Development of a Non-Targeted Method to Study Petroleum Polyaromatic Hydrocarbons in Soil by Ultrahigh Resolution Mass Spectrometry Using Multiple Ionization Methods

Abstract A non-targeted method for analyzing petroleum hydrocarbons in soil was established by extracting a wide range of PAC compounds using a model system. Here, sand was spiked with a heavy crude oil, which simulated a hydrocarbon contaminated soil of low soil organic matter (SOM). Soxhlet and supercritical fluid extraction (SFE) was optimized for PAXHs analysis in soil. A concept of using multidimensional ionization for ultrahigh resolution mass spectrometry was employed for detection using electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo ionization (APPI) for the analysis of polycyclic aromatic hydrocarbons (PAHs) and polycyclic aromatic heterocycles. Fourier transform mass spectrometry (FT-MS) with its ultrahigh mass resolution and high mass accuracy enables an unambiguous assignment of the detected ions obtained from different ionization methods, which provides a secure and comprehensive view of contaminants in soil on a molecular level. Over 95% of the spiked crude oil could be recovered by Soxhlet extraction using toluene, dichloromethane and acetone:n-hexane (1:1, v:v). In comparison, when using SFE with supercritical CO2 the recovery was only about 50% but showed more polar compounds. Detailed analyses showed that besides 16 PAHs, a wide range with more than 10,000 PAXHs could also be successfully extracted.


Sample preparation
The Soxhlet extractions were performed using 5.00 g of spiked sand and additionally 5.00 g of anhydrous sodium sulfate (99.5%, Acros Organics) with 100 mL of toluene (Tol), dichloromethane (DCM), acetone:n-hexane (1:1, v:v, AceHex) or methanol (MeOH) at the boiling point of the solvents for around 300 cycles. All solvents used were HPLC-grade and purchased from Sigma-Aldrich (Germany) or Acros Organics (Germany). The extracts were concentrated in a rotary evaporator and dried under gentle argon stream.
The SFE experiments were performed using a lab-built SFE system (for detailed description see Figure  S1). 5.00 g of spiked sand were added into a stainless steel column and heated to 40 C. Then samples were extracted at 180 bar and 40 C with supercritical CO 2 (99.995%, Air Liquide, Germany) at a flow rate of 2 mL min À1 for 4 h. The SFE extracts were rotary-evaporated and dried under gentle argon stream. The SFE residues were Soxhlet extracted using dichloromethane with the procedure described above.

Mass spectrometric measurements and data analysis
Mass analysis was performed on a research type linear quadrupole ion-trap (LTQ) FT Orbitrap MS (Thermo Fisher, Bremen, Germany) with LTQ Tune Plus 2.7.0 data processing system (Thermo Fisher, Bremen, Germany). The mass spectra were collected from 200 to 1200 Da using the spectral stitching method with 30 Da mass windows with 5 Da overlap. 15,49,50 Additionally positive/negative mode ESI or positive mode APCI/APPI with a mass resolution of 960,000 at m/z 400 was applied. For ESI measurements samples were diluted to 250 lg mL À1 in toluene:methanol (1:1, v:v), and injected at a flow rate of 5 lL min À1 with a spray voltage of 4/À3.5 kV for the positive/negative mode. Sheath, auxiliary and sweep gases were set to 5, 2, and 1 arbitrary unit for both polarities. Samples for APCI or APPI were diluted to 250 lg mL À1 in toluene and ionized under the following conditions: flow rate ¼ 20 lL min À1 , vaporizer temperature ¼ 350 C, sheath gas ¼ 20 arbitrary unit, auxiliary gas ¼ 5, and sweep gas ¼ 2. The corona discharge for the APCI was set to 5 lA and a Krypton VUV lamp (10.0 and 10.6 eV, Syagen, Tustin, CA, U.S.A.) was used for the APPI.
The DBE value for a given elemental composition C c H h N n S s O o was calculated with the following equation:

Recovery of crude oil in spiked sand
A non-targeted analysis of petroleum hydrocarbons in soil is an adventure due to the high complexity of compounds present in such a sophisticated system. As one of the most important sample preparation steps, the extraction of petroleum based contaminants from soil needs to be investigated in detail.
Therefore, pure sand spiked with a heavy crude oil served as a model sample for a better understanding of the extraction step. Here, we compared the traditional Soxhlet extraction using different solvents with supercritical CO 2 extraction. Toluene, dichloromethane and a 1:1 (v:v) mixture of acetone and n-hexane are commonly used solvents for the Soxhlet extraction of 16 EPA PAHs in soils on a routine scale. 36,[41][42][43][44]51 Using these three solvent systems a recovery of over 95% for crude oil in the spiked sand was achieved (see Figure 1), where dichloromethane with 97% ± 1% recovery delivered the highest extraction efficiency. Methanol was applied as a mild extraction solvent to study the solubility in polar solvents and therefore, the bioavailability of hydrocarbons in soil. 52 Here, a lower recovery of 73% ± 4% compared to other Soxhlet extraction solvents was obtained. This implies the complex character of the heavy crude oil.
When using supercritical CO 2 extraction 47% ± 1% of petroleum components were extracted. Reports in the literature showed a wide range of results for the extraction using SFE. A good recovery of over 80% was reported for lighter jet-diesel oil and fuel oil from spiked sand after the SFE at 65 C and 400 bar. 46 However, Hawthorne and Miller showed that at a comparable extraction temperature of 50 C and accelerated pressure of 405 bar, lower extraction efficiency was found for higher molecular weight and heteroatom containing PAHs. 47 This explained the lower extraction efficiency gained in our study, thus the heavy crude oil contained to a proper portion high molecular weight and heteroatom containing PAXHs.
Although SFE is more environmental friendly and time saving, it cannot extract all contaminants from the soil, especially compounds with higher molecular weight. In contrast to that, the traditionally Soxhlet extraction with dichloromethane offers a simple and feasible way to extract petroleum hydrocarbons from the soil for the non-targeted analysis, albeit the large volume and long extraction time needed.

Mass spectra
Because of the sample complexity, the selectivity of different ionization methods and polarities can be used to provide a comprehensive view of the sample. 21 This is presented in the recombined mass spectra, which was recorded using spectral stitching method, 15,49,50 for the original crude oil (Figure 2, left column). For all ionization methods applied ions were detected throughout the whole mass range starting from 200 to 1200 Da. In the zoomed in mass spectra between m/z 400.00 and 400.50 ( Figure 2, right column) up to 42 elemental compositions were detected, which was depended on the selected ionization method. Compounds containing basic nitrogen dominated in the ESI(þ) spectrum. Acidic oxygen classes and neutral nitrogen containing components are well ionized in ESI(À). Nonpolar classes such as pure hydrocarbons and sulfur containing PAHs appear in both APCI and APPI measurements. To resolve isobaric peaks of C 26 13 CH 43 S þ (m/z 400.31135) and C 30 H 40 .þ (m/z 400.31245) with a Dm ¼ 0.00110 Da, a resolving power higher than 400,000 is needed. The result shows the necessity of combining different ionization methods with ultrahigh resolution MS for a non-targeted analysis of complex petroleum hydrocarbons.
In Figure 3, the zoomed in positive mode APPI mass spectra between m/z 400.00 and 400.50 for the original crude oil, Soxhlet extract of the spiked sand using dichloromethane, SFE extract and residue of the spiked sand were compared. In total, 42 elemental compositions were found at this mass range for the original crude oil (Table S1). Here, radical cations of pure hydrocarbons and PASHs dominated the mass spectra. C 30 H 40 and C 27 H 44 S with a double bond equivalent (DBE) of 11 and 6 were the first and second highest signal detected in the mass spectra for the original crude oil, Soxhlet and SFE extracts. By decreasing m/z value the DBE value of isobaric pure hydrocarbon ions (C 29 H 52 þ , C 30 H 40 þ , C 31 H 28 þ , C 32 H 16 þ , highlighted in red from right to left in Figure 3), which have a Dm ¼ 0.09390 Da between each other, increases from 4 to 25. The extraction efficiency of SFE decreases the higher the aromaticity is. As can be seen in the spectra, C 29 H 52 þ (DBE ¼ 4) and C 30 H 40 þ (DBE ¼ 11) were to a great degree extracted using SFE, while C 31 H 28 þ and C 32 H 16 þ with a DBE of 18 and 25 remained mainly in the residue. This is also applicable for S class compositions shown in Figure 3.
Furthermore, positive and negative mode ESI mass spectra for the original crude oil, the extract and residue after SFE reveal that basic and acidic crude oil related contaminants could also be extracted from the spiked sand ( Figures S2-S4 are documented in the supporting information). Compounds with lower DBE were preferentially extracted than compounds with higher DBE. Using API-FT MS a detailed comparison between Soxhlet and SFE on a molecular level can be fulfilled.

Class distribution
Due to the high data load obtained from the non-targeted analysis using ultrahigh resolution FT MS, a suitable, concise and explicit data interpretation is obligatory. This can be done by sorting each signal from the mass spectrum into elemental compositions, which then can be categorized into classes according to their counts of heteroatoms. Different classes such as pure hydrocarbon or heteroatom (N, S, and O) containing classes from different ionization techniques or samples can be compared in a bar chart with their corresponding relative abundances or numbers of assigned compositions.
In Figure 4, a summary of intensity based class distributions for the Soxhlet extract using dichloromethane is shown. Protonated basic nitrogen containing classes such as N, N 2 , NS, and NS 2 dominate the ESI(þ) spectrum with few compounds form radical ions as well as oxygenated sulfur containing class ionized with sodium adduct. In ESI(À) the mass spectrum consists of mainly oxygenated classes especially O 2 and O 4 , which can correspond to molecules contain one or two carboxylic acids. 53 Other oxidized heteroatom containing classes such as NO x and SO x were also present with a lower intensity. The selective ionization of acidic components in the sample makes it a perfect method for examining polar transformation products, which are created during the chemical/biological oxidation of PAHs contaminated soil. [54][55][56] Both APCI and APPI form radical cations [M] þ and protonated molecules [M þ H] þ of predominantly nonpolar pure hydrocarbons (HC), S x , and N classes. In particular APPI by exciting the double bonds in a molecule which suits the best for the targeted and non-targeted analyses of pure PAHs and PAXHs (X ¼ S, N, O). 21,57 Results show how complex such a system, which is simulating a petroleum hydrocarbon contaminated soil, can be. Additionally, when analyzing petroleum hydrocarbons in soil in a non-targeted way, ion suppression and discrimination effects can occur. 58 This consolidates the need of multidimensional ionization techniques for the deep and comprehensive analysis of petroleum hydrocarbon contaminated soils.
For the targeted analysis, only the 16 EPA PAHs are of interest. But by using positive mode APPI FT MS other high molecular weight and heteroatom containing PAHs present as contaminants can be efficiently ionized and analyzed as well. 21 For the original crude oil the highest number of assigned compositions was detected by APPI(þ) (25,943), followed by APCI(þ) (22,802), ESI(þ) (15,264), and finally ESI(À) (12,221) (Figures 5-7). Results confirmed the nonpolar character of the spiked contamination source. For a comparison the extraction efficiency of Soxhlet extraction using different solvents and SFE is summarized in Figure 5. Here, both the relative abundance and the number of assigned compositions for each detected class were considered.
Among these 25,943 assigned compositions by APPI(þ) around 4000 were detected as either protonated or radical hydrocarbons in the mass spectrum, which display a relative intensity of around 35%. With an increase in polarity from toluene to methanol, the number of assigned compositions in the HC class decreases from 4082 to 3629. For the polycyclic aromatic heterocycles, such as compounds in S, S 2 , and N classes, over 95% of the assigned compositions from the original crude oil could be found in the Soxhlet extracts using toluene, dichloromethane, and acetone-n-hexane solvent mixture. For methanol as extraction solvent a recovery of over 90% of the assigned compositions was observed.
In comparison, less than 67% (17,367 out of 25,943 compositions from the original crude oil) were present in the SFE extract, which consisted of predominantly pure hydrocarbons (41%), followed by S (32%), S 2 (6%), O (5%), and N (4%) classes. As a result of extraction, the relative intensities of HC and S classes found in the SFE residue decreased dramatically to 21% and 14%, respectively.
However, the numbers of assigned compositions between the original crude oil and SFE residue did not differ too much, which indicates lower extraction efficiency for the complex sample when SFE was applied. The slightly higher amount of HC class compounds and new classes found in the SFE residue could be explained, as there was discrimination and ion suppression occurred during ionization for the original crude oil. 58 Additionally, SFE separated the sample into two fractions which led to an increasing analysis depth. 59 Similar results were observed by APCI(þ) studies (see Figure 6). There, compositions from pure hydrocarbon und heteroatom containing classes such as S, S 2 , and N could be extracted well by Soxhlet extraction using toluene, dichloromethane and acetone/n-hexane solvent mixture. Only a portion (%70%) of the compositions in the above mentions classes could be extracted using SFE.
Results from APPI(þ) and APCI(þ) were mutually supportive and implied a better extraction efficiency for nonpolar compounds by Soxhlet extraction using toluene, dichloromethane and acetone-n-hexane solvent mixture than SFE. In ESI(þ) the N class with the highest intensity of 60% in the original crude oil consists of mainly basic pyridine-like azaarenes (see Figure 7, left). 13 Out of 2701 assigned compositions as either protonated or radical cations for the N class in original crude oil 1951, 2002, 1569, and 2259 compositions were recovered using toluene, dichloromethane and acetone-n-hexane solvent mixture and methanol, respectively. For the second and third highest intensity classes in the original crude oil, namely NS and N 2 , same trend was observed. As the most polar solvent investigated methanol could extract over 80% of the assigned compositions for these two classes, whereas for other solvent systems the recovery was between   60% and 70%. The appearance of [SO þ Na] þ class with relative high intensity for the Soxhlet extracts was probably a result of sodium salt added for the removal of water. 60 Compared to the Soxhlet extraction also a good extraction of polar N class compounds from the spiked sand was achieved when supercritical CO 2 was used. Here, a decrease of relative abundance from 74% (SFE E) to 14% (SFE R) was observed.
Compared to ESI(þ) with 15,264 assignments, in ESI(À) 12,221 compositions were detected for the original crude oil, which comprise polar compounds containing acidic functional groups (Figure 7, right). Unlike in the positive mode the assigned compositions in ESI(À) were distributed to O 2-4 , N 0-2 , and S 2-4 quite equally. A comparable high recovery was achieved by Soxhlet extraction using dichloromethane and methanol. In comparison to this, SFE could only partially extract polar compositions in each class.
These findings obtained from each ionization method showed, Soxhlet extraction is an easy and efficient method, which can be applied prior to the non-targeted analysis of crude oil related contaminants in soil and gives the best representation of the sample.

DBE distribution
The absolute intensity within a given DBE was plotted against compound classes for the APPI(þ) measurement of the samples (Figure 8). The radical cation and protonated molecule classes are denoted as M and M[H], respectively.
A DBE range of 1-50 was observed for the pure hydrocarbon and heteroatom containing classes, such as S 1-3 and N, found in the original crude oil, Soxhlet extract and SFE residue. However, in the SFE extract compounds with DBE only up to 37 were detected. Considering a benzene ring has a DBE value of 4 and by adding another aromatic ring into the system to form naphthalene, anthracene, and so on, the DBE increases by 3 every time with up to 12 aromatic rings. Similar results were observed using APCI(þ) as shown in Figure S5. Since predominant compounds with lower DBE could be extracted using SFE, this reduces the sample complexity and increases the intensity of compounds with lower abundance (Figure 5), 59 which explains the additional classes found in the SFE residue.
As summarized in Table 1, the mean m/z value for the SFE extract was shifted to the lower mass range compared to the original crude oil sample. Besides, the DBE value decreased from 19 to 15 while H/C increased from 1.23 to 1.38. Accordingly for the SFE residue the mean DBE value increased to 20 while H/C decreased to 1.17. These indicate that SFE favors low aromatized hydrocarbons, which is in agreement with previous studies. 40 Moreover, polar compounds such as basic pyridine-like or neutral pyrrole-like nitrogen containing compounds and oxygenated classes, analyzed using ESI(þ/À), could also be extracted with a maximum DBE of 35 ( Figures S6 and S7).

DBE vs. carbon count/intensity plot
A closer look into the radical HC class on a molecular level is shown in left column of Figure 9 using the so-called Kendrick plot. 14 Here, each detected composition was visualized by its DBE value and number of carbon atoms per molecule. In addition, the total monoisotopic abundance as a sum of all compositions within one given DBE was displayed in line.
2107 and 1515 out of in total 2173 radical cations found in the original crude oil could be fully or partially extracted using Soxhlet or SFE, respectively. For the original crude oil, Soxhlet and SFE extracts pure hydrocarbons with DBE of 12 exhibited the highest intensity, which correspond to PAHs containing 4 rings, such as pyrene or fluoranthene with different length of side chains. Instead, in the SFE residue the maximum intensity of DBE shifted to 15, indicating PAHs involving 5 aromatic rings, such as benzo[a]anthracene, benzo[b]fluoranthene or benzo[k]fluoranthene, were dominant in the mass spectrum. Furthermore, the maximum intensity at DBE 15 in the SFE residue (10 5 ) was reduced by one order of magnitude compared to the value in the extract (10 6 ).
The PASHs in the original crude oil, Soxhlet, and SFE extracts showed a maximum intensity at DBE of 9, which correspond to mostly dibenzothiophene and its alkylated derivatives ( Figure  S8, left column). 61 However, in the SFE residue compounds with DBE ¼ 19 gained the highest intensity. Also for PANHs a shift of the maximum intensity from DBE ¼ 12 to DBE ¼ 17 was observed after SFE ( Figure S8, right column). Overall, the results from APPI(þ) measurements showed that SFE as compared to Soxhlet extracts only contains nonpolar PAHs and PAXHs with DBE below 35 and carbon number below 90.  As an example for ESI(þ) measurements, basic nitrogen containing compounds are shown in the middle column of Figure 9. From 2134 polar pyridine-like compounds 1538 and 1101 could be extracted using Soxhlet and SFE, which contains mainly 1-5 aromatic rings. However, as the length of alkyl side chains increases, the extraction efficiency decreases. The neutral pyrrole-like nitrogen containing compounds were analyzed using ESI(À) (Figure 9, right column). Since in the original crude oil N class compounds with only DBE up to 36 were detected, they can all be extracted to a certain extent using both methods. Comparing the SFE extract with its corresponding residue, neutral PANHs could be largely (about 90%) extracted through SFE.

Conclusion
In this work, different organic solvents, which are commonly used for the quantitative analysis of 16 EPA PAHs by Soxhlet extraction, were extended to extract other PAHs and PAXHs containing N or S and their alkylated or oxygenated derivatives. The analysis on a molecular level showed that nonpolar compounds could be extracted quite well by the solvents applied, whereas for polar compounds the sample extracted using dichloromethane and methanol exhibited the highest recovery. Results demonstrated that prior to mass spectrometric analysis the Soxhlet extraction could be used for extracting a large variety of hydrocarbon related contaminants from the soil, which are co-occurring with these 16 PAHs.
Additionally, SFE using supercritical CO 2 , which is usually referred to as a green extraction method, was studied in detail for extracting petroleum hydrocarbons from spiked sand. Compared to the standard Soxhlet extraction SFE could extract a range of compounds from nonpolar to polar hydrocarbons with a DBE and carbon count lower than 35 and 90, respectively.
In our study, a non-targeted method was developed to examine oil contamination in soil by using ultrahigh resolution mass spectrometry with different ionization methods. A heavy crude soil sample was spiked into pure sand to produce a simple model simulating a hydrocarbon contaminated soil sample with low organic content. Results showed that the selectivity of ionization methods applied offers sometime comparable but more importantly complementary data, each of which reflects only a part of the real sample. Therefore, for the non-targeted analysis of petroleum hydrocarbons in soil, a multidimensional ionization is indispensable. In addition, the coupling to FT MS instrument after ionization ensures a distinct assignment of the signals for such a complex sample.