is a joint venture between Applera Corporation and MDS Inc. Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free: Fax: For Research Use Only. Not for use in diagnostic procedures. Analyst, Applied Biosystems, and QSTAR are registered trademarks and AB (Design), iscience and iscience (Design), API 2000, API 3000, API 4000, and Applera are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. Figure 9. Comparison of ambient air samples at three sites: rural, urban and traffic tunnel. PhotoSpray is a trademark and Q TRAP is a registered trademark of Applied Biosystems/MDS Sciex, a joint venture between Applera Corporation and MDS, Inc. All other trademarks are the sole property of their respective owners Applied Biosystems. All Rights Reserved. Information subject to change without notice. Printed in the USA, 10/04, P+s, Publication 114AP22-01
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