Analysis of PAH Compounds Using LC/Single Quadrupole MS with a Field Free APCI Source Avinash Dalmia; Thomas White ; Daniel Pentek; PerkinElmer, Inc., Shelton, CT 1 Atmospheric Pressure Chemical Ionization Source Overview Purpose – A study was conducted to compare sample throughput and detection limits between conventional reverse phase HPLC/MS and UHPLC/MS using a novel “field free” APCI source. The study was performed on a 16 component mixture of PAHs. Mass Spectrum of Benzo[k]fluoranthene Conventional LC/MS Chromatogram of a 16 Component PAH Mixture 13 500 ng injection 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Naphthalene [M ]·+ Naphthalene Acenaphtylene Acenaphthene Fluorene Anthracene Phenanthrene Fluoranthene Pyrene Benzo [a] anthracene Chrysene Benzo [a] pyrene Benzo [b] fluoranthene Benzo [k] fluoranthene Dibenzo [a,h] anthracene Benzo [g,h,i] perylene Indeno[1,2,3-cd] pyrene [M+H]+ 15 2 Figure 1a Figure 1b Figure 1 : Figure 1a represents a schematic of most commonly used APCI configuration. Note that the heated gas exiting the vaporizer assembly experiences rapid expansion minimizing the exposure of the analyte to the corona discharge needle. Figure 1b illustrates the Field Free APCI configuration where the corona discharge needle is contained within the grounded vaporizer assembly shielding the needle electric field. This configuration allows the high electric field in the corona discharge region to be separated from the ion focusing region into vacuum. Methods A field free APCI source was interfaced to a PerkinElmer Flexar FX-10 UHPLC system and coupled to a Flexar SQ 300 MS detector. A PerkinElmer Brownlee Analytical DB PAH Column: 3.2 X 150 mm, 5 µm, was used for the conventional reverse phase LC. A sixteen component mixture of various PAHs was injected on-column at a solvent flow rate of 1.0 ml/min. A gradient was run from 50% to 60% ACN in water for 6 minutes, from 60% to 100% ACN over 6 minutes and then 100% ACN for an additional 6 minutes. For the UHPLC analysis, a PerkinElmer Brownlee Analytical DB PAH Column: 2.1 X 50 mm, 1.9 µm, was run at a solvent flow rate of 0.6 ml/min. The same PAH mixture was injected and a gradient was run from 50% to 100% ACN over 5 minutes then held at 100% ACN for 1 minute. The optimized field free APCI source parameters used were held constant for both the conventional reverse phase LC and the UHPLC separations. The corona current was set to 5 µA at the APCI vaporizer temperature was set to 350°C. Naphthalene m/z = 128 Acenaphthylene m/z = 152 Anthracene m/z = 178 Phenanthrene m/z = 178 Benzo [a] anthracene m/z = 228 Benzo [k] fluoranthene m/z = 252 Fluorene m/z = 166 Acenaphthene m/z = 154 Fluoranthene m/z = 202 Chrysene m/z = 228 Benzo [a] pyrene m/z = 252 Pyrene m/z = 202 Benzo [b] fluoranthene m/z = 252 11 Figure 7 : Mass spectrum for benzo[k] fluoranthene. Note that two different species were detected, one being the molecular radical cation and the other being the protonated species. For all PAH’s analyzed, the radical cation was the most intense ion detected. Consequently, the radical cation was used to acquire all SIM data in order to determine the detection limits and linearity response of the PAH’s. Linearity Response for Benzo[k]fluoranthene (0.05 ng-500 ng) 1E+09 100000000 UHPLC/MS Acquisition Parameters Analyte Ion Type Ion (m/z) Capillary Exit Voltage/V Dwell Time/ms Retention Window/mi n Retention Time/min Naphthalene M+ 128 120 50 0-3.2 1.23 Acenaphthylene M+ 152 200 50 0-3.2 1.48 Acenaphthene M+ 154 110 50 0-3.2 1.85 Fluorene M+ 166 110 50 0-3.2 1.95 Anthracene M+ 178 170 50 0-3.2 2.21 Phenanthrene M+ 178 170 50 0-3.2 2.43 Fluoranthene M+ 202 180 50 0-3.2 2.73 Pyrene M+ 202 180 50 0-3.2 2.90 Benzo[a] anthracene M+ 228 180 75 3.2-6.0 3.57 Chrysene M+ 228 180 75 3.2-6.0 3.66 Benzo [a] pyrene M+ 252 200 75 3.2-6.0 4.26 Benzo [b] fluoranthene M+ 252 200 75 3.2-6.0 4.47 Benzo [k] fluoranthene M+ 252 200 75 3.2-6.0 4.66 Benzo [ghi] perylene M+ 276.1 200 75 3.2-6.0 5.30 Indeno [1,2,3-cd] pyrene M+ 276.1 200 75 3.2-6.0 5.43 Dibenzo [a,h] anthracene M+ 278.1 180 75 3.2-6.0 5.10 Conventional LC/MS Acquisition Parameters Ion Type Ion (m/z) Capillary Exit Voltage/V Dwell Time/ms Retention Window/min Retention Time/min Naphthalene M+ 128 120 100 0-8.2 4.83 Acenaphthylene M+ 152 200 100 0-8.2 5.80 Acenaphthene M+ 154 110 100 0-8.2 7.40 Fluorene M+ 166 110 100 0-8.2 7.75 Anthracene M+ 178 170 100 8.2-16.5 8.61 Phenanthrene M+ 178 170 100 8.2-16.5 9.32 Fluoranthene M+ 202 180 100 8.2-16.5 10.02 Pyrene M+ 202 180 100 8.2-16.5 10.49 Benzo[a] anthracene M+ 228 180 100 8.2-16.5 11.87 Chrysene M+ 228 180 100 8.2-16.5 12.15 Benzo [a] pyrene M+ 252 200 100 8.2-16.5 13.09 Benzo [b] fluoranthene M+ 252 200 100 8.2-16.5 13.51 Benzo [k] fluoranthene M+ 252 200 100 8.2-16.5 13.98 Benzo [ghi] perylene M+ 276.1 200 100 8.2-16.5 15.32 Indeno [1,2,3-cd] pyrene M+ 276.1 200 100 8.2-16.5 15.74 Dibenzo [a,h] anthracene M+ 278.1 180 100 8.2-16.5 14.68 Figure 3 : Acquisition parameters used for the conventional HPLC/MS separation. In order to find the retention times of the 16 PAH compounds the mass spectrometer was operated in scan mode. 15 7 1 2 3 4 5 10 11 12 0.5 5 50 500 Benzo [k] fluoranthene Amount/ng Figure 8 : 4 orders of magnitude linear response for benzo[k]fluoranthene is demonstrated with the SQ 300 MS operating in SIM mode. On-Column Detection Limits 5 9 1000 Analyte HPLC SIM Detection Limit (ng) UHPLC SIM Detection Limit (ng) Naphthalene 15.015 0.459 Acenaphthylene 2.492 0.390 Acenaphthene 4.144 0.285 Fluorene 4.288 1.502 Anthracene 1.502 0.258 Phenanthrene 0.291 0.111 Fluoranthene 0.601 0.099 Pyrene 0.180 0.060 Benzo[a] anthracene 0.366 0.075 Chrysene 0.655 0.105 Benzo [a] pyrene 0.661 0.150 Benzo [b] fluoranthene 0.372 0.123 Benzo [k] fluoranthene 0.021 0.006 Benzo [ghi] perylene 0.051 0.021 Indeno [1,2,3-cd] pyrene 0.111 0.036 Dibenzo [a,h] anthracene 0.114 0.048 Decrease in Detection Limit 32.7 X 6.4 X 14.5 X 2.9 X 5.8 X 2.6 X 6.1 X 3.0 X 4.9 X 6.2 X 4.4 X 3.0 X 3.5 X 2.4 X 3.1 X 2.4 X Figure 9 : The SIM detection limits for HPLC/MS and UHPLC/MS analyses are presented. Note that increased sensitivity was achieved with increasing molecular weight. Naphthalene Acenaphtylene Acenaphthene Fluorene Anthracene Phenanthrene Fluoranthene Pyrene Benzo [a] anthracene Chrysene Benzo [a] pyrene Benzo [b] fluoranthene Benzo [k] fluoranthene Dibenzo [a,h] anthracene Benzo [g,h,i] perylene Indeno[1,2,3-cd] pyrene 6 10000 1 0.05 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 100000 10 UHPLC/MS Chromatogram of a 16 Component PAH Mixture 150 ng injection 1000000 100 Figure 5 : The acquisition parameters used for the UHPLC/MS separation. The retention times of the 16 PAH compounds were determined using scan mode. Note that the retention times for all 16 components are considerably shorter when compared to the separation using conventional HPLC. In addition the pulse counting dwell times were shortened to increase the sampling rate necessary for narrower eluting peaks do to the use of the 1.9 µm particle size column. Results Analyte R² = 0.9999 10000000 Benzo [ghi] perylene Indeno [1,2,3-cd] pyrene Dibenzo [a,h] anthracene m/z = 276 m/z = 276 m/z = 278 Figure 2 : The chemical structures of the 16 different PAH’s are shown that were separated by conventional reverse phase HPLC and UHPLC before analysis by a field free APCI source coupled to a Flexar SQ 300 MS detector. 4 3 4 10 Figure 4 : The total ion chromatogram for the conventional HPLC/MS separation is shown. In this experiment 500 ng was injected on column. A single analysis required 18 minutes of run time. Chemical Structures of the 16 Polyaromatic Hydrocarbons Introduction Methods for LCMS analysis of polycyclic aromatic hydrocarbons (PAHs) were developed using conventional reverse phase UHPLC coupled to a single quadrupole mass spectrometer featuring a proprietary “field free” Atmospheric Pressure Chemical Ionization (APCI) source. PAHs are a group of semi-volatile organic compounds that consist of one or more aromatic rings. They are common byproducts of fossil fuel combustion which lead to atmospheric pollutants. These non-polar compounds are usually not ionizable by ESI and are of interest since they have been identified as carcinogenic, mutagenic and teratogenic. In this work, a comparison between conventional reverse phase HPLC/MS to UHPLC/MS was performed to determine the sample throughput and detection limits for a 16 component mixture of PAHs. 3 1 8 7 5 16 Response/Counts Results – Going from a 5 µm to a 1.9 µm particle size improved the detection limits by an average factor of 6.5 for the 16 component PAH mixture and decreased the total run time by a factor of 3. The detection limits varied from 1.5 ng to 6.0 pg. 6 2 8 Methods – A recently introduced PerkinElmer “field free” APCI source was interfaced to a PerkinElmer Flexar TM FX-10 UHPLC system coupled to a Flexar SQ 300 MS detector. 12 9 14 14 16 Figure 6 : The total ion chromatogram for the UHPLC/MS separation. Note that the peak widths are narrower when compared to the conventional HPLC/MS separation. This allowed a lower injection volume of 150 ng as compared to 500 ng due to the increase in sensitivity with the UHPLC method. In addition, the total run time to achieve the separation was decreased by a factor of 3 increasing the sample throughput time. Conclusions For the conventional HPLC/MS analysis, all sixteen PAHs were separated with detection limits ranging between 15 ng to 21 pg. To complete this experiment a total run time of 18 minutes was needed. For the UHPLC/MS analysis the on-column detection limits achieved were between 1.5 ng to 6 pg. In order to complete one UHPLC/MS run, a total run time of 6 minutes was needed. From these experiments the results showed that going from a 5 µM particle size to a 1.9 µm particle size can lower detection limits substantially with a significant increase in throughput. The detection limits were decreased by an average factor of 6.5 for the 16 components, and the total run time for the analysis was decreased by a factor of 3. PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com