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Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of aromatic amines

Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of... Some aromatic amines (AA) have been classified as carcinogens to humans. After entering the body, mainly through tobacco smoke, they can be detected in urine. Thus, their trace analysis as biomarkers in biofluids is of high relevance and can be achieved with gas chromatography (GC–MS), usually after derivatization. This study compares three gas chromatographic methods for the analysis of ten iodinated derivatives of AA: GC–MS in single-ion monitoring (SIM) mode with (1) electron ionization (GC-EI-MS) and (2) negative chemical ionization (GC-NCI-MS), and (3) GC-EI-MS/MS in multiple reaction monitoring (MRM) mode using electron ionization. All methods and most analytes showed good coefficients of determination (R > 0.99) for broad linear ranges covering three to five orders of magnitude in the picogram-per-liter to nanogram-per-liter range, with one and two exceptions for (1) and (2) respectively. Excellent limits of detection (LODs) of 9–50, 3.0–7.3, and 0.9–3.9 pg/L were observed for (1), (2), and (3) respectively, and good precision was achieved (intra-day repeatability < 15% and inter-day repeatability < 20% for most techniques and concentration levels). On average, recoveries between 80 and 104% were observed for all techniques. Urine samples of smokers and non-smokers were successfully analyzed, and p-toluidine and 2-chloroaniline could be found at significantly (α = 0.05) higher concentrations among smokers. Keywords Gas chromatography-mass spectrometry (GC–MS) · Gas chromatography-tandem mass spectrometry (GC–MS/ MS) · Negative chemical ionization (NCI) · Aromatic amines · Derivatization · Urine Introduction substances, but also the general public is at risk: the main source of exposure to some aromatic amines, such as the carcinogenic Several aromatic amines (AA) have been classified as possible, 2-naphthylamine and ortho-toluidine, and the probable carcino- probable, or certain carcinogens by the International Agency genic aniline and 4-chloro-o-toluidine, is cigarette smoke [2]. for Research on Cancer (IARC) [1], and most, if not all AA, AA enter the blood during the smoking process and are are believed to have carcinogenic potential [2]. However, they transported into the liver, where they can be metabolized are still widely used, for example, for the production of phar- and further transported, for example, to the bladder, where maceuticals, pesticides, dyes, or rubber [1]. Unfortunately, not they can react with DNA and proteins to form adducts that only the workers in these industries can get in contact with these can lead to cancer, and can be excreted in the urine [3]. AA have been suggested as the main cause for the excess risk of bladder cancer in smokers [4]. * Torsten C. Schmidt The concentrations of AA in different matrices have torsten.schmidt@uni-due.de been studied in several steps of the aforementioned pro- Instrumental Analytical Chemistry, University of Duisburg- cess, for example, in smoke [5–9] (e.g., aniline, toluidines, Essen, Universitätsstrasse 5, 45141 Essen, Germany or dimethylanilines), as DNA [10–13] and protein adducts Institute of Analytical Chemistry and Food Chemistry, Graz [12, 14–17] in cells/blood (e.g., 4-aminobiphenyl), or as University of Technology, Stremayrgasse 9/II 8010, Graz, free AA and metabolites in urine [3, 18–31] (e.g., naphthy- Austria lamines, chloroanilines). Because the intake of substances Centre for Water and Environmental Research, University during smoking varies depending on the individual smok- of Duisburg-Essen, Universitätsstrasse 5, 45141 Essen, ing topography [32], and the amount of DNA and protein Germany 4 adducts is typically extremely small [33], this study focuses IWW Water Centre, Moritzstrasse 26, on urine samples. There, not only free aromatic amines can 45476 Mülheim an Der Ruhr, Germany Vol.:(0123456789) 1 3 Lorenzo-Parodi N. et al. be found but also their metabolites, such as N-acetylaryl- compounds (Table 1), with a purity of 97% or more, were amine, N-glucuronide arylamine, or hemoglobin and DNA purchased from Merck KGaA (Darmstadt, Germany). adducts, which can be hydrolyzed and converted back to Concentrated hydrochloric acid (HCl, 37%) from VWR; the free aromatic amines [14, 34]. ethyl acetate (99.9%) from Carl Roth (Karlsruhe, Germany); Direct analysis of AA is possible using liquid chroma- diethyl ether (99.5%) from ChemLab (Zedelgem, Belgium); tography (LC) [19, 21, 24, 25, 35–37]. However, its low sodium hydroxide (NaOH, 99%), alizarin red S (98%), hydri- peak capacity [38, 39] hinders its use for the analysis of odic acid (unstabilized, 55%), and sodium nitrite (99%) from complex urine samples. Due to its high sensitivity, short Merck KGaA; and sodium sulfite (≥ 98%) and sulfamic acid analysis time, and high resolving power [40], gas chro- (≥ 99%) from Fluka (Buchs, Switzerland) were used. matography (GC) was used for this study. In order to reduce the polarity of the AA and facilitate Preparation of stock and standard solutions their analysis, they are typically derivatized. In this study, they were iodinated via a Sandmeyer-like reaction as All the stock and intermediate solutions were prepared in reported by [18, 22, 39]. This derivatization procedure offers methanol. Individual stock solutions of each of the analytes the advantage that the reagents used do not need strictly were prepared at 1 g/L. An intermediate standard solution anhydrous conditions, as is the case for the commonly used was prepared at 1 mg/L for the iodinated aromatic com- acylation [39] and silylation derivatizations [40]. pounds. Working solutions were prepared by diluting the This derivatization step enables their analysis with dif- intermediate standard solutions in methanol and were used ferent types of GC systems, such as GC–MS [18, 27, 30, 31, within 1 month. One working solution was prepared for each 41, 42], GC × GC–MS [22], GC-NCI-MS [8, 29, 43–45], or concentration tested. All the solutions were stored at 7 °C. GC–MS/MS [3, 9]. However, a comparison of the different techniques, namely GC–MS, GC-NCI-MS, and GC–MS/ Sample preparation MS, has not been previously reported for these analytes. The aim of this study is, therefore, the comparison of Glass-covered stirring bars (VWR International GmbH) different GC detection techniques for the determination were placed in 20-mL crimp vials, which were then filled of aromatic amines in urine after derivatization to the with 5 mL of the samples and closed with magnetic caps corresponding iodinated benzenes, namely GC-EI-MS, with Silicone/PTFE septa. GC-NCI-MS, and GC-EI-MS/MS. To that end, all studied For the validation experiments, the samples were pre- methods were validated and used for the analysis of real pared by adding 10 µL of the corresponding iodinated work- urine samples from smokers and non-smokers. ing solution to 5 mL deionized water. Urine samples from seven donors (four smokers and three non-smokers) were collected in 1-L Schott bottles and stored Materials and methods at 7 °C for up to 1 month. The analytes are likely stable under those conditions based on a recent study by Mazumder Chemicals and reagents et al. [46], who found no marked decrease in concentration with samples at similar temperatures during the total time Methanol ≥ 99.9%, HiPerSolv Chromanorm for LC–MS studied, which, however, was limited to 10 days. The urine (VWR International GmbH, Darmstadt, Germany), was used samples were prepared according to Lamani et al. [22], with for the preparation of standard solutions. Iodinated aromatic a few modifications. First, 20 mL of urine was hydrolyzed Table 1 Iodinated compounds Analyte Abbreviation Aromatic amine precursor CAS Nr Purity (%) used, including the abbreviation by which they are referred to 4-Iodotoluene 4IMB p-Toluidine 624–31-7 99 in the text, their corresponding Iodopentafluorobenzene IPFB Pentafluoroaniline 827–15-6 99 aromatic amine precursor, CAS 2-Iodo-1,3-dimethylbenzene 2I13DMB 2,6-Dimethylaniline 608–28-6 97 Number (CAS Nr), and purity Iodobenzene IB Aniline 591–50-4 98 1-Chloro-2-iodobenzene 1C2IB 2-Chloroaniline 615–41-8 99 3-Chloro-4-fluoroiodobenzene 3C4FIB 3-Chloro-4-fluoroaniline 156150–67-3 98 2,4,5-Trichloroiodobenzene 245TCIB 2,4,5-Trichloroaniline 7145–82-6 98 2,4-Dichloroiodobenzene 24DCIB 2,4-Dichloroaniline 29898–32-6 98 1-Bromo-4-iodobenzene 1B4IB 4-Bromoaniline 589–87-7 98 2,4-Difluoroiodobenzene 24DFIB 2,4-Difluoroaniline 2265–93-2 98 1 3 Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… with 10 mL of HCl (37%) at 80 °C and 200 rpm stirring were extracted from the headspace with a 65-μm PDMS/ speed, for 12 h in order to convert metabolized AA into DVB SPME fiber (1 cm length, Stableflex, 23 Ga, Merck free AA. All heating and stirring steps were done on an MR KGaA) for 30 min before injection into the GC system. The 3001 K stirring plate from Heidolph Instruments GmbH & SPME fiber remained at least for 5 min in the injector in Co. KG (Schwabach, Germany). Once the sample reached order to condition the fiber after injection, except for the room temperature, it was basified by adding 20 mL of 10 M GC-EI-MS/MS measurements, where the fiber was pre- NaOH to the solution. Afterward, the amines were extracted conditioned for 2 min in a dedicated conditioning station at two times into 5 mL of diethyl ether. The organic fractions 280 °C, and remained for 2 min in the injector. were then mixed and cleaned with 2 mL of 0.1 M NaOH. The extraction efficiencies of the three SPME fibers used The amines were subsequently back-extracted into 10 mL of (one for each technique) were compared after the methods water, previously acidified with 200 μL concentrated HCl were validated, using GC-NCI-MS and a iodinated deriva- (37%). Any remaining diethyl ether in the aqueous frac- tives solution (1 ng/L, Fig. S2 and SI). Furthermore, a SPME tion was evaporated by nitrogen blowing on the samples test mix (200 ng/L) was analyzed regularly in order to ensure for 20 min. the integrity of the fiber and the performance of the system, The aqueous extracts were then derivatized by substitut- by adding 20 µL of a stock solution in a vial with a stir- ing the nitrogen for an iodine atom in order to decrease the rer. A list of the analytes included in the mix (minimum polarity of the extracted amines (see Supplementary infor- purity 95%, different providers) can be seen in Table S1 (SI). mation (SI), Fig. S1). This was achieved by adding 200 μL Because the mix included mostly analytes that are not ion- hydriodic acid (55%) and 400 μL sodium nitrite (50 g/L) izable by GC-NCI-MS, it was not used for this technique. and stirring at 200 rpm for 20 min (step 1), adding 1 mL of A significantly lower intensity was observed with GC-EI- sulfamic acid (50 g/L) and stirring at 200 rpm for another MS/MS during the first use of the corresponding SPME fiber 45 min (step 2), then heating the sample to 95 °C for 5 min (see Fig. S3, SI). Although the manufacturer instructions (step 3), and finally adding 800 μL sodium sulfite (120 g/L) were followed, it has been previously reported that it might (step 4) and 200 μL of alizarin red S (1% w/v) (step 5), and be insufficient conditioning [47, 48]. Therefore, the GC-EI- adjusting the pH to 5 with NaOH and HCl solutions (step 6). MS/MS results were normalized according to the SPME Mix This way, (step 1) the aromatic amines are diazotized and the intensities, which, as expected, also showed a similar trend diazonium ions are further substituted by iodine; (step 2) the (see Table S1, SI). surplus of nitrite is destroyed; (step 3) the unreacted diazo- nium ions are transformed into phenols, and the excess sul- GC–MS analysis famic acid is destroyed; (step 4) the iodine residue is reduced; (step 5) a pH indicator is added for easy identification of the In order to facilitate the comparison of the different tech- correct pH; and (step 6) a pH value suitable for subsequent niques, as many parameters as possible were kept constant SPME is achieved. throughout the different devices. Helium 5.0 (Linde, Höll- Finally, 5 mL was transferred into a 20-mL vial with a stir- riegelskreuth, Germany) was used as carrier gas for all rer, crimped, and then placed in the autosampler for further techniques. treatment, namely SPME and injection into the GC. For GC- A GCMS-QP2010 Ultra (Shimadzu) equipped with a ZB- NCI-MS and GC-EI-MS/MS, the samples were diluted 1:10. Wax 20  m × 0.18  mm × 0.18  µm (Phenomenex, CA, USA) was used for the GC-EI-MS analysis. The linear velocity SPME extraction was set to 45 cm/s, which corresponds to a column flow of 1.03 mL/min. The samples were injected in splitless mode, All the SPME fibers were conditioned prior to their first use, and after a sampling time of 1 min, the split ratio was set to as recommended by the supplier (i.e., 250 °C for 30 min). 10. The injector temperature was set to 250 °C, the interface The SPME extraction was done automatically by different temperature to 230 °C, and the ion source temperature to autosamplers, namely HTX PAL (CTC Analytics, Zwingen, 200 °C. The oven program started at a temperature of 40 °C, Switzerland) for the GC-EI-MS measurements, AOC-6000 was held for 1 min, ramped at a rate of 10 °C/min to 240 °C, (Shimadzu, Kyoto, Japan) for the measurements with the and held for 1 min. The final oven temperature was lower GC-EI-MS/MS, and AOC-5000 Plus for GC-NCI-MS (Shi- than in other instruments due to the different column used. madzu). All the autosamplers were controlled with PAL The acquisition was made in SIM mode, with an event time Cycle Composer except for the autosampler used in com- of 0.2 s. Twenty channels were looked into (Table S2 (SI)), bination with the GC-EI-MS/MS, which was directly con- typically corresponding to the molecular ions and the frag- trolled by the GCMS Real Time Analysis software (Shi- ment resulting from the loss of iodine. madzu). The samples were incubated for 5 min at 60 °C and A GCMS-TQ8050 (Shimadzu) with a 30 m × 0.25 mm × 500 rpm in a single magnet mixer (SMM). Afterward, they 0.25  µm Rxi-5MS (Restek, PA, USA) was used for the 1 3 Lorenzo-Parodi N. et al. GC-EI-MS/MS analysis. The injector, interface, and ion the calibration curves were up to five orders of magnitude source temperatures were set to 270, 280, and 200 °C respec- (1–100,000 pg/L) broad, and the points were equidistant only tively. The injection was done in splitless mode, and a split in the logarithmic scale, a normal linear fit would be very ratio of 10 was applied after a sampling time of 1 min. The heavily influenced by the higher calibration levels. There- linear velocity was 35 cm/s. The oven starting temperature fore, in order to accurately determine lower concentrations, was 40 °C, which was held for 1 min, ramped to 280 °C at a we limited the number of calibration levels in this and the rate of 10 °C/min, and held for 1 min. The MS was operated following sections, so that there would be at least 5 points in multiple reaction monitoring (MRM) mode, using argon per curve and up to 7 levels. Furthermore, the concentration 5.0 (Linde) as the collision gas. The optimal collision ener- to be determined was, if possible, kept in the middle of the gies (CE) were found by directly injecting 1 µL of a mixture levels selected. Afterward, the limits were calculated accord- of the iodinated analytes in 50:50 methanol:ethyl acetate ing to the Eurachem Guide [49], with a constant (k, equal (0.5 mg/L), at different CE, and comparing the intensities. to 3 for LODs and 10 for LOQs) multiplied by the standard The transitions monitored, and their corresponding CE, can deviation of the replicate concentrations and divided by the be seen in Table S3 (SI). The event time was set to 0.3 s for degrees of freedom (n − 1 = 9). all transitions. The next step was to calculate the intra-day and inter-day GC-NCI-MS measurements were done on a GCMS- repeatability. In order to do that, at least three calibration QP2010 Plus system (Shimadzu) equipped with a points (equidistant in the logarithmic scale and well distrib- 30  m × 0.25  mm × 0.25  µm Rxi-5Sil MS column (Restek). uted within the linear range) were measured in triplicate over The interface temperature was set to 250 °C and the ion three consecutive days. The concentration levels tested were source temperature to 160 °C. All other GC parameters were 1, 10, and 100 ng/L for GC-EI-MS; 0.1, 1, and 10 ng/L for the same as for the GC–MS/MS. MS acquisition was per- GC-NCI-MS, and, because of the broad range that could be formed in SIM mode, with an event time of 0.3 s, and moni- analyzed with GC-EI-MS/MS, four concentrations, namely toring the ions corresponding to chlorine (35, 37), bromine 0.01, 0.1, 1, and 10 ng/L, were studied with that technique. (79, 81), and iodine (127). The ionization gas was isobutane The recovery was calculated from the repeatability 3.5 (Linde), set to a pressure of 0.7 bar. experiments. For each instrument and concentration level, A comparison of the chromatograms obtained with the the recovery was calculated by dividing the average of the aforementioned parameters can be seen in Fig. S4 (SI). concentrations obtained (n = 9) by the expected theoretical concentration and multiplying the result by 100. Method validation The validation was done mostly according to the Eurachem Results and discussion Guide [49]. The raw data were evaluated with GCMSsolu- tion (Shimadzu) without applying smoothing, and the cal- Ten aromatic amine derivatives (i.e., iodinated aromatic culations were performed in Excel (Microsoft). compounds) were measured directly, without further sam- First of all, the linear ranges were studied with the aim ple treatment, in order to facilitate the direct comparison of of seeing not only how sensitive the instruments can be, but the methods. The studied analytes were selected as model also whether they have a linear response at the concentration compounds due to their diverse chemical structures and levels expected for real samples. Therefore, a very broad properties. Furthermore, most of them have been previously range was studied, and, subsequently, a logarithmic scale studied and found in smoke [5–9], blood/tissue [10, 14–16], was used in order to have equidistant calibration levels, as and/or urine matrixes [3, 21–23, 26, 28–30]. Three of the recommended by the DIN 38402–51 [50]. Concentrations most comprehensive papers in terms of AA studied [8, 22, from 1 pg/L to 500 ng/L were tested, with three concentra- 28] found aniline, p-toluidine, 2,6-dimethylaniline, 2-chlo- tion levels per order of magnitude. Exemplary calibration roaniline, 2,4,5-trichloroaniline, and 2,4-dichloroaniline, curves for each technique can be seen in Fig. S5 (SI). which are also included in this research. Afterward, the limits of detection (LODs) and of quantifi- cation (LOQs) were studied by repeating ten times the analy- Linear range sis of a calibration level where most of the analytes showed a signal to noise ratio (S/N) between 6 and 15 (200 pg/L The linear ranges observed can be found in Table 2. For the for GC-EI-MS, 100 pg/L for GC-NCI-MS, and 10 pg/L for GC-NCI-MS experiments, a plateau in the linear curve could GC-EI-MS/MS). This was done so that the concentrations be observed at concentrations of 50 or 100 ng/L, depending used for the calculation of the limits were not extremely on the compound. Excellent goodness of fit was achieved high in comparison with the limits themselves, and to conse- for all the methods tested, with coefficients of determination quently avoid obtaining overestimated sensitivities. Because (R ) above 0.99 for all cases except for 1B4IB and 245TCIB 1 3 Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… Table 2 Limits of detection (LOD), quantification (LOQ), and linear NCI-MS, and 1–100,000  pg/L for GC-EI-MS/MS. LODs and LOQs ranges in picograms per liter, obtained for the iodinated, aromatic were calculated with concentrations where most analytes had S/N compounds with the studied GC methods. The concentration ranges between 6 and 15, namely 200 pg/L for GC-EI-MS, 100 pg/L for GC- tested were 20–500,000 pg/L for GC-EI-MS, 2–100,000 pg/L for GC- NCI-MS, and 10 pg/L for GC-EI-MS/MS GC-EI-MS GC-NCI-MS GC-EI-MS/MS LOD (pg/L) LOQ (pg/L) Linear range (pg/L) LOD* (pg/L) LOQ* (pg/L) Linear range (pg/L) LOD (pg/L) LOQ (pg/L) Linear range (pg/L) IPFB 30 99 100–500,000 7.3 19 50–50,000 0.9 2.9 5–100,000 24DFIB 50 167 200–500,000 4.7 31 10–20,000 0.9 2.9 2–100,000 IB 25 84 100–500,000 6.8 23 20–50,000 2.1 7 5–100,000 4IMB 21 71 100–500,000 5.2 31 20–50,000 1.1 3.5 1–100,000 3C4FIB 14 47 100–500,000 6.3 21 10–20,000 1.3 4 2–100,000 1C2IB 26 86 100–500,000 3.0 15 5–20,000 0.8 2.5 2–100,000 2I13 DMB 21 71 50–500,000 5.6 28 10–50,000 0.5 1.7 1–100,000 1B4IB - - 10,000–500,000 4.9 14 20–50,000 2.0 7 10–100,000 24DCIB 9* 29* 50–500,000 4.8 20 10–20,000 1.2 4.0 2–100,000 245 TCIB 28 93 200–500,000 6.3 13 10–50,000 3.9 13 10–100,000 *Outliers found with Dixon’s Q test (α = 0.05, Q = 0.412) not included in the calculations Critical LODs and LOQs were calculated according to the Eurachem Guide [49], as a constant (3 and 10, respectively) multiplied by the standard devia- tion of the concentration from tenfold replicates, and divided by the degrees of freedom (n − 1 = 9). Smoothing was set to “none” when measured with GC-NCI-MS (0.988 and 0.989 respec- GC-EI-MS/MS can be explained by the fact that in the tively, data not shown). first quadrupole, only the ions selected are trapped, which The determination of 1B4IB with the GC-EI-MS method decreases the background noise, and consequently increases was hindered by an interfering signal covering the peak (see the sensitivity significantly. Fig. S6, SI), which led to the analyte being identified only in LODs and LOQs for the analysis of aromatic amines concentration levels of 10 ng/L or above. A different column in urine are generally reported in the nanogram-per-liter was used with this instrument, which could have led to a dif- range (see Table 3). The best LODs reported (< 5 ng/L) ferent elution pattern and may explain why the interference were achieved with MS/MS detectors [20, 24, 27] and was not observed in the other systems. A different set of m/z GC-NCI-MS [29] systems, while the worst (> 50 ng/L) may be used to study this compound, such as 155 and 157, were observed with EI-MS detectors [21, 30]. A simi- which corresponds to the fragment without iodine. lar trend can be observed in this study (Table 2), where When compared with literature (Table  3), the results GC-EI-MS shows worse limits than the other methods are similar or better than those typically reported. In most tested. Nonetheless, the results obtained were better cases, a linear range of approximately 3 orders of mag- than most of those found in literature, with LODs of nitude is reported [20–24, 27, 29, 30]. The results pre- 9–50  pg/L for GC-EI-MS, 3.0–7.3  pg/L for GC-NCI- sented here show a linear range of 4 orders of magnitude MS, and 0.5–3.9  pg/L for GC-EI-MS/MS. The lowest for GC-EI-MS and GC-NCI-MS and of 5 for GC-EI-MS/ limit reliably reported for aromatic amines in urine is MS. The broader the linear range, the higher the likelihood 0.89  ng/L [27], which is between 120 and 1800 times that analytes at very low concentrations can be accurately worse than those reported here for the iodinated deriva- quantified, and that there is no further dilution needed for tives with GC-NCI-MS and GC-EI-MS/MS. The reason samples with very high concentrations, saving both sample for the higher sensitivity achieved here is most likely volume and time. the combination of a pre-concentration step like SPME with very sensitive measurement techniques and the fact LODs and LOQs that iodinated derivatives were measured directly. Taking into account that during a similar derivatization proce- As expected, GC-EI-MS/MS shows the lowest LODs and dure, for most analytes an estimated loss of 10% was LOQs, followed by GC-NCI-MS and GC-EI-MS (see observed [38], it would be expected that the limits found Table  2), which on average have 3 and 12 times higher with these instruments, including the complete sample LODs, respectively. The high sensitivity achieved with preparation, would still be comparable if not better than GC-NCI-MS can be attributed to the high selectivity of those found in literature. this technique for halogenated compounds, which have a Other factors can affect the sensitivity of the method, such high electron affinity. The even better results obtained with as the amount of sample used (typically within 5–20 mL), 1 3 Lorenzo-Parodi N. et al. 1 3 Table 3 Figures of merit of most recent literature regarding the analysis of aromatic amines from urine samples. Ranges reported correspond to the minimum and maximum values from differ - ent analytes and/or concentration levels. Data in parentheses indicate missing experimental information needed for its interpretation Der. reagent Injection Volume/SPME fiber Instrument Calibration LOD (ng/L) Recovery (%) Intra-day pre- Inter-day Concentration in real Ref technique range (ng/L) cision (%) precision samples (%) HI HS-SPME 110 μm PDMS/DVB GC–MS 100–1200 3–12 n.r 3–12 n.r NS: n.d., S: [18] n.d.–243 ng/L 2 a No LI 5 µL LC–MS/MS (MRM) 100–50,000 25–500 75–114 1.6–11.7 2.1–15.9 U: n.d.–1.5, S: [19] n.d.–3.47 µg/L TMA-HCl, PFPA LI 1 µL GC–MS/MS (EI, 482–1280 1.8–111.2 > 85 1.1–6.3 2.6–6.3 n.r [3] MRM) No HS-SPME 80 µm, JUC-Z2 GC–MS/MS (MRM) 50–100,000 (0.010–0.012) 95–101 (7.1–7.7) n.r NS: n.d., S: [20] 68.4–123.1 ng/L PFPA, Pyr LI 5 µL LC–MS (SIM) 1000– 1000 n.r n.r n.r NS: 1–1.13, S: [21] 1,000,000 1.46–2.33 µg/L HI HS-SPME 65 μm PDMS/DVB GC × GC–MS 1–500 5.2–24.4 n.r n.r n.r n.r [22] No LI 1 µL GC-FID 30–100,000 (7–10) 93.0–99.9 2.5–5.9 4.7–7.3 NS: n.d.–1.2, S: [23] 2–14.5 µg/L No LI 10 µL LC–MS/MS (MRM) 5–10,000 (1.5–5) 88–111 6.1–8.9 9.0–9.9 NS: 1.11–12.32, S: [24] 5.39–67.02 ng/24 h No LI 3 µL UFLC (UV–Vis) 5000–500,000 (DI: 39,600–94,400, 89–105 0.6–7.9 2.4–10 U: n.d.–12.8 µg/L [25] AE: 880–1300) 2 a No HS-SPME PEG/CNTs GC-FID 1–105 (0.5–50) 63.7–97.0 3.2–9.1 5.5–12.0 NS: n.d.–940, S: [26] 1140–50,960 ng/L TMA-HCl, PFPA LI 1 µL GC–MS/MS (EI, 50–25,000 0.89 (20–25) 1.7–6.7 7.5–8.4 NS: 1.30–2.07, S: [27] MRM) 7.43–10.16 pg/mg Cr No LI 1 µL GC–MS (SIM) 5–60,000 2–26 94–104 4.5–6.2 6.0–6.8 U: n.d.–690 ng/L [28] PFPA, Pyr LI 0.2 µL GC–MS (NCI, SIM) 10–2500 (1–4) 94–107 (2.7–4.6) (5.1–7.0) NS: 9.6–105.2, S: [29] 15.3–204.2 ng/24 h PFPA, Pyr LI 1 µL GC–MS (SIM) 100–100,000 (50–2000) 70–125 1.8–14 7.5–19 U: n.d.–3.5 µg/L [30] Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… 1 3 Table 3 (continued) Der. reagent Injection Volume/SPME fiber Instrument Calibration LOD (ng/L) Recovery (%) Intra-day pre- Inter-day Concentration in real Ref technique range (ng/L) cision (%) precision samples (%) PFPI LI 1 µL GC–MS (SIM) n.r (0.05 ng) (82.3–96.8) n.r n.r NS: n.d.–1073.4, S: [31] 3.6–2119.8 ng/24 h HI HS-SPME 65 μm PDMS/DVB GC-EI-MS 0.05–500 0.009–0.05 93–116 2.8–11 1.8–46 NS: n.d.–64, S: This n.d.–173 ng/L study GC-NCI-MS 0.005–50 0.003–0.007 71–104 2.1–12 3.7–40 8 c GC-EI-MS/MS 0.001–100 0.001–0.004 66–117 0.2–13 4.0–35 Abbreviations: AE after extraction, Cr creatinine, Der. derivatization, DI direct injection, HS-SPME headspace solid-phase microextraction, JUC-Z2 two-dimensional porous organic framework, LI liquid injection, LOD limit of detection, n.d. not detected, n.r. not reported, NS non-smoker, PDMS/DVB polydimethylsiloxane/divinylbenzene, PEG/CNTs poly(ethylene glycol) modified with multi-walled carbon nanotubes, PFPA pentafluoropropionic anhydride, PFPI pentafluoropropionyl-imidazol, Pyr pyridine, Ref reference, S smoker, TMA-HCl trimethylamine hydrochlo- ride, U unknown smoking status, UFLC ultra-fast liquid-chromatography Considering 37 of the 41 analytes studied. Batch-to-batch precision. Excluding 24TCIB (28%) and IB (19%) at a concentration level of 10 pg/L LOD calculations: according to CLSI EP17-A S/N (signal to noise ratio) = 3 not repor ted according to DIN 32645 according to FDA guideline SD low-quality-control samples LOD = 3*SD /b ( = regression) y/x y/x LOD = 3*SD/(n − 1), (n = degrees of freedom), according to Eurachem Guide [49] Lorenzo-Parodi N. et al. Table 4 Average intra-day and inter-day repeatability, and recovery M (medium) = 10  ng/L, and H (high) = 100  ng/L; for GC-NCI-MS, (%) results obtained for each of the techniques studied. The concen- L = 0.1  ng/L, M = 1  ng/L, and H = 10  ng/L; and for GC-EI-MS/MS, tration levels tested were as follows: for GC-EI-MS, L (low) = 1 ng/L, L = 0.01 ng/L, M-L = 0.1 ng/L, M-H = 1 ng/L, and H = 10 ng/L GC-EI-MS GC-NCI-MS GC-EI-MS/MS L M H L M H L M-L M-H H Intra-day repeatability (%, n = 9) 7.1 7.8 5.2 5.6 5.5 3.7 12 4.0 2.8 2.1 Intra-day repeatability (%, n = 9)* 3.8 5.7 2.0 3.9 2.9 1.6 10.3 3.5 1.5 1.0 Inter-day repeatability (%, n = 3) 25 15 7.7 13 27 24 15 16 21 15 Inter-day repeatability (%, n = 3)* 12.6 8.7 4.5 5.3 7.4 2.9 13.2 9.7 9.6 7.7 Recovery (%, n = 9) 102 104 96 83 94 80 92 89 88 80 *Results obtained after internal standard-equivalent correction (explained in SI) the concentration level studied, the steps of the sample concentration. If the same concentration level is compared preparation procedure included, the use of matrix-matched across methods, for example, 1 ng/L, the average intra-day calibrations, and the equations used for the calculations (sig- repeatabilities of the methods are 7.1% for GC-EI-MS (with- nal to noise ratio, standard deviation, etc.). Unfortunately, out IB4IB), 5.5% for GC-NCI-MS, and 2.8% for GC-EI-MS/ in several occasions, information was lacking for a proper MS. The individual intra-day repeatabilities of each analyte interpretation of the results. For example, when the limits can be seen in Table S4, SI. were calculated based on the S/N ratio, the concentrations The majority of the inter-day repeatability results used or if smoothing was applied was usually not reported. (reported as RSDs) are below 20%; however, there are some If too-high concentrations are used, this can lead to too-low exceptions. This could be due to the fact that n is smaller (3 LODs, which seems to be the case for the lowest limit found vs 9), and the typical errors introduced during sample prepa- in literature [20], where the extrapolated limit reported is ration and measurement have a bigger effect the smaller the more than three orders of magnitude lower than the linear number of samples measured. range. If the different replicates are studied over time (as exem- plified for 10 ng/L in Fig. S7, SI), a clear pattern appears Precision: intra‑day and inter‑day repeatability for the GC-NCI-MS results. This decrease over time can be explained by the fact that the ionization gas used (isobu- Intra-day repeatability (reported as relative standard tane 3.5) is not as pure as the gases typically used for gas deviations or RSDs) values were on average below 15% chromatography (5.0 or above), leading to the ion source for all analytes, concentration levels, and measuring becoming dirty with a corresponding decrease of the result- techniques (Table 4). These results are in agreement with ing signals. Unfortunately, to the best of our knowledge there literature, as seen in Table 3, despite the fact that gener- is no purer isobutane commercially available, and the other ally lower concentrations are used in this study, and a gases that are typically used present other disadvantages decrease in precision can be expected at lower concen- (namely, methane induces harder ionization, and ammonia tration levels. results in more maintenance needed). Therefore an equiva- For all three methods, as expected, the repeatability lent to an internal standard correction, based on the averaged improves with increased concentration. For GC-EI-MS, it response of all the analytes instead of one specific standard, is not apparent at first; however, the method is not sensitive was made (as explained in SI and exemplified in Table S5), enough to detect 1B4IB at the lower concentration, which and much better precision results (below 15% in all cases) would significantly worsen the average repeatability of that were achieved (Table 4 and Fig. S8, SI). Because of how Table 5 Total number of tentatively identified derivatized aromatic account for the GC-NCI-MS and GC-EI-MS/MS techniques, and only amines with each technique, in urine samples from three NS = non- peaks with a loss of 127 were included in the GC-EI-MS calculations smokers and four S = smokers. All peaks found were taken into NS1 NS2 NS3 S1 S2 S3 S4 GC-EI-MS 37 39 38 41 41 42 45 GC-NCI-MS 55 55 49 61 79 74 68 GC-EI-MS/MS 13 15 14 16 16 16 16 1 3 Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… fast the intensity decreases, GC-NCI-MS would not be rec- was observed, and, in some cases, cut off due to the window ommended, even if an internal standard is used, for larger length (see Fig. S9, SI). Therefore, especially for the higher sample batches. concentration levels, a worse recovery can be observed. This could be avoided by increasing the observed window, or, Recoveries alternatively, by using a higher split ratio. The overall recovery range found in the literature is On average, recoveries between 80 and 120% were obtained between 64 and 125%, although generally, it is between 80 for all techniques and concentration levels studied, with and 110% (Table 3). Despite the fact that the concentration RSDs between 3 and 14% (see Table 4 and Table S6 (SI) levels used in the literature are typically higher than in this for a more detailed table with recoveries for each analyte). study, the recoveries observed are in agreement. As mentioned in “LODs and LOQs,” it was not possible to always select the calibration curve used so that the con- Real samples centrations studied were in the middle. This could explain why some recoveries for the lower and higher levels appear GC-NCI-MS and GC-EI-MS/MS show extremely good sen- to be worse. However, it needs to be kept in mind that up sitivities, which allows for the analysis of derivatized AA to 4 different levels were tested for recoveries, when typi- in the picogram-per-liter range. A few derivatized AA can cally only one is reported. This was the case because the be often found in higher concentrations, which could lead overall performance of the three instruments was to be com- to some analytes being outside of the calibration curves. If pared. In the case of real samples, it is recommended that a these AA were the main interest, this could be easily solved smaller calibration curve is used, with more points per order by diluting the samples before measuring them, which would of magnitude. provide the added advantage of reducing the matrix interfer- During the method optimization for GC-EI-MS/MS, the ence and therefore increasing the robustness of the analysis. measuring windows were set relatively narrow in order to This could also be an advantage when measuring archived have better selectivity. However, because during later experi- samples, since instead of diluting after the sample prepara- ments the intensity of the peaks increased, as explained in tion is done, less urine sample could be used to start with. “SPME extraction” and the SI, a higher tailing than expected Alternatively, if high- and low-concentration AA need to be Fig. 1 Chromatogram compari- a) GC-EI-MS son of pink = NS1, blue = S4, and black = 100 ng/L for a GC- EI-MS or 10 ng/L for b GC- NCI-MS, and c GC-EI-MS/MS, zoomed. The m/z shown are a the quantifier and qualifier ions reported in Table S2 (SI), b 127 6.57.5 8.59.5 10.5 11.5 12.5 13.5 14.5 and c the transitions reported in NS1 S4 100ng/L Table S3 (SI). NS1 and S4 were diluted 1:10 for b and c b) GC-NCI-MS 6.58.5 10.5 12.5 14.5 16.5 18.5 NS1 S4 10 ng/L c) GC-EI-MS/MS 78 910111213141516 NS1 S4 10 ng/L 1 3 Lorenzo-Parodi N. et al. analyzed, the GC-EI-MS/MS method could be adjusted by smokers’ samples, as determined by either Welch’s two- changing the Q1 or Q3 resolutions so that the sensitivity in sided t-test or the two-variable t-test (α = 0.05) [51, 52], and those highly concentrated compounds is lower compared to thus may be good candidates for future biomarker studies. those of the rest of the compounds. The three techniques show comparable results, most of In this study, the validated methods were used and the the time within the same order of magnitude. Despite the samples were diluted to avoid saturation. In most cases, IB extra dilution step, and because of the high sensitivity of the still showed concentrations above the highest calibration technique, IPFB, 24DFIB, and 1B4IB could only be detected point. This analyte is typically found in both smokers and with GC-EI-MS/MS in most samples (Table S7, SI). 1C2IB non-smokers in high concentrations, which means there is shows the highest similarities between the three techniques, another source of exposure besides tobacco smoke. There- with RSDs below 20% for all samples. 2,4DCIB could not fore, when analyzing the concentrations of aromatic amines be detected with GC-NCI-MS but also showed RSDs below in relation to smoking status, and in order to avoid satura- 20% for the other two techniques. In several cases, the higher tion of the detector in scan methods, this analyte could be deviation was due to co-elutions present with some of the left out. techniques (see SI). Depending on the analytical require- With all three techniques, more aromatic amines could ments, the GC parameters could be optimized to resolve be tentatively identified in the samples from smokers than specific co-elutions. Furthermore, the use of internal stand- non-smokers (see Table 5). As expected, with GC-NCI-MS, ards could have a positive effect minimizing the deviations the most aromatic amines could be tentatively identified. between the techniques. This is due to the fact that with this technique, m/z = 127 was one of the monitored ions. This ion corresponds to the loss of iodine and, due to the derivatization process, is to be Conclusion expected in all the aromatic amines in the sample. Because the GC-EI-MS analysis was done in SIM mode, only those The most promising technique for the analysis of the iodi- compounds with the studied m/z (Table S2, SI) could be nated derivatives of aromatic amines in urine is GC-EI- detected. This technique is the least specific, as also non- MS/MS. Despite showing slightly worse recoveries than aromatic compounds are detected, and therefore have to be GC-EI-MS, the obtained results are still within accept- filtered out manually. Finally, GC-EI-MS/MS in MRM mode able ranges. Furthermore, as expected, the sensitivity is the most selective technique, and the best option among and selectivity of the method are significantly better, those tested for target screening, as it only detects molecules so that GC-EI-MS/MS would be the method of choice with defined transitions within defined measuring windows. for further analysis. GC-NCI-MS shows a slightly worse Nonetheless, a few isomers could still be detected. An exem- behavior than GC-EI-MS/MS, with the addition of the plarily chromatogram from a smoker’s and non-smoker’s significant loss in sensitivity over time due to the ioni- sample can be seen in Fig. 1. zation gas purity. Nonetheless, for qualitative/non-target Six of the analytes studied could also be quantified with analysis, GC-NCI-MS offers the advantage that all the at least two techniques in most samples (Table  6). Great derivatized iodinated amines can be easily identified. variability could be observed, as expected due to the nature Finally, GC-EI-MS shows the worst results in terms of of the samples, which could be partially accounted for by sensitivity and selectivity. However, it has the advantage normalizing to creatinine and thereby correcting urinary of being the most widespread and least expensive of the output differences. Nonetheless, the averaged concentra- three techniques studied. This technique could therefore tions in samples from smokers were higher than in samples be especially interesting when low concentrations are not from non-smokers for all six analytes. A similar trend can of interest, or for screening purposes. be observed in literature (Table 3). Furthermore, 4IMB and One of the main drawbacks of GC-EI-MS/MS in MRM 1C2IB were found at significantly higher concentrations in mode is that the analytes need to be defined in advance. Table 6 Calculated NS1 NS2 NS3 NS S1 S2 S3 S4 S concentrations in urine samples from three NS = non-smokers 4IMB 31 64 25 40 78 173 130 145 132 and four S = smokers, in 3C4FIB 0.6 0.6 0.5 0.6 0.6 0.4 1.0 0.9 0.8 nanograms per liter, based 1C2IB 13 17 13 15 22 19 34 29 26 on the average of the three techniques studied. Average 2I13DMB 2.5 3.9 1.6 3 5.2 24 21 46 24 NS and S concentrations are 24DCIB 0.2 0.2 0.3 0.3 0.3 0.4 0.5 0.8 0.5 presented in bold 245TCIB 0.5 0.7 1.2 0.8 2.9 0.8 0.8 0.5 1.2 1 3 Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… need to obtain permission directly from the copyright holder. To view a This could be problematic when measuring real samples, copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . since approximately 150 different AA have previously been identie fi d in smokers’ urine [ 22]. 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J Chromatogr A. 2017;1507:37–44. https://doi. or g/ Food Chemistry for their help during the experimental part of this 10. 1016/j. chroma. 2017. 05. 056. project, especially Dorothea Leis, Nina Haar, Sigrid Hager, and Claudia 6. Bie Z, Lu W, Zhu Y, Chen Y, Ren H, Ji L. Rapid determination of six Koraimann for the introduction to the laboratory. carcinogenic primary aromatic amines in mainstream cigarette smoke by two-dimensional online solid phase extraction combined with liq- Author contribution Nerea Lorenzo Parodi: conceptualization, meth- uid chromatography tandem mass spectrometry. J Chromatogr A. odology, validation, investigation, writing the original draft, visualiza- 2017;1482:39–47. https:// doi. org/ 10. 1016/j. chroma. 2016. 12. 060. tion. Erich Leitner: resources, writing—review and editing. Torsten C. 7. Zhang J, Bai R, Zhou Z, Liu X, Zhou J. 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Statistics and chemometrics for analytical 46. Mazumder S, Ahamed RA, Seyler TH, Wang L. Short- and long- chemistry. 6 ed. Harlow, England: Pearson Education Limited; term stability of aromatic amines in human urine. Int J Env Res 2010. https://eli bra ry.p earson. de /bo ok/9 9.150 005/978 12921 867 26. Public Health. 2023;20:4135. 47. Domínguez I, Arrebola FJ, Gavara R, Martínez Vidal JL, Fren- Publisher's note Springer Nature remains neutral with regard to ich AG. Automated and simultaneous determination of priority jurisdictional claims in published maps and institutional affiliations. 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Analytical and Bioanalytical Chemistry Springer Journals

Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of aromatic amines

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Abstract

Some aromatic amines (AA) have been classified as carcinogens to humans. After entering the body, mainly through tobacco smoke, they can be detected in urine. Thus, their trace analysis as biomarkers in biofluids is of high relevance and can be achieved with gas chromatography (GC–MS), usually after derivatization. This study compares three gas chromatographic methods for the analysis of ten iodinated derivatives of AA: GC–MS in single-ion monitoring (SIM) mode with (1) electron ionization (GC-EI-MS) and (2) negative chemical ionization (GC-NCI-MS), and (3) GC-EI-MS/MS in multiple reaction monitoring (MRM) mode using electron ionization. All methods and most analytes showed good coefficients of determination (R > 0.99) for broad linear ranges covering three to five orders of magnitude in the picogram-per-liter to nanogram-per-liter range, with one and two exceptions for (1) and (2) respectively. Excellent limits of detection (LODs) of 9–50, 3.0–7.3, and 0.9–3.9 pg/L were observed for (1), (2), and (3) respectively, and good precision was achieved (intra-day repeatability < 15% and inter-day repeatability < 20% for most techniques and concentration levels). On average, recoveries between 80 and 104% were observed for all techniques. Urine samples of smokers and non-smokers were successfully analyzed, and p-toluidine and 2-chloroaniline could be found at significantly (α = 0.05) higher concentrations among smokers. Keywords Gas chromatography-mass spectrometry (GC–MS) · Gas chromatography-tandem mass spectrometry (GC–MS/ MS) · Negative chemical ionization (NCI) · Aromatic amines · Derivatization · Urine Introduction substances, but also the general public is at risk: the main source of exposure to some aromatic amines, such as the carcinogenic Several aromatic amines (AA) have been classified as possible, 2-naphthylamine and ortho-toluidine, and the probable carcino- probable, or certain carcinogens by the International Agency genic aniline and 4-chloro-o-toluidine, is cigarette smoke [2]. for Research on Cancer (IARC) [1], and most, if not all AA, AA enter the blood during the smoking process and are are believed to have carcinogenic potential [2]. However, they transported into the liver, where they can be metabolized are still widely used, for example, for the production of phar- and further transported, for example, to the bladder, where maceuticals, pesticides, dyes, or rubber [1]. Unfortunately, not they can react with DNA and proteins to form adducts that only the workers in these industries can get in contact with these can lead to cancer, and can be excreted in the urine [3]. AA have been suggested as the main cause for the excess risk of bladder cancer in smokers [4]. * Torsten C. Schmidt The concentrations of AA in different matrices have torsten.schmidt@uni-due.de been studied in several steps of the aforementioned pro- Instrumental Analytical Chemistry, University of Duisburg- cess, for example, in smoke [5–9] (e.g., aniline, toluidines, Essen, Universitätsstrasse 5, 45141 Essen, Germany or dimethylanilines), as DNA [10–13] and protein adducts Institute of Analytical Chemistry and Food Chemistry, Graz [12, 14–17] in cells/blood (e.g., 4-aminobiphenyl), or as University of Technology, Stremayrgasse 9/II 8010, Graz, free AA and metabolites in urine [3, 18–31] (e.g., naphthy- Austria lamines, chloroanilines). Because the intake of substances Centre for Water and Environmental Research, University during smoking varies depending on the individual smok- of Duisburg-Essen, Universitätsstrasse 5, 45141 Essen, ing topography [32], and the amount of DNA and protein Germany 4 adducts is typically extremely small [33], this study focuses IWW Water Centre, Moritzstrasse 26, on urine samples. There, not only free aromatic amines can 45476 Mülheim an Der Ruhr, Germany Vol.:(0123456789) 1 3 Lorenzo-Parodi N. et al. be found but also their metabolites, such as N-acetylaryl- compounds (Table 1), with a purity of 97% or more, were amine, N-glucuronide arylamine, or hemoglobin and DNA purchased from Merck KGaA (Darmstadt, Germany). adducts, which can be hydrolyzed and converted back to Concentrated hydrochloric acid (HCl, 37%) from VWR; the free aromatic amines [14, 34]. ethyl acetate (99.9%) from Carl Roth (Karlsruhe, Germany); Direct analysis of AA is possible using liquid chroma- diethyl ether (99.5%) from ChemLab (Zedelgem, Belgium); tography (LC) [19, 21, 24, 25, 35–37]. However, its low sodium hydroxide (NaOH, 99%), alizarin red S (98%), hydri- peak capacity [38, 39] hinders its use for the analysis of odic acid (unstabilized, 55%), and sodium nitrite (99%) from complex urine samples. Due to its high sensitivity, short Merck KGaA; and sodium sulfite (≥ 98%) and sulfamic acid analysis time, and high resolving power [40], gas chro- (≥ 99%) from Fluka (Buchs, Switzerland) were used. matography (GC) was used for this study. In order to reduce the polarity of the AA and facilitate Preparation of stock and standard solutions their analysis, they are typically derivatized. In this study, they were iodinated via a Sandmeyer-like reaction as All the stock and intermediate solutions were prepared in reported by [18, 22, 39]. This derivatization procedure offers methanol. Individual stock solutions of each of the analytes the advantage that the reagents used do not need strictly were prepared at 1 g/L. An intermediate standard solution anhydrous conditions, as is the case for the commonly used was prepared at 1 mg/L for the iodinated aromatic com- acylation [39] and silylation derivatizations [40]. pounds. Working solutions were prepared by diluting the This derivatization step enables their analysis with dif- intermediate standard solutions in methanol and were used ferent types of GC systems, such as GC–MS [18, 27, 30, 31, within 1 month. One working solution was prepared for each 41, 42], GC × GC–MS [22], GC-NCI-MS [8, 29, 43–45], or concentration tested. All the solutions were stored at 7 °C. GC–MS/MS [3, 9]. However, a comparison of the different techniques, namely GC–MS, GC-NCI-MS, and GC–MS/ Sample preparation MS, has not been previously reported for these analytes. The aim of this study is, therefore, the comparison of Glass-covered stirring bars (VWR International GmbH) different GC detection techniques for the determination were placed in 20-mL crimp vials, which were then filled of aromatic amines in urine after derivatization to the with 5 mL of the samples and closed with magnetic caps corresponding iodinated benzenes, namely GC-EI-MS, with Silicone/PTFE septa. GC-NCI-MS, and GC-EI-MS/MS. To that end, all studied For the validation experiments, the samples were pre- methods were validated and used for the analysis of real pared by adding 10 µL of the corresponding iodinated work- urine samples from smokers and non-smokers. ing solution to 5 mL deionized water. Urine samples from seven donors (four smokers and three non-smokers) were collected in 1-L Schott bottles and stored Materials and methods at 7 °C for up to 1 month. The analytes are likely stable under those conditions based on a recent study by Mazumder Chemicals and reagents et al. [46], who found no marked decrease in concentration with samples at similar temperatures during the total time Methanol ≥ 99.9%, HiPerSolv Chromanorm for LC–MS studied, which, however, was limited to 10 days. The urine (VWR International GmbH, Darmstadt, Germany), was used samples were prepared according to Lamani et al. [22], with for the preparation of standard solutions. Iodinated aromatic a few modifications. First, 20 mL of urine was hydrolyzed Table 1 Iodinated compounds Analyte Abbreviation Aromatic amine precursor CAS Nr Purity (%) used, including the abbreviation by which they are referred to 4-Iodotoluene 4IMB p-Toluidine 624–31-7 99 in the text, their corresponding Iodopentafluorobenzene IPFB Pentafluoroaniline 827–15-6 99 aromatic amine precursor, CAS 2-Iodo-1,3-dimethylbenzene 2I13DMB 2,6-Dimethylaniline 608–28-6 97 Number (CAS Nr), and purity Iodobenzene IB Aniline 591–50-4 98 1-Chloro-2-iodobenzene 1C2IB 2-Chloroaniline 615–41-8 99 3-Chloro-4-fluoroiodobenzene 3C4FIB 3-Chloro-4-fluoroaniline 156150–67-3 98 2,4,5-Trichloroiodobenzene 245TCIB 2,4,5-Trichloroaniline 7145–82-6 98 2,4-Dichloroiodobenzene 24DCIB 2,4-Dichloroaniline 29898–32-6 98 1-Bromo-4-iodobenzene 1B4IB 4-Bromoaniline 589–87-7 98 2,4-Difluoroiodobenzene 24DFIB 2,4-Difluoroaniline 2265–93-2 98 1 3 Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… with 10 mL of HCl (37%) at 80 °C and 200 rpm stirring were extracted from the headspace with a 65-μm PDMS/ speed, for 12 h in order to convert metabolized AA into DVB SPME fiber (1 cm length, Stableflex, 23 Ga, Merck free AA. All heating and stirring steps were done on an MR KGaA) for 30 min before injection into the GC system. The 3001 K stirring plate from Heidolph Instruments GmbH & SPME fiber remained at least for 5 min in the injector in Co. KG (Schwabach, Germany). Once the sample reached order to condition the fiber after injection, except for the room temperature, it was basified by adding 20 mL of 10 M GC-EI-MS/MS measurements, where the fiber was pre- NaOH to the solution. Afterward, the amines were extracted conditioned for 2 min in a dedicated conditioning station at two times into 5 mL of diethyl ether. The organic fractions 280 °C, and remained for 2 min in the injector. were then mixed and cleaned with 2 mL of 0.1 M NaOH. The extraction efficiencies of the three SPME fibers used The amines were subsequently back-extracted into 10 mL of (one for each technique) were compared after the methods water, previously acidified with 200 μL concentrated HCl were validated, using GC-NCI-MS and a iodinated deriva- (37%). Any remaining diethyl ether in the aqueous frac- tives solution (1 ng/L, Fig. S2 and SI). Furthermore, a SPME tion was evaporated by nitrogen blowing on the samples test mix (200 ng/L) was analyzed regularly in order to ensure for 20 min. the integrity of the fiber and the performance of the system, The aqueous extracts were then derivatized by substitut- by adding 20 µL of a stock solution in a vial with a stir- ing the nitrogen for an iodine atom in order to decrease the rer. A list of the analytes included in the mix (minimum polarity of the extracted amines (see Supplementary infor- purity 95%, different providers) can be seen in Table S1 (SI). mation (SI), Fig. S1). This was achieved by adding 200 μL Because the mix included mostly analytes that are not ion- hydriodic acid (55%) and 400 μL sodium nitrite (50 g/L) izable by GC-NCI-MS, it was not used for this technique. and stirring at 200 rpm for 20 min (step 1), adding 1 mL of A significantly lower intensity was observed with GC-EI- sulfamic acid (50 g/L) and stirring at 200 rpm for another MS/MS during the first use of the corresponding SPME fiber 45 min (step 2), then heating the sample to 95 °C for 5 min (see Fig. S3, SI). Although the manufacturer instructions (step 3), and finally adding 800 μL sodium sulfite (120 g/L) were followed, it has been previously reported that it might (step 4) and 200 μL of alizarin red S (1% w/v) (step 5), and be insufficient conditioning [47, 48]. Therefore, the GC-EI- adjusting the pH to 5 with NaOH and HCl solutions (step 6). MS/MS results were normalized according to the SPME Mix This way, (step 1) the aromatic amines are diazotized and the intensities, which, as expected, also showed a similar trend diazonium ions are further substituted by iodine; (step 2) the (see Table S1, SI). surplus of nitrite is destroyed; (step 3) the unreacted diazo- nium ions are transformed into phenols, and the excess sul- GC–MS analysis famic acid is destroyed; (step 4) the iodine residue is reduced; (step 5) a pH indicator is added for easy identification of the In order to facilitate the comparison of the different tech- correct pH; and (step 6) a pH value suitable for subsequent niques, as many parameters as possible were kept constant SPME is achieved. throughout the different devices. Helium 5.0 (Linde, Höll- Finally, 5 mL was transferred into a 20-mL vial with a stir- riegelskreuth, Germany) was used as carrier gas for all rer, crimped, and then placed in the autosampler for further techniques. treatment, namely SPME and injection into the GC. For GC- A GCMS-QP2010 Ultra (Shimadzu) equipped with a ZB- NCI-MS and GC-EI-MS/MS, the samples were diluted 1:10. Wax 20  m × 0.18  mm × 0.18  µm (Phenomenex, CA, USA) was used for the GC-EI-MS analysis. The linear velocity SPME extraction was set to 45 cm/s, which corresponds to a column flow of 1.03 mL/min. The samples were injected in splitless mode, All the SPME fibers were conditioned prior to their first use, and after a sampling time of 1 min, the split ratio was set to as recommended by the supplier (i.e., 250 °C for 30 min). 10. The injector temperature was set to 250 °C, the interface The SPME extraction was done automatically by different temperature to 230 °C, and the ion source temperature to autosamplers, namely HTX PAL (CTC Analytics, Zwingen, 200 °C. The oven program started at a temperature of 40 °C, Switzerland) for the GC-EI-MS measurements, AOC-6000 was held for 1 min, ramped at a rate of 10 °C/min to 240 °C, (Shimadzu, Kyoto, Japan) for the measurements with the and held for 1 min. The final oven temperature was lower GC-EI-MS/MS, and AOC-5000 Plus for GC-NCI-MS (Shi- than in other instruments due to the different column used. madzu). All the autosamplers were controlled with PAL The acquisition was made in SIM mode, with an event time Cycle Composer except for the autosampler used in com- of 0.2 s. Twenty channels were looked into (Table S2 (SI)), bination with the GC-EI-MS/MS, which was directly con- typically corresponding to the molecular ions and the frag- trolled by the GCMS Real Time Analysis software (Shi- ment resulting from the loss of iodine. madzu). The samples were incubated for 5 min at 60 °C and A GCMS-TQ8050 (Shimadzu) with a 30 m × 0.25 mm × 500 rpm in a single magnet mixer (SMM). Afterward, they 0.25  µm Rxi-5MS (Restek, PA, USA) was used for the 1 3 Lorenzo-Parodi N. et al. GC-EI-MS/MS analysis. The injector, interface, and ion the calibration curves were up to five orders of magnitude source temperatures were set to 270, 280, and 200 °C respec- (1–100,000 pg/L) broad, and the points were equidistant only tively. The injection was done in splitless mode, and a split in the logarithmic scale, a normal linear fit would be very ratio of 10 was applied after a sampling time of 1 min. The heavily influenced by the higher calibration levels. There- linear velocity was 35 cm/s. The oven starting temperature fore, in order to accurately determine lower concentrations, was 40 °C, which was held for 1 min, ramped to 280 °C at a we limited the number of calibration levels in this and the rate of 10 °C/min, and held for 1 min. The MS was operated following sections, so that there would be at least 5 points in multiple reaction monitoring (MRM) mode, using argon per curve and up to 7 levels. Furthermore, the concentration 5.0 (Linde) as the collision gas. The optimal collision ener- to be determined was, if possible, kept in the middle of the gies (CE) were found by directly injecting 1 µL of a mixture levels selected. Afterward, the limits were calculated accord- of the iodinated analytes in 50:50 methanol:ethyl acetate ing to the Eurachem Guide [49], with a constant (k, equal (0.5 mg/L), at different CE, and comparing the intensities. to 3 for LODs and 10 for LOQs) multiplied by the standard The transitions monitored, and their corresponding CE, can deviation of the replicate concentrations and divided by the be seen in Table S3 (SI). The event time was set to 0.3 s for degrees of freedom (n − 1 = 9). all transitions. The next step was to calculate the intra-day and inter-day GC-NCI-MS measurements were done on a GCMS- repeatability. In order to do that, at least three calibration QP2010 Plus system (Shimadzu) equipped with a points (equidistant in the logarithmic scale and well distrib- 30  m × 0.25  mm × 0.25  µm Rxi-5Sil MS column (Restek). uted within the linear range) were measured in triplicate over The interface temperature was set to 250 °C and the ion three consecutive days. The concentration levels tested were source temperature to 160 °C. All other GC parameters were 1, 10, and 100 ng/L for GC-EI-MS; 0.1, 1, and 10 ng/L for the same as for the GC–MS/MS. MS acquisition was per- GC-NCI-MS, and, because of the broad range that could be formed in SIM mode, with an event time of 0.3 s, and moni- analyzed with GC-EI-MS/MS, four concentrations, namely toring the ions corresponding to chlorine (35, 37), bromine 0.01, 0.1, 1, and 10 ng/L, were studied with that technique. (79, 81), and iodine (127). The ionization gas was isobutane The recovery was calculated from the repeatability 3.5 (Linde), set to a pressure of 0.7 bar. experiments. For each instrument and concentration level, A comparison of the chromatograms obtained with the the recovery was calculated by dividing the average of the aforementioned parameters can be seen in Fig. S4 (SI). concentrations obtained (n = 9) by the expected theoretical concentration and multiplying the result by 100. Method validation The validation was done mostly according to the Eurachem Results and discussion Guide [49]. The raw data were evaluated with GCMSsolu- tion (Shimadzu) without applying smoothing, and the cal- Ten aromatic amine derivatives (i.e., iodinated aromatic culations were performed in Excel (Microsoft). compounds) were measured directly, without further sam- First of all, the linear ranges were studied with the aim ple treatment, in order to facilitate the direct comparison of of seeing not only how sensitive the instruments can be, but the methods. The studied analytes were selected as model also whether they have a linear response at the concentration compounds due to their diverse chemical structures and levels expected for real samples. Therefore, a very broad properties. Furthermore, most of them have been previously range was studied, and, subsequently, a logarithmic scale studied and found in smoke [5–9], blood/tissue [10, 14–16], was used in order to have equidistant calibration levels, as and/or urine matrixes [3, 21–23, 26, 28–30]. Three of the recommended by the DIN 38402–51 [50]. Concentrations most comprehensive papers in terms of AA studied [8, 22, from 1 pg/L to 500 ng/L were tested, with three concentra- 28] found aniline, p-toluidine, 2,6-dimethylaniline, 2-chlo- tion levels per order of magnitude. Exemplary calibration roaniline, 2,4,5-trichloroaniline, and 2,4-dichloroaniline, curves for each technique can be seen in Fig. S5 (SI). which are also included in this research. Afterward, the limits of detection (LODs) and of quantifi- cation (LOQs) were studied by repeating ten times the analy- Linear range sis of a calibration level where most of the analytes showed a signal to noise ratio (S/N) between 6 and 15 (200 pg/L The linear ranges observed can be found in Table 2. For the for GC-EI-MS, 100 pg/L for GC-NCI-MS, and 10 pg/L for GC-NCI-MS experiments, a plateau in the linear curve could GC-EI-MS/MS). This was done so that the concentrations be observed at concentrations of 50 or 100 ng/L, depending used for the calculation of the limits were not extremely on the compound. Excellent goodness of fit was achieved high in comparison with the limits themselves, and to conse- for all the methods tested, with coefficients of determination quently avoid obtaining overestimated sensitivities. Because (R ) above 0.99 for all cases except for 1B4IB and 245TCIB 1 3 Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… Table 2 Limits of detection (LOD), quantification (LOQ), and linear NCI-MS, and 1–100,000  pg/L for GC-EI-MS/MS. LODs and LOQs ranges in picograms per liter, obtained for the iodinated, aromatic were calculated with concentrations where most analytes had S/N compounds with the studied GC methods. The concentration ranges between 6 and 15, namely 200 pg/L for GC-EI-MS, 100 pg/L for GC- tested were 20–500,000 pg/L for GC-EI-MS, 2–100,000 pg/L for GC- NCI-MS, and 10 pg/L for GC-EI-MS/MS GC-EI-MS GC-NCI-MS GC-EI-MS/MS LOD (pg/L) LOQ (pg/L) Linear range (pg/L) LOD* (pg/L) LOQ* (pg/L) Linear range (pg/L) LOD (pg/L) LOQ (pg/L) Linear range (pg/L) IPFB 30 99 100–500,000 7.3 19 50–50,000 0.9 2.9 5–100,000 24DFIB 50 167 200–500,000 4.7 31 10–20,000 0.9 2.9 2–100,000 IB 25 84 100–500,000 6.8 23 20–50,000 2.1 7 5–100,000 4IMB 21 71 100–500,000 5.2 31 20–50,000 1.1 3.5 1–100,000 3C4FIB 14 47 100–500,000 6.3 21 10–20,000 1.3 4 2–100,000 1C2IB 26 86 100–500,000 3.0 15 5–20,000 0.8 2.5 2–100,000 2I13 DMB 21 71 50–500,000 5.6 28 10–50,000 0.5 1.7 1–100,000 1B4IB - - 10,000–500,000 4.9 14 20–50,000 2.0 7 10–100,000 24DCIB 9* 29* 50–500,000 4.8 20 10–20,000 1.2 4.0 2–100,000 245 TCIB 28 93 200–500,000 6.3 13 10–50,000 3.9 13 10–100,000 *Outliers found with Dixon’s Q test (α = 0.05, Q = 0.412) not included in the calculations Critical LODs and LOQs were calculated according to the Eurachem Guide [49], as a constant (3 and 10, respectively) multiplied by the standard devia- tion of the concentration from tenfold replicates, and divided by the degrees of freedom (n − 1 = 9). Smoothing was set to “none” when measured with GC-NCI-MS (0.988 and 0.989 respec- GC-EI-MS/MS can be explained by the fact that in the tively, data not shown). first quadrupole, only the ions selected are trapped, which The determination of 1B4IB with the GC-EI-MS method decreases the background noise, and consequently increases was hindered by an interfering signal covering the peak (see the sensitivity significantly. Fig. S6, SI), which led to the analyte being identified only in LODs and LOQs for the analysis of aromatic amines concentration levels of 10 ng/L or above. A different column in urine are generally reported in the nanogram-per-liter was used with this instrument, which could have led to a dif- range (see Table 3). The best LODs reported (< 5 ng/L) ferent elution pattern and may explain why the interference were achieved with MS/MS detectors [20, 24, 27] and was not observed in the other systems. A different set of m/z GC-NCI-MS [29] systems, while the worst (> 50 ng/L) may be used to study this compound, such as 155 and 157, were observed with EI-MS detectors [21, 30]. A simi- which corresponds to the fragment without iodine. lar trend can be observed in this study (Table 2), where When compared with literature (Table  3), the results GC-EI-MS shows worse limits than the other methods are similar or better than those typically reported. In most tested. Nonetheless, the results obtained were better cases, a linear range of approximately 3 orders of mag- than most of those found in literature, with LODs of nitude is reported [20–24, 27, 29, 30]. The results pre- 9–50  pg/L for GC-EI-MS, 3.0–7.3  pg/L for GC-NCI- sented here show a linear range of 4 orders of magnitude MS, and 0.5–3.9  pg/L for GC-EI-MS/MS. The lowest for GC-EI-MS and GC-NCI-MS and of 5 for GC-EI-MS/ limit reliably reported for aromatic amines in urine is MS. The broader the linear range, the higher the likelihood 0.89  ng/L [27], which is between 120 and 1800 times that analytes at very low concentrations can be accurately worse than those reported here for the iodinated deriva- quantified, and that there is no further dilution needed for tives with GC-NCI-MS and GC-EI-MS/MS. The reason samples with very high concentrations, saving both sample for the higher sensitivity achieved here is most likely volume and time. the combination of a pre-concentration step like SPME with very sensitive measurement techniques and the fact LODs and LOQs that iodinated derivatives were measured directly. Taking into account that during a similar derivatization proce- As expected, GC-EI-MS/MS shows the lowest LODs and dure, for most analytes an estimated loss of 10% was LOQs, followed by GC-NCI-MS and GC-EI-MS (see observed [38], it would be expected that the limits found Table  2), which on average have 3 and 12 times higher with these instruments, including the complete sample LODs, respectively. The high sensitivity achieved with preparation, would still be comparable if not better than GC-NCI-MS can be attributed to the high selectivity of those found in literature. this technique for halogenated compounds, which have a Other factors can affect the sensitivity of the method, such high electron affinity. The even better results obtained with as the amount of sample used (typically within 5–20 mL), 1 3 Lorenzo-Parodi N. et al. 1 3 Table 3 Figures of merit of most recent literature regarding the analysis of aromatic amines from urine samples. Ranges reported correspond to the minimum and maximum values from differ - ent analytes and/or concentration levels. Data in parentheses indicate missing experimental information needed for its interpretation Der. reagent Injection Volume/SPME fiber Instrument Calibration LOD (ng/L) Recovery (%) Intra-day pre- Inter-day Concentration in real Ref technique range (ng/L) cision (%) precision samples (%) HI HS-SPME 110 μm PDMS/DVB GC–MS 100–1200 3–12 n.r 3–12 n.r NS: n.d., S: [18] n.d.–243 ng/L 2 a No LI 5 µL LC–MS/MS (MRM) 100–50,000 25–500 75–114 1.6–11.7 2.1–15.9 U: n.d.–1.5, S: [19] n.d.–3.47 µg/L TMA-HCl, PFPA LI 1 µL GC–MS/MS (EI, 482–1280 1.8–111.2 > 85 1.1–6.3 2.6–6.3 n.r [3] MRM) No HS-SPME 80 µm, JUC-Z2 GC–MS/MS (MRM) 50–100,000 (0.010–0.012) 95–101 (7.1–7.7) n.r NS: n.d., S: [20] 68.4–123.1 ng/L PFPA, Pyr LI 5 µL LC–MS (SIM) 1000– 1000 n.r n.r n.r NS: 1–1.13, S: [21] 1,000,000 1.46–2.33 µg/L HI HS-SPME 65 μm PDMS/DVB GC × GC–MS 1–500 5.2–24.4 n.r n.r n.r n.r [22] No LI 1 µL GC-FID 30–100,000 (7–10) 93.0–99.9 2.5–5.9 4.7–7.3 NS: n.d.–1.2, S: [23] 2–14.5 µg/L No LI 10 µL LC–MS/MS (MRM) 5–10,000 (1.5–5) 88–111 6.1–8.9 9.0–9.9 NS: 1.11–12.32, S: [24] 5.39–67.02 ng/24 h No LI 3 µL UFLC (UV–Vis) 5000–500,000 (DI: 39,600–94,400, 89–105 0.6–7.9 2.4–10 U: n.d.–12.8 µg/L [25] AE: 880–1300) 2 a No HS-SPME PEG/CNTs GC-FID 1–105 (0.5–50) 63.7–97.0 3.2–9.1 5.5–12.0 NS: n.d.–940, S: [26] 1140–50,960 ng/L TMA-HCl, PFPA LI 1 µL GC–MS/MS (EI, 50–25,000 0.89 (20–25) 1.7–6.7 7.5–8.4 NS: 1.30–2.07, S: [27] MRM) 7.43–10.16 pg/mg Cr No LI 1 µL GC–MS (SIM) 5–60,000 2–26 94–104 4.5–6.2 6.0–6.8 U: n.d.–690 ng/L [28] PFPA, Pyr LI 0.2 µL GC–MS (NCI, SIM) 10–2500 (1–4) 94–107 (2.7–4.6) (5.1–7.0) NS: 9.6–105.2, S: [29] 15.3–204.2 ng/24 h PFPA, Pyr LI 1 µL GC–MS (SIM) 100–100,000 (50–2000) 70–125 1.8–14 7.5–19 U: n.d.–3.5 µg/L [30] Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… 1 3 Table 3 (continued) Der. reagent Injection Volume/SPME fiber Instrument Calibration LOD (ng/L) Recovery (%) Intra-day pre- Inter-day Concentration in real Ref technique range (ng/L) cision (%) precision samples (%) PFPI LI 1 µL GC–MS (SIM) n.r (0.05 ng) (82.3–96.8) n.r n.r NS: n.d.–1073.4, S: [31] 3.6–2119.8 ng/24 h HI HS-SPME 65 μm PDMS/DVB GC-EI-MS 0.05–500 0.009–0.05 93–116 2.8–11 1.8–46 NS: n.d.–64, S: This n.d.–173 ng/L study GC-NCI-MS 0.005–50 0.003–0.007 71–104 2.1–12 3.7–40 8 c GC-EI-MS/MS 0.001–100 0.001–0.004 66–117 0.2–13 4.0–35 Abbreviations: AE after extraction, Cr creatinine, Der. derivatization, DI direct injection, HS-SPME headspace solid-phase microextraction, JUC-Z2 two-dimensional porous organic framework, LI liquid injection, LOD limit of detection, n.d. not detected, n.r. not reported, NS non-smoker, PDMS/DVB polydimethylsiloxane/divinylbenzene, PEG/CNTs poly(ethylene glycol) modified with multi-walled carbon nanotubes, PFPA pentafluoropropionic anhydride, PFPI pentafluoropropionyl-imidazol, Pyr pyridine, Ref reference, S smoker, TMA-HCl trimethylamine hydrochlo- ride, U unknown smoking status, UFLC ultra-fast liquid-chromatography Considering 37 of the 41 analytes studied. Batch-to-batch precision. Excluding 24TCIB (28%) and IB (19%) at a concentration level of 10 pg/L LOD calculations: according to CLSI EP17-A S/N (signal to noise ratio) = 3 not repor ted according to DIN 32645 according to FDA guideline SD low-quality-control samples LOD = 3*SD /b ( = regression) y/x y/x LOD = 3*SD/(n − 1), (n = degrees of freedom), according to Eurachem Guide [49] Lorenzo-Parodi N. et al. Table 4 Average intra-day and inter-day repeatability, and recovery M (medium) = 10  ng/L, and H (high) = 100  ng/L; for GC-NCI-MS, (%) results obtained for each of the techniques studied. The concen- L = 0.1  ng/L, M = 1  ng/L, and H = 10  ng/L; and for GC-EI-MS/MS, tration levels tested were as follows: for GC-EI-MS, L (low) = 1 ng/L, L = 0.01 ng/L, M-L = 0.1 ng/L, M-H = 1 ng/L, and H = 10 ng/L GC-EI-MS GC-NCI-MS GC-EI-MS/MS L M H L M H L M-L M-H H Intra-day repeatability (%, n = 9) 7.1 7.8 5.2 5.6 5.5 3.7 12 4.0 2.8 2.1 Intra-day repeatability (%, n = 9)* 3.8 5.7 2.0 3.9 2.9 1.6 10.3 3.5 1.5 1.0 Inter-day repeatability (%, n = 3) 25 15 7.7 13 27 24 15 16 21 15 Inter-day repeatability (%, n = 3)* 12.6 8.7 4.5 5.3 7.4 2.9 13.2 9.7 9.6 7.7 Recovery (%, n = 9) 102 104 96 83 94 80 92 89 88 80 *Results obtained after internal standard-equivalent correction (explained in SI) the concentration level studied, the steps of the sample concentration. If the same concentration level is compared preparation procedure included, the use of matrix-matched across methods, for example, 1 ng/L, the average intra-day calibrations, and the equations used for the calculations (sig- repeatabilities of the methods are 7.1% for GC-EI-MS (with- nal to noise ratio, standard deviation, etc.). Unfortunately, out IB4IB), 5.5% for GC-NCI-MS, and 2.8% for GC-EI-MS/ in several occasions, information was lacking for a proper MS. The individual intra-day repeatabilities of each analyte interpretation of the results. For example, when the limits can be seen in Table S4, SI. were calculated based on the S/N ratio, the concentrations The majority of the inter-day repeatability results used or if smoothing was applied was usually not reported. (reported as RSDs) are below 20%; however, there are some If too-high concentrations are used, this can lead to too-low exceptions. This could be due to the fact that n is smaller (3 LODs, which seems to be the case for the lowest limit found vs 9), and the typical errors introduced during sample prepa- in literature [20], where the extrapolated limit reported is ration and measurement have a bigger effect the smaller the more than three orders of magnitude lower than the linear number of samples measured. range. If the different replicates are studied over time (as exem- plified for 10 ng/L in Fig. S7, SI), a clear pattern appears Precision: intra‑day and inter‑day repeatability for the GC-NCI-MS results. This decrease over time can be explained by the fact that the ionization gas used (isobu- Intra-day repeatability (reported as relative standard tane 3.5) is not as pure as the gases typically used for gas deviations or RSDs) values were on average below 15% chromatography (5.0 or above), leading to the ion source for all analytes, concentration levels, and measuring becoming dirty with a corresponding decrease of the result- techniques (Table 4). These results are in agreement with ing signals. Unfortunately, to the best of our knowledge there literature, as seen in Table 3, despite the fact that gener- is no purer isobutane commercially available, and the other ally lower concentrations are used in this study, and a gases that are typically used present other disadvantages decrease in precision can be expected at lower concen- (namely, methane induces harder ionization, and ammonia tration levels. results in more maintenance needed). Therefore an equiva- For all three methods, as expected, the repeatability lent to an internal standard correction, based on the averaged improves with increased concentration. For GC-EI-MS, it response of all the analytes instead of one specific standard, is not apparent at first; however, the method is not sensitive was made (as explained in SI and exemplified in Table S5), enough to detect 1B4IB at the lower concentration, which and much better precision results (below 15% in all cases) would significantly worsen the average repeatability of that were achieved (Table 4 and Fig. S8, SI). Because of how Table 5 Total number of tentatively identified derivatized aromatic account for the GC-NCI-MS and GC-EI-MS/MS techniques, and only amines with each technique, in urine samples from three NS = non- peaks with a loss of 127 were included in the GC-EI-MS calculations smokers and four S = smokers. All peaks found were taken into NS1 NS2 NS3 S1 S2 S3 S4 GC-EI-MS 37 39 38 41 41 42 45 GC-NCI-MS 55 55 49 61 79 74 68 GC-EI-MS/MS 13 15 14 16 16 16 16 1 3 Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… fast the intensity decreases, GC-NCI-MS would not be rec- was observed, and, in some cases, cut off due to the window ommended, even if an internal standard is used, for larger length (see Fig. S9, SI). Therefore, especially for the higher sample batches. concentration levels, a worse recovery can be observed. This could be avoided by increasing the observed window, or, Recoveries alternatively, by using a higher split ratio. The overall recovery range found in the literature is On average, recoveries between 80 and 120% were obtained between 64 and 125%, although generally, it is between 80 for all techniques and concentration levels studied, with and 110% (Table 3). Despite the fact that the concentration RSDs between 3 and 14% (see Table 4 and Table S6 (SI) levels used in the literature are typically higher than in this for a more detailed table with recoveries for each analyte). study, the recoveries observed are in agreement. As mentioned in “LODs and LOQs,” it was not possible to always select the calibration curve used so that the con- Real samples centrations studied were in the middle. This could explain why some recoveries for the lower and higher levels appear GC-NCI-MS and GC-EI-MS/MS show extremely good sen- to be worse. However, it needs to be kept in mind that up sitivities, which allows for the analysis of derivatized AA to 4 different levels were tested for recoveries, when typi- in the picogram-per-liter range. A few derivatized AA can cally only one is reported. This was the case because the be often found in higher concentrations, which could lead overall performance of the three instruments was to be com- to some analytes being outside of the calibration curves. If pared. In the case of real samples, it is recommended that a these AA were the main interest, this could be easily solved smaller calibration curve is used, with more points per order by diluting the samples before measuring them, which would of magnitude. provide the added advantage of reducing the matrix interfer- During the method optimization for GC-EI-MS/MS, the ence and therefore increasing the robustness of the analysis. measuring windows were set relatively narrow in order to This could also be an advantage when measuring archived have better selectivity. However, because during later experi- samples, since instead of diluting after the sample prepara- ments the intensity of the peaks increased, as explained in tion is done, less urine sample could be used to start with. “SPME extraction” and the SI, a higher tailing than expected Alternatively, if high- and low-concentration AA need to be Fig. 1 Chromatogram compari- a) GC-EI-MS son of pink = NS1, blue = S4, and black = 100 ng/L for a GC- EI-MS or 10 ng/L for b GC- NCI-MS, and c GC-EI-MS/MS, zoomed. The m/z shown are a the quantifier and qualifier ions reported in Table S2 (SI), b 127 6.57.5 8.59.5 10.5 11.5 12.5 13.5 14.5 and c the transitions reported in NS1 S4 100ng/L Table S3 (SI). NS1 and S4 were diluted 1:10 for b and c b) GC-NCI-MS 6.58.5 10.5 12.5 14.5 16.5 18.5 NS1 S4 10 ng/L c) GC-EI-MS/MS 78 910111213141516 NS1 S4 10 ng/L 1 3 Lorenzo-Parodi N. et al. analyzed, the GC-EI-MS/MS method could be adjusted by smokers’ samples, as determined by either Welch’s two- changing the Q1 or Q3 resolutions so that the sensitivity in sided t-test or the two-variable t-test (α = 0.05) [51, 52], and those highly concentrated compounds is lower compared to thus may be good candidates for future biomarker studies. those of the rest of the compounds. The three techniques show comparable results, most of In this study, the validated methods were used and the the time within the same order of magnitude. Despite the samples were diluted to avoid saturation. In most cases, IB extra dilution step, and because of the high sensitivity of the still showed concentrations above the highest calibration technique, IPFB, 24DFIB, and 1B4IB could only be detected point. This analyte is typically found in both smokers and with GC-EI-MS/MS in most samples (Table S7, SI). 1C2IB non-smokers in high concentrations, which means there is shows the highest similarities between the three techniques, another source of exposure besides tobacco smoke. There- with RSDs below 20% for all samples. 2,4DCIB could not fore, when analyzing the concentrations of aromatic amines be detected with GC-NCI-MS but also showed RSDs below in relation to smoking status, and in order to avoid satura- 20% for the other two techniques. In several cases, the higher tion of the detector in scan methods, this analyte could be deviation was due to co-elutions present with some of the left out. techniques (see SI). Depending on the analytical require- With all three techniques, more aromatic amines could ments, the GC parameters could be optimized to resolve be tentatively identified in the samples from smokers than specific co-elutions. Furthermore, the use of internal stand- non-smokers (see Table 5). As expected, with GC-NCI-MS, ards could have a positive effect minimizing the deviations the most aromatic amines could be tentatively identified. between the techniques. This is due to the fact that with this technique, m/z = 127 was one of the monitored ions. This ion corresponds to the loss of iodine and, due to the derivatization process, is to be Conclusion expected in all the aromatic amines in the sample. Because the GC-EI-MS analysis was done in SIM mode, only those The most promising technique for the analysis of the iodi- compounds with the studied m/z (Table S2, SI) could be nated derivatives of aromatic amines in urine is GC-EI- detected. This technique is the least specific, as also non- MS/MS. Despite showing slightly worse recoveries than aromatic compounds are detected, and therefore have to be GC-EI-MS, the obtained results are still within accept- filtered out manually. Finally, GC-EI-MS/MS in MRM mode able ranges. Furthermore, as expected, the sensitivity is the most selective technique, and the best option among and selectivity of the method are significantly better, those tested for target screening, as it only detects molecules so that GC-EI-MS/MS would be the method of choice with defined transitions within defined measuring windows. for further analysis. GC-NCI-MS shows a slightly worse Nonetheless, a few isomers could still be detected. An exem- behavior than GC-EI-MS/MS, with the addition of the plarily chromatogram from a smoker’s and non-smoker’s significant loss in sensitivity over time due to the ioni- sample can be seen in Fig. 1. zation gas purity. Nonetheless, for qualitative/non-target Six of the analytes studied could also be quantified with analysis, GC-NCI-MS offers the advantage that all the at least two techniques in most samples (Table  6). Great derivatized iodinated amines can be easily identified. variability could be observed, as expected due to the nature Finally, GC-EI-MS shows the worst results in terms of of the samples, which could be partially accounted for by sensitivity and selectivity. However, it has the advantage normalizing to creatinine and thereby correcting urinary of being the most widespread and least expensive of the output differences. Nonetheless, the averaged concentra- three techniques studied. This technique could therefore tions in samples from smokers were higher than in samples be especially interesting when low concentrations are not from non-smokers for all six analytes. A similar trend can of interest, or for screening purposes. be observed in literature (Table 3). Furthermore, 4IMB and One of the main drawbacks of GC-EI-MS/MS in MRM 1C2IB were found at significantly higher concentrations in mode is that the analytes need to be defined in advance. Table 6 Calculated NS1 NS2 NS3 NS S1 S2 S3 S4 S concentrations in urine samples from three NS = non-smokers 4IMB 31 64 25 40 78 173 130 145 132 and four S = smokers, in 3C4FIB 0.6 0.6 0.5 0.6 0.6 0.4 1.0 0.9 0.8 nanograms per liter, based 1C2IB 13 17 13 15 22 19 34 29 26 on the average of the three techniques studied. Average 2I13DMB 2.5 3.9 1.6 3 5.2 24 21 46 24 NS and S concentrations are 24DCIB 0.2 0.2 0.3 0.3 0.3 0.4 0.5 0.8 0.5 presented in bold 245TCIB 0.5 0.7 1.2 0.8 2.9 0.8 0.8 0.5 1.2 1 3 Comparison of gas chromatographic techniques for the analysis of iodinated derivatives of… need to obtain permission directly from the copyright holder. To view a This could be problematic when measuring real samples, copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . since approximately 150 different AA have previously been identie fi d in smokers’ urine [ 22]. Most GC-EI-MS/MS oe ff r the possibility of doing scan/MRM, which could enable References qualitative non-target screening and the sensitive and selec- tive quantification of specific target compounds. Another 1. IARC Working Group on the Evaluation of Carcinogenic Risks to alternative would be to combine the derivatization method Humans. Chemical agents and related occupations. Lyon, France: International Agency for Research on Cancer; 2012;100F. https:// presented here with GC-EI-MS/MS in neutral loss mode. publi catio ns. iarc. fr/ Book- And- Report- Series/ Iarc- Monog raphs- Finally, the high sensitivity and selectivity obtained for On- The- Ident ifica tion- Of- Car ci nog en ic- Hazar ds- To- Humans/ the analysis of the iodinated derivatives with HS-SPME Chemi cal- Agents- And- Relat ed- Occup ations- 2012. GC-EI-MS/MS are a great advantage over other methods 2. Pereira L, Mondal PK, Alves M. Aromatic amines sources, envi- ronmental impact and remediation. In: Lichtfouse E, Schwarz- found in literature. Especially for the analysis of valuable bauer J, Robert D, editors. Pollutants in buildings, water and liv- samples, such as archived samples (for example, from ing organisms. Cham: Springer International Publishing; 2015. cohort studies), since it can enable a considerable reduc- pp. 297–346. https:// doi. org/ 10. 1007/ 978-3- 319- 19276-5_7. tion of sample volume needed. This could be used to foster 3. Mazumder S, Ahamed RA, McGahee E, Wang L, Seyler TH. A new automated method for the analysis of aromatic amines in our understanding of the interactions between the smoking human urine by GC-MS/MS. J Anal Toxicol. 2019;43:25–35. status, the concentration of aromatic amines, and the risk https:// doi. org/ 10. 1093/ jat/ bky045. of developing smoking-related diseases. 4. Besaratinia A, Tommasi S. Genotoxicity of tobacco smoke-derived aromatic amines and bladder cancer: current state of knowledge Supplementary Information The online version contains supplemen- and future research directions. FASEB J. 2013;27:2090–100. tary material available at https://doi. or g/10. 1007/ s00216- 023- 04713-8 . https:// doi. org/ 10. 1096/ fj. 12- 227074. 5. Deng H, Yang F, Li Z, Bian Z, Fan Z, Wang Y, Liu S, Tang G. Acknowledgements We gratefully acknowledge the financial support Rapid determination of 9 aromatic amines in mainstream ciga- provided by Evonik Industries AG, Essen and the Open Access Pub- rette smoke by modified dispersive liquid liquid microextraction lication Fund of the University of Duisburg-Essen. The first author and ultraperformance convergence chromatography tandem mass would also like to thank the Institute of Analytical Chemistry and spectrometry. J Chromatogr A. 2017;1507:37–44. https://doi. or g/ Food Chemistry for their help during the experimental part of this 10. 1016/j. chroma. 2017. 05. 056. project, especially Dorothea Leis, Nina Haar, Sigrid Hager, and Claudia 6. Bie Z, Lu W, Zhu Y, Chen Y, Ren H, Ji L. Rapid determination of six Koraimann for the introduction to the laboratory. carcinogenic primary aromatic amines in mainstream cigarette smoke by two-dimensional online solid phase extraction combined with liq- Author contribution Nerea Lorenzo Parodi: conceptualization, meth- uid chromatography tandem mass spectrometry. J Chromatogr A. odology, validation, investigation, writing the original draft, visualiza- 2017;1482:39–47. https:// doi. org/ 10. 1016/j. chroma. 2016. 12. 060. tion. Erich Leitner: resources, writing—review and editing. Torsten C. 7. Zhang J, Bai R, Zhou Z, Liu X, Zhou J. Simultaneous analysis of Schmidt: writing—review and editing, supervision. nine aromatic amines in mainstream cigarette smoke using online solid-phase extraction combined with liquid chromatography- Funding Open Access funding enabled and organized by Projekt tandem mass spectrometry. Anal Bioanal Chem. 2017;409:2993– DEAL via the Open Access Publication Fund of the University of 3005. https:// doi. org/ 10. 1007/ s00216- 017- 0245-6. Duisburg-Essen. This work received financial support provided by 8. Smith CJ, Dooly GL, Moldoveanu SC. New technique using solid- Evonik Industries AG, Essen. The findings and conclusions in this phase extraction for the analysis of aromatic amines in mainstream report are those of the authors and do not represent the official views cigarette smoke. J Chromatogr A. 2003;991:99–107. https:// doi. or positions of the supporting companies. org/ 10. 1016/ S0021- 9673(03) 00205-X. 9. Stabbert R, Schäfer KH, Biefel C, Rustemeier K. 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Journal

Analytical and Bioanalytical Chemistry – Springer Journals

Published: Jul 1, 2023

Keywords: Gas chromatography-mass spectrometry (GC–MS); Gas chromatography-tandem mass spectrometry (GC–MS/MS); Negative chemical ionization (NCI); Aromatic amines; Derivatization; Urine

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