SFC-APLI-(TOF)MS: Hyphenation of Supercritical Fluid

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SFC-APLI-(TOF)MS – a novel hyphenation of Supercritical Fluid Chromatography to Atmospheric Pressure Laser Ionization Mass Spectrometry Dennis Klink, and Oliver Johannes Schmitz Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04402 • Publication Date (Web): 03 Dec 2015 Downloaded from http://pubs.acs.org on December 7, 2015

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Analytical Chemistry

SFC-APLI-(TOF)MS – a novel hyphenation of Supercritical Fluid Chromatography to Atmospheric Pressure Laser Ionization Mass Spectrometry Dennis Klink 1)†, Oliver Johannes Schmitz1,2)* 1)

Institute for Pure and Applied Mass Spectrometry, University of Wuppertal, Gaussstr. 20, 42119 Wuppertal, Germany 2)

Applied Analytical Chemistry, University of Duisburg-Essen, Universitaetsstr. 5-7, 45141 Essen

Corresponding Author * Prof. Dr. Oliver J. Schmitz Applied Analytical Chemistry, Faculty of Chemistry, University of Duisburg-Essen, Universitaetsstr. 5-7, 45141 Essen, Germany tel.: +49 202 439 2492 fax: + 49 202 439 3915 email: [email protected] Present Addresses †Dr. Dennis Klink Alicestr. 18a, 68623 Lampertheim, Germany tel.: +49 151 4190 2055 email: [email protected]

KEYWORDS Atmospheric Pressure Laser Ionization, APLI, Mass Spectrometry, Supercritical Fluid Chromatography, SFC, 1-hydroxypyrene, urine

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ABSTRACT Atmospheric-pressure laser ionization mass spectrometry (APLI-MS) is a powerful method for the analysis of polycyclic aromatic hydrocarbon (PAH) molecules, which are ionized in a selective and highly sensitive way via resonance-enhanced multi-photon ionization. APLI was presented in 2005 and has been hyphenated successfully to chromatographic separation techniques like high performance liquid chromatography (HPLC) and gas chromatography (GC). In order to expand the portfolio of chromatographic couplings to APLI, a new hyphenation setup of APLI and supercritical-fluid chromatography (SFC) was constructed and aim of this work. Here, we demonstrate the first hyphenation of SFC and APLI in a simple designed way with respect to different optimization steps to ensure a sensitive analysis. The new setup permits qualitative and quantitative determination of native and also more polar PAH molecules. As a result of the altered ambient characteristics within the source enclosure, the quantification of 1-hydroxypyrene (1-HP) in human urine is possible without prior derivatization. The limit of detection for 1-HP by SFC-APLI-TOF(MS) was found to be 0.5 µg L-1, which is lower than the 1-HP concentrations found in exposed persons.

INTRODUCTION The outstanding sensitivity of high performance liquid chromatography (HPLC) and gas chromatography (GC) separation systems hyphenated with an atmospheric pressure laser ionization (APLI) source to a time-of-flight mass spectrometer (TOF-MS) has been realized and described recently1–3. In APLI a bench top fixed-frequency ultra-violet (UV) KrF*-excimer laser (λ =248 nm) is used as the radiation source for ionization4. Such laser systems provide high light power densities in the order of 106 W cm-2 without any optical focusing devices5,6. In contrast to atmospheric pressure photo ionization (APPI), where ionization is realized via vacuum-UV


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(VUV) radiation and a one-step ionization process, the powerful ionization of APLI utilizes a near resonant two-photon excitation process (1+1 REMPI, resonance enhanced multi photon ionization) at ambient pressure which allows selective ionization and ultra-sensitive analysis of polycyclic aromatic hydrocarbon (PAH) molecules. As a result of high molecular absorption coefficients in the near UV and favorable located excited states, combined with their relatively long excitation lifetime, PAH molecules are very efficiently ionized via 1+1-REMPI in contrast to common solvent compounds or atmospheric gas molecules1,4,7. PAHs were found to be detectable with APLI-MS in two or three orders of magnitude more sensitive than with other atmospheric pressure ionization (API) methods or with classical GC-EI-MS8. Observed limits of detection (LOD) were in the amol region for the GC coupling2,5. Therefore, APLI should probably be the ionization technique of choice for ultra-sensitive and selective analysis of PAH compounds which are known to be ubiquitous environmental pollutants with a suspected high carcinogenicity and mutagenicity potential9–11. Additionally, also non-aromatic compounds can be ionized with APLI after derivatization with ionization labels and analyzed with outstanding sensitivity 12,13. HPLC and GC are the most common techniques to separate complex PAH mixtures and are used for this purpose for a long time11,14-16. Furthermore, other methods are also very useful to separate PAH mixtures. Among these, supercritical fluid chromatography (SFC) has to be mentioned. It is well known that for a couple of reasons SFC is often classified as a so-called hybrid-technique between HPLC and GC17–20. It combines their benefits and clears the drawbacks of both methods. HPLC for example shows a higher selectivity to separate even critical isomer pairs but is least efficient than GC21. On the other hand, thermally labile substances cannot be separated with GC but with HPLC22. SFC in contrast gives respect to both.


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SFC utilizes a mobile phase mostly above its supercritical point. The most commonly used supercritical fluid is carbon dioxide (CO2) which shows a liquid-like density combined with a gas-like viscosity in a favorable temperature and pressure range23. The density is highly responsible for the solvation power of a supercritical fluid and is a function of pressure and temperature24. Both properties, high eluting power and low viscosity, allow a fast separation with supercritical fluids and implement an efficient separation method, even for thermally labile substances combined with a high degree of selectivity. Nevertheless, several high performance columns for HPLC are commercial available, which allows even a faster separation of PAH (eg. the YMC PAH column, which allows the separation of EPA 610 (16 PAHs) in less than 4 min). A lot of excellent reviews about SFC were published such as the one from Lesellier and West25. SFC is known as a fast and relatively cheap separation method for different applications in analytical and preparative purposes. Depending on the stationary phase and mobile phase combination, SFC may act like a normal or reverse phase separation technique. Together with the most common used eluent CO2, several organic solvents (e.g. methanol, acetonitrile, isopropanol and so forth) can be used as organic modifiers in order to widen the applicative areas and/or separation power of SFC26,27. Furthermore, water or even more polar additives may be added to the modifier for the same reason28,29. With SFC the analysis of enantiomers is possible30,31 and also non-polar substances such as PAHs can be separated very well. Heaton et al. successfully demonstrated a very fast separation of a 16 compound PAH-mixture (US EPA PAH-mixture) within 6 minutes by applying a pressure, temperature and eluent gradient32. The separation efficiency of SFC hyphenated to


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APLI-(TOF)MS promises a powerful, fast and sensitive method to analyze PAH compounds in a fast, efficient and sensitive way, which will be described in more detail. Today, commercially available SFC instruments allow the hyphenation to MS analyzers, often using electrospray (ESI) or atmospheric pressure chemical ionization (APCI) as ionization technique. The most demanding challenge in coupling SFC to MS ionization stages working at AP is to avoid precipitation of the analytes in combination with the realization of a proper restriction for the high-pressure effluent (up to around 40 MPa) used in SFC. The restrictor keeps the mobile phase supercritical and is therefore a vital component of the system18. To restrict the effluent, different types of restrictors are used. A nice work about the SFC-MS interface was done by Guillarme’s group33. Common restrictor types are needle valves or fused-silica (fs) capillaries with a small inner diameter (ID). Needle valves enable adjustment of the applied back pressure independently from the eluent flow. These restrictor types are heated to prevent condensation inside the valve. The back pressure of rigid restrictors on the other hand depends on the eluent flow rate, its composition and temperature or density, respectively. The flow through a fixed restrictor capillary, through which the effluent reaches the ion source, depends on the fluid viscosity. As the BPR keeps the backpressure in the system constant, the flow would change as the composition of the mobile phase changes. Since the sensitivity of the method depends on the flow, stable isotopic standards have to be used for a quantitative analysis by applying a gradient in SFC based on a fixed restrictor capillary. The system described in here originally operates with a needle valve restrictor, used to control the system pressure. Additionally a capillary restrictor is installed to couple the SFC instrument to the AP ionization source.


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EXPERIMENTAL SECTION Chemicals, Samples and Materials. 1-Hydroxypyrene (1-HP, 98 %) was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and certified, deuterated and nondeuterated PAH standard solutions (US EPA PAH-mixture) were from Dr. Ehrenstorfer GmbH (Augsburg, Germany) as well as a certified solution of a deuterated PAH mixture containing five deuterated PAH compounds (d8-naphthalene, d10-acenaphthene, d10-phenanthrene, d12chrysene, and d12-perylene). A stock solution of 1-HP was prepared by dissolving the pure substance in 100 mL of acetonitrile (HPLC gradient grade, Fisher Scientific, Loughborough, United Kingdom) using volumetric flasks. PAH stock solutions (both mixtures) were prepared by diluting the delivered standard solution in an appropriate solvent also using volumetric flasks. Final PAH standard solutions used for injection were dissolved in a mixture of methanol and deionized water (80:20). Methanol (HPLC gradient grade) as solvent and for chromatographic purposes was obtained from Karl Roth GmbH (Karlsruhe, Germany) and the deionized water was obtained from a TKA GenPure 08.2207 water treatment system (Niederelbert, Germany). Carbon dioxide 4.5 and nitrogen 5.0 were purchased from AirLiquide GmbH (Düsseldorf, Germany). All compounds were used as received and without further purification. Mixed solutions were stored in glass vessels. Urine was sampled from a health volunteer as a natural sample and spiked with an 1-HP solution (1 mg L-1 1-HP in acetonitrile) to give a urinary concentration of 500 ng L-1 prior to enrichment, which was performed with 25 mL urine (native and spiked, respectively) using a pre-conditioned C18-SPE phase (SEP-PAK C18, Waters Corp., Milford, MA, United States). The retained compounds have been eluted with HPLC gradient grade acetonitrile. The eluate was subsequently dried under a gentle flow of nitrogen 5.0 and redissolved in 1.5 mL acetonitrile (enrichment factor 16.7).


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Intrumentation. All analyses were performed on an Agilent 1260 Infinity SFC-System equipped with an Aurora A5 Fusion system (Agilent Technologies GmbH, Waldbronn, Germany) using carbon dioxide 4.5 and methanol as the eluent in gradient or isocratic mode. For 1-HP measurements a 150 mm x 4.6 mm, 5 µm particle size Agilent Zorbax RX-SIL and for PAH analyses a 250 mm x 4.6 mm, 5 µm particle size Agilent Zorbax Eclipse PAH were used as separation columns (both Agilent Technologies Inc., Santa Clara, CA, United States). The column temperature was set to 60 °C. All injections were performed using a fixed volume (5 µL) sample loop built in the autosampler of the SFC-system. The system back pressure was set to 275 bar at the back pressure regulator (BPR) valve kept at 60 °C. A T-piece between the outlet of the separation column and the BPR was used to connect the SFC-system to the MS using a combination of two fused-silica capillaries (first: 1250 to 750 mm x 0.05 mm, second: 330 mm x 0.18 mm, 0.25 mm or 0.53 mm; SGE, Analytical Science, Melbourne, Australia and BGB Analytik AG, Boeckten, Switzerland) as restriction line. The fs-capillaries were connected via a SGE capillary column connector for 0.25 mm capillaries (SGC, Analytical Science, Melbourne, Australia) or a deactivated glass connector (CS Chromatographieservice, Langerwehe, Germany) and fed through an Agilent GC 7890 A (Agilent Technologies Inc. Shanghai, China) GC-oven (held at various temperatures for optimization; ideal temperature was found to be 200 °C) and a temperature controlled rigid transfer line (iGenTraX UG, Haan, Germany) heated with an offset of 5 to 50 °C (related to the GC oven) fixed in the GC-oven. The mass spectrometric data was obtained from a Bruker micrOTOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) and treated with the Bruker software package “DataAnalysis”, ver. 3.4 and 4.0 including the calculation of the signal-to-noise (S/N) ratios for individual chromatographic peaks for extracted ion chromatograms (EIC) rounded to the


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nominal mass ± 0.5. The instrument was equipped with a custom built temperature controlled multi purpose ion source (TC-MPIS, set to 110 °C from iGenTraX UG, Haan, Germany) featuring an identical APCI vaporization stage as used with the original Bruker source. An ATLEX 300 SI KrF* excimer-laser (wavelength 248 nm, pulse width (FWHM) ca. 5 ns; ATL Lasertechnik GmbH, Wermelskirchen, Germany) was used as the light source, operated with an internally generated pulse frequency of 100 Hz and stabilized to an output energy of 5 mJ pulse-1. In all cases the dry gas flow rate was set to 0.0 L/min. Other MS ion source parameters have been set to a spray shield voltage of -400 V, a capillary voltage of -400 V, a nebulizer temperature of 300 °C, and an optimized nebulizer pressure of 0.0 bar.

RESULTS AND DISCUSSION Comparison of different SFC-MS settings. Due to the improved sensitivity of GC-APLI compared to the HPLC coupling option, the GC-setup was chosen to serve as basis for extending the experimental system setup with an SFC instrument. The GC-APLI setup consists of an Agilent GC 7890 A gas chromatograph coupled to a Bruker Daltonics micrOTOF mass spectrometer via a transfer line and ion source from iGenTraX2,3. The allocated SFC instrument was an Agilent 1260 Infinity system equipped with an Aurora A5 Fusion restrictor system using a temperature-controlled needle valve restrictor. As known from a technical overview for SFCMS34 coupling of the Agilent 1260 Infinity SFC instrument to a mass spectrometer can be realized in two ways, either useful for qualitative or quantitative analysis. In common, both options couple the outlet of the needle valve restrictor, called back-pressure regulator (BPR), to the vaporizer of an ion source operating with electrospray or chemical ionization at AP. Obviously it is deemed necessary to heat the transfer line between BPR and ion source to prevent


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freezing as a consequence upon the expansion of CO2. To improve sensitivity and repeatability of retention times and peak areas, it is recommended to use an additional HPLC-pump prior to the BPR – coupled via a T-piece – to supplementary deliver a make-up flow consisting of organic solvent34. On the other hand, operating the described SFC-MS setup without such an additional pump is also possible, but results in a higher variability to the MS response34. There are two major reasons against utilization of an additional pump: investment costs and a decrease of sensitivity. Purchasing another HPLC pump increases the investment costs for an SFC-MS system and additionally leads to consumption of pure and expensive organic solvent, which is contrary to the idea of an environmentally friendly and “green” SFC system. Furthermore, the usage of a liquid requires operating the ion source in the less sensitive LCAPLI mode. To avoid the limitation when using the LC mode, we figured out an approach where no additional pump for a make-up flow is required and where the effluent can be transferred to the ion source operated in the GC mode.

Improvement of sensitivity in SFC-APLI. Pretests with an identical experimental setup using a flame ionization detector (FID) with a homologous series of hydrocarbons between C6 and C16 showed a dramatic dependence of the detector response and peak shape on the carbon atom number. With increasing carbon atom number or equivalent decreasing vapor pressure, the detector response was minimized and the peaks lost their narrow shape. For these experiments the Aurora A5 needle valve outlet was directly coupled through a heated transfer line and split valve to a FID. Obviously, the final target analyte, the PAH molecules, have much lower vapor pressures than the aliphatics and an analysis with transferring them from the BPR to the ion source would be hard to perform. As a result, the effluent has to be transferred to the MS while


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keeping it at an elevated pressure, which can be done with an additional restriction line prior to the BPR. A T-piece between the column outlet and the BPR can be used to provide this opportunity. The T-piece is coupled to a fused silica capillary of a specific length and inner diameter (ID) of 50 µm as a restriction line via a short stainless steel capillary. The capillary provides a system pressure drop and carries a split part of the effluent to the ion source. Various optimization experiments showed that it is necessary to heat the capillary to avoid freezing out of the analytes on their way to the ion source. The GC-APLI setup provides some benefits: It is capable to hold the capillary at an elevated temperature and also enables an easy insertion into the TC-MPIS via its rigid transfer line. Additionally, the elevated temperature leads to a lower viscosity of the effluent accompanied with a higher effluent split part and therefore more analytes transferred to the ion source. The experimental setup is shown schematically in figure 1.

Figure 1

Schematic overview of the entire SFC-APLI-(TOF)MS setup with a custom built TC-MPIS (a) and transfer line (b), capillary connectors (c and d), t-piece (e)


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The schematic overview shows two different kinds of capillary connectors. The connector d simply couples the stainless steel capillary with the fused silica restriction capillary. The second connector c is a capillary connector, connecting a second fused silica capillary with a different inner diameter. This extension element is necessary, because the fluid dynamic velocity of the SFC effluent streaming out of the restriction capillary within the ion source is around 3 orders of magnitude higher than it is in the case of GC-APLI. A predicted reason for this fact is that the exit velocity out of capillaries like a GC column or a restriction capillary depends on the diameter of the exit area and the volume flow through it. The volume flow depends in turn on the length and inner diameter of the capillary. The 100-fold higher volume flow (1 to 2 mL min-1 for GC and approx. 100 to 200 mL min-1 for SFC) and the five times smaller inner diameter of the capillary (assuming an ID of 250 µm for GC capillaries and a 50 µm for the SFC restriction line) are responsible for the extraordinary increase of effluent speed in SFC. The analyte molecules in a GC effluent are slow enough to be lit up with a sufficient number of laser shots (typically between 100 and 200 Hz) within the time frame of travelling the gap between the capillary end and the mass spectrometer inlet to be ionized efficiently. In the initial SFC-APLI experiments, the molecules are assumed to be too fast for a sufficient ionization. Table 1 gives an overview of the approximated values for the velocities out of a GC column and the SFC restriction capillary used in the experiments as well as the related travelling time within the gap between the capillary end and the mass spectrometer inlet. Please note that the excimer laser maybe operated with a repetition rate between 50 and 250 Hz, which is convertible to one light impulse each 2 x 10-2 s to 4 x 10-3 s.

Table 1

Comparison of numeral flow characteristics for the GC- and SFC-APLI-(TOF)MS setup


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gas volume stream

exit area

exit velocity

travelling time

(mL min-1)

(m2)

(m s-1)

(s)

GC

1-2

5 x 10-8

0.34 - 0.68

1.5 x 10-2 - 7.4 x 10-3

SFC

100 - 200

2 x 10-9

849 – 1698

5.9 x 10-6 - 2.9 x 10-6

It may be assumed that at a given length an additional capillary downstream possessing a larger inner diameter leads to a decrease in flow velocity. As a result, such extension would lead to a more efficient ionization in SFC-APLI. To verify this assumption three different additional capillaries with inner diameters of 180, 250, and 530 µm and a length of 330 mm have been coupled using the capillary connector c in figure 1. With each experimental setup the same analyte mixture was separated under constant conditions (isocratic separation with a 75:25 mixture

of

CO2/methanol)

with

SFC-APLI-(TOF)MS.

The

resulting

extracted

ion

chromatograms (EIC) and the summarized mass signals (m/z values ± 0.5) for the analytes d10phenanthrene, d12-chrysene, and d12-perylene (50 µg L-1 each, injection volume: 5 µL) are shown in figure 2.


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Figure 2

SFC-APLI-(TOF)MS chromatograms (summarized EIC) for four different compositions of the restriction setup. Each setup uses a 1250 mm x 50 µm restriction capillary, without (black line) and combined with an extension capillary of 330 mm length and inner diameters of 180 µm (red line), 250 µm (blue line), and 530 µm (green line).

The given chromatograms show a triplicate increase in signal intensity. The related signal-tonoise (S/N) ratio achieves the same growth as only the analyte signal rises, not the noise. Furthermore, despite the wider inner diameter, the peaks do not broad up as it might be expected. In fact, the given turbulence in consequence of the fast eluent stream is stabilizing the peak width. Due to this improvement, further analyses were performed using the 530 µm extension downstream the restriction capillary.


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As known from Hagen-Poiseuille’s equation, the volume flow through a capillary is inversely proportional to its length as its back-pressure decreases with a shorter capillary. This reveals a basic phenomenon and points out to another possibility to improve the sensitivity of SFC-APLI. The restriction capillary length of 1250 mm – given from initial experiments - can be shortened in order to increase the effluent flow to the ion source and hereby amplify the signal intensity. Attention should be paid to the stability of the BPR pressure. The restriction line prior to the BPR opens the otherwise sealed system and creates the pressure drop. However, pressure drops prior to the BPR which are too high lead to an unstable system pressure measured with the BPR’s pressure sensor and may prompt the SFC system to switch into an error mode. To discover the minimal length at which the BPR pressure remains stable to ensure a proper analysis, the length of the restriction capillary has been reduced step by step. At a capillary length of 650 mm the BPR pressure (setpoint was 275 bar) showed a noisy baseline for some time. Therefore, a minimal length of 750 mm was chosen to ensure stable conditions for analysis. The consequences of using shorter capillaries are presented with EIC in figure 3 for the abovementioned analytes using the same chromatographic conditions. To demonstrate the impact of length, the restriction capillary was used in a length of 1250 mm, 1000 mm and 750 mm, respectively, and was coupled in each case to the above mentioned 330 mm x 530 µm extension capillary.


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Figure 3

SFC-APLI-(TOF)MS chromatograms (summarized EIC) for three different restriction capillary lengths. Black line: 1250 mm, red line 1000 mm, and blue line 750 mm. Each setup uses an extension capillary of 330 mm length and an inner diameters 530 µm.

Once again the signal intensities and the peak areas could be enhanced with the altered setup. The enhancement accounts for 50 to 70 % this time. Additionally the S/N could be increased in the same scale for equal reasons like before. Both improvements base on the optimized flow and restriction characteristics of the capillary restriction unit. Anyway, further improvements could be achieved with an optimized and analyte-specific GC oven- and transfer line temperature as well as with varying the gas flows within the ion source (data not shown). A lower vapor pressure requires a higher temperature and vice versa. Despite the variable vapor pressures for different analytes within the related temperature range it is possible to find an optimum


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temperature at which many analytes can be detected with a sufficient signal intensity. An ideal temperature set used for further analyses was 200 °C for the GC oven and 250 °C for the transfer line temperature. At this temperature e.g. phenanthrene as well as perylene give intense signals with decent peak shapes.

Separation of a complex PAH-mixture. In order to demonstrate the entire capability of SFCAPLI-(TOF)MS including the improvements made so far, two different applications have been evaluated. The first is pictured in figure 4 and shows the summarized extracted ion chromatogram (m/z values ± 0.5) of a complex PAH-mixture (perdeuterated US EPA PAHmixture). An elution gradient of CO2 and methanol starting at a ratio of 100:0 and ramped up to 60:40 was used for this separation in less than 11 minutes, which is similar to an Agilent HPLCapplication note with the same column and a flow rate of 2 mL/min. After increasing the flow rate to 4 mL/min the analysis time was reduced to less than 5 min35. But with such high flow rates the coupling to a mass spectrometer is only possible with a flow split, which reduce the sensitivity.


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Figure 4

SFC-APLI-(TOF)MS chromatogram (summarized EIC) of a 10 µg L-1 perdeuterated US EPA PAH-mixture (injection volume: 5 µL).

Using the above mentioned conditions, several analyses have been performed on the SFCAPLI-(TOF)MS system to determine the limits of detection (LOD). A non-deuterated US EPA PAH-mixture in a concentration range between 2 ng L-1 and 50 µg L-1 (for each analyte) has been used for this purpose. Injections were performed with a 5 µL sample loop and result in a quantity of analyte on column between 0.01 pg and 250 pg. The determined detection limits for 14 analytes were primarily in the range of sub µg L-1 (mean value: 592 ng L-1, median: 75 ng L-1) and only three analytes were found to have a LOD above 1 µg/L. Phenanthrene (Phe), benz[a]anthracene (BaA) and chrysene (Chry) showed the lowest LOD values and were found to


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be 20 ng L-1, resulting in analyte amounts on column of 0.56 fmol for Phe, and 0.44 fmol for BaA and Chry. The LODs found for several PAH compounds with SFC-APLI-(TOF)MS were compared to the LODs of the same mixture analyzed with HPLC-APLI-(TOF)MS (data not shown). Except for the late eluting compounds (BP, dibenzo[a,h]anthracene, and indeno[1,2,3cd]pyrene) LODs found with SFC were generally lower by a factor of 5 to 10 than with HPLC. For the same PAH mixture Cai et al. determined the limit of detection by using a more sensitive triplequad-MS and atmospheric pressure photoionization (APPI) between 1.7 and 158 pg PAH on column36. Therefore, the detection limit could probably further improved with APLItriplequad-MS instead of APLI-TOF-MS.

Determination of urinary 1-hydroxypyrene. The observed sensitivity of SFC-APLI(TOF)MS and the properties associated to the ion source setup and the flow characteristics indicate the possibility of a second area of application for this technique: the determination of 1-hydroxypyrene (1-HP). The knowledge of human exposure to carcinogens like PAH compounds, which are known to be genotoxic, are of medical and toxicological importance. A quantitative evidence of their exposure to humans is therefore an interesting and relevant task. PAH molecules are metabolized in the human body to more polar structures, mainly to hydroxy species. Depending on the parent structure, the excreted species diversifies in multiple forms, e.g. polyhydroxy compounds. Additionally, a mixture of PAHs always appears with at least more than 100 different individual structures, including isomer forms37,38. Pyrene is an exception as it occurs as a highly abundant compound within a complex mixture of PAHs and is mainly metabolized to only one specific species (1-HP) which nearly exclusively (to approx. 90 %) is excreted as glucuronide adduct in urine37–39. These facts combined with a good correlation of the


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amount of 1-HP to PAH exposure suggest 1-HP as a perfect biomarker for PAH exposure. A gas or liquid chromatographic separation and quantitative determination of 1-HP often follows extraction and enzymatic or acidic digestion of the glucuronide. The first determination of 1-HP with APLI-MS was reported by Schiewek et. al. in 20072. GC was used as chromatographic stage and the analyte was derivatized prior to analysis. The derivatization was performed in order to minimize a loss of sensitivity through gas phase reactions with residues of water. Analyzing 1-HP under watery conditions, like in separations using RP-HPLC, results in a high degree (60 to 70 %) of analyte loss due to fragmentation. The conditions within the ion source enclosure in SFC-APLI-(TOF)MS were found to be slightly different compared to HPLC-APLI-(TOF)MS as the effluent consist of CO2 and MeOH without water, acting as a gas phase reactant. Furthermore, the gas velocities and streamlines apparently support the assumption that the feasibility of water to react with the analyte is confined. Consequently, the analysis of primary 1-HP without derivatization or sensitivity loss due to gas phase reactions should be possible and is shown in figure 5 with the analysis of an urine sample, spiked with 1-HP and enriched via SPE on a C18 phase. The concentration of the 1-HP spike was chosen in a level comparable to the lower concentrations found in urines of non-exposed persons38. The analysis was performed using an elution gradient and a normal phase separation column. The gradient started at a CO2:methanol-ratio of 95:5 and was ramped to 60:40 within 3 minutes.


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Figure 5

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SFC-APLI-(TOF)MS chromatograms (EIC 218 ± 0.5) of urine with (black line) and without (red line) spiking with 1-hydroxypyrene (500 ng L-1) after enrichment using SPE

Prior experiments (data not shown) exploring the linearity showed a confident relationship (r2 = 0.996) of the 1-HP signal and the concentration within a range of 1 µg L-1 to 200 µg L-1. This method is therefore useful for the determination of urinary 1-HP for both cases, exposed and unexposed person’s urine.

CONCLUSIONS Different optimization steps have been performed to enhance the sensitivity of the novel hyphenation of SFC and APLI-(TOF)MS. The new setup allows a qualitative and quantitative analysis of PAH compounds in the sub-µg L-1 range and shows good linearity with successful


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peak shapes for a lot of PAH compounds. Compared to HPLC, SFC-APLI-(TOF)MS shows lower or equal LODs for a lot of analytes. The systems’ sensitivity combined with the basic setup properties allows the determination of 1-hydroxypyrene in human urine in relevant concentrations.

ACKNOWLEDGMENT The authors gratefully acknowledge SIM Scientific Instruments Manufacturer GmbH, Oberhausen, Germany for providing the SFC instrument. Further on, it is a great pleasure to honor Thorsten Benter and his research group at the University of Wuppertal for support, knowledge, lab space, lasers, and mass spectrometers. DK is thankful to H. W. Kling for allocating an office, lab space and some financial support. ABBREVIATIONS SFC, supercritical fluid chromatography; APLI, atmospheric pressure laser ionization; MS, mass spectrometry; PAH, polycyclic aromatic hydrocarbons; 1-HP, 1-hydroxypyrene. REFERENCES

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SFC-APLI-(TOF)MS: Hyphenation of Supercritical Fluid

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