The atmospheric pressure photoioniza- tion 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 mole- cules are efficiently ionized through secondary reactions initiated by the charged dopant. This new technique was used to develop an analytical method for polycyclic aromatic hydro- carbons (PAHs), comparing the difference under reverse phase and normal phase chromatographic condi- tions. 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 differ- ent 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 process- ing and as important indicators of gas-phase atmospheric processes. Certain carcinogenic PAHs may be carried by ultrafine particles in ambi- ent 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 separations, and complicated MS methods. Atmospheric pressure pho- toionization 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 chro- matographic 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 acetoni- trile/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 The Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) by LC/MS/MS Using an Atmospheric Pressure Photoionization Source Application Note Mass Spectrometry www.appliedbiosystems.com Figure 1. PAH structures.
Transcript
The atmospheric pressure photoioniza-tion source, PhotoSpray™ ion source
(Robb, Covey and Bruins1) uses photons to ionize large quantities of adopant molecule added along with thevaporized mobile phase. Analyte mole-cules are efficiently ionized through
secondary reactions initiated by thecharged dopant. This new technique
was used to develop an analyticalmethod for polycyclic aromatic hydro-carbons (PAHs), comparing thedifference under reverse phase andnormal phase chromatographic condi-
tions. A total of 16 PAHs were analysedincluding the determination of unknownquantities in atmospheric samples fromboth rural and urban areas.
IntroductionPolycyclic aromatic hydrocarbons(PAHs) are a group of over 100 differ-ent chemicals, typically formed duringincomplete combustion, which are
present in a variety of environmentalmatrices. Airborne PAHs, usuallyfound in aerosol particles, are implicatedin heterogeneous atmospheric process-ing and as important indicators ofgas-phase atmospheric processes.
Certain carcinogenic PAHs may becarried by ultrafine particles in ambi-
ent air into the lungs, and are often
the targets of rigorous environmentalmonitoring. Currently, environmentalPAHs are most commonly analysed byHPLC or GC/EI-MS; techniques
which require long, high-resolution
separations, and complicated MSmethods. Atmospheric pressure pho-toionization is a new technique that
uses the energy of photons to generateions from the vaporized eluent of anLC. This study compares the analysisof PAHs by LC/MS/MS using both
reverse phase and normal phase chro-matographic conditions.
Materials and methods A 20 ppm stock standard solution
containing 16 common PAHs’ (see Figure 1). Under optimized
LC/MS/MS conditions for bothreversed phase and normal phase chromatographic conditions, the performance, linearity and LOD levels
were determined. The most abundantsingle positive transition for each PAH
compound was used to construct an+MRM method. Concentrations fromambient air for several PAHs weredetermined from samples collected on
high volume filters at both urban andrural sites. Standard solutions wereprepared by serial dilution in acetoni-
trile/water and iso-octane for reversedphase and normal phase conditions,respectively.
Spectrometer: API 3000™ LC/MS/MSSystem with Analyst® Software
PhotoSpray™ Source (see Figure 2 and 3) HPLC:Perkin-Elmer™ 200 Series Autosampler
and Micro pumps
The Analysis of Polycyclic Aromatic Hydrocarbons(PAHs) by LC/MS/MS Using an AtmosphericPressure Photoionization Source
Application Note Mass Spectrometry
www.appliedbiosystems.com
Figure 1. PAH structures.
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LC conditions: Acetonitrile/water
(gradient mode) flow at 1.0 mL/min
Column: Hypersil PAH, 100 x 4.6mm; 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 4.6mm; 5 µm
Dopant: Toluene at a flowrate of 100 µl/min.
ResultsThe source uses a modified housing of a conventional APCI source. The corona discharge needle has been removed and replaced with aconnection for the lamp gas (nitrogen)
which protects the MagnesiumFluoride optical window from thevaporised 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 2. Schematic diagram of the PhotoSpray™ source
Figure 5. Normal phase chromatography.
Figure 3. PhotoSpray™ source.
Figure 4. Reversed phase chromatography.
Normal phase PAH separation wasaccomplished with 100% iso-octanethrough a Spherisorb Silica column,250 x 4.6 mm; 5 µm. IndividualMRM transitions were monitored for16 common PAHs (see Figure 1 forcorresponding PAH). Figure 5 is a 20 µL injection of a 5 ng/µL solution(100 ng on column).
The observed response for PAHsunder normal phase conditions showan increase on the order of 10 to 70times of that observed under reversedphase conditions (Figure 6).
The post-column addition of dopantflow can have an effect on the overallresponse. This will vary from com-pound to compound. As shown inFigure 7, the response of naphthaleneis increased by a factor of 7, while theresponse of benzo(a)pyrene is relativelyunchanged.
Under normal phase conditions, 5000 pg/µL fell out of the calibrationrange for most PAHs, exceptIndeno(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).
Ambient air samples were collected
on high volume filters(subsequentlysolvent extracted, dried to 0.2 ml)from three locations, rural, urban and a traffic tunnel. Sample chro-
matogramsare shown in Figure 9, andcalculated ambient concentrations areshown in Table 2.
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Figure 6. Response comparison for normal phase and reversed phase conditions.
Figure 7. Effect of dopant on response.
Figure 8. PAH calibrations under normal phase conditions.
Conclusions• The new PhotoSpray™ source pro-
vides a sensitive ionization techniquefor analysis of polycyclic aromatichydrocarbons by LC/MS/MS.
• Normal phase conditions provide anincrease in sensitivity by a factor ofat least 10 and as much as 70 overreversed phase conditions.
• The APPI LC/MS/MS normal
phase method provides adequatesensitivity to be useful in the analy-sis of atmospheric aerosol samplesfor PAHs.
References1Robb, D. B., Covey, T. R., Bruins, A. P., Anal. Chem. 2000, 72, 3653-3659.
AcknowledgementsThe authors wish to thank Satoshi Irei(York University, Toronto, Canada) forsupplying PAH standards and theambient air samples for analysis.
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Table 1. Limits of detection for 16 common PAHs under normal phase and reversed phase conditions.
Table 2. Ambient concentrations at three sites: rural, urban, and traffic tunnel. *Not shown in Figure 9.
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