A methodological approach to the selection of liquid reagents for chemical ionization ion trap-gas chromatography mass spectrometry: A case study of GBL and 1,4-BD

International Journal of Mass Spectrometry 388 (2015) 34–39

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A methodological approach to the selection of liquid reagents for chemical ionization ion trap-gas chromatography mass spectrometry: A case study of GBL and 1,4-BD Luca Guandalini a , Giacomo Soldani a , Luca Rosi b , Luca Calamai c , Gianluca Bartolucci a a NEUROFARBA – Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino Sezione Scienze Farmaceutiche e Nutraceutiche, Università di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino (FI), Italy b Dipartimento di Chimica “Ugo Schiff”, Università di Firenze, Via della Lastruccia 3-13, 50019 Sesto Fiorentino (FI), Italy c Dipartimento di Scienze Produzioni Agroalimentari e dell’Ambiente (DISPAA), Università di Firenze, Piazzale delle Cascine 28, 50144 Firenze, Italy

a r t i c l e

i n f o

Article history: Received 4 May 2015 Received in revised form 22 July 2015 Accepted 22 July 2015 Available online 1 August 2015 Keywords: Club drugs Gamma-hydroxybutyric acid Chemical ionization In situ ion trap

a b s t r a c t A new approach is proposed for the selection of reagent ion species in a gas chromatography-chemical ionization mass spectrometry (GC-PICI-MS) method for GBL and 1,4-BD determination, the two “prodrugs” of gamma-hydroxybutyric acid (GHB), a drug associated with sexual assault. The GC-PICI-MS is often the best technique to avoid an extended fragmentation occurring in EI source and it preserves the information on molecular ions. Ion-trap mass spectrometry (IT-MS) is a valuable tool in chemical ionization experiments, commonly affording reaction times 104 –105 higher than those in conventional CI sources. This feature allows the use of either vapors from liquid reagents, or many reactant species that are difficult to generate and employ in the conventional CI experiments. In this research acetone, acetonitrile, methanol and diethylamine were evaluated to generate vapors of the chemical ionization species. The use of liquid CI reagent offers a wide range of chemical–physical properties that can greatly affect the specificity, with the possibility to modulate the detection of the analyte in comparison with background or matrix interferences. The experimental data using different CI liquid reagents and reaction times were compared through calibration curves of GBL and 1,4-BD (ranging from 10 to 1000 ng/mL). The linear regression curves obtained were used to calculate the sensitivity (slope) and limit of detection (LOD) of the method. Methanol resulted in the most efficient reagent for the determination of studied analytes. However, its employment as ionization agent of the 1,4-BD favors the hydride abstraction mechanism or hydrogen loss from protonated-molecule ions. These phenomena can be considered as a possible sources of uncertainty or errors. Therefore, acetonitrile can be employed as a good compromise between sensitivity and reliability of signal for both analytes. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Mass Spectrometry (MS) is a well recognized, highly sensitive and selective tool for analysis in many fields. The easy coupling with gas chromatography (GC) offers a third-dimension separation feature (i.e. mass spectrum) allowing the detection of analytes in complex matrices even at ultra-trace levels [1]. In benchtop GC–MS, the electron ionization (EI) source is the most common ion generation system. This ionization source, at a given electron energy, produces a characteristic and reproducible mass spectrum which allows a tentative compounds identification through the comparison with spectral databases. However, in some cases, EI mode is not always the most suitable ionization method since the associated extended analyte fragmentation may lead to a loss of information concerning the molecular http://dx.doi.org/10.1016/j.ijms.2015.07.018 1387-3806/© 2015 Elsevier B.V. All rights reserved.

ion [2]. As a matter of fact, the molecular weight evaluation of analytes is often carried out using chemical ionization (CI), which allows a better control of the internal energy deposited during the ionization processes [1]. In addition, selectivity and/or sensitivity can be increased, for selected compound classes, by using suitable CI reagents with different proton affinities and ion-molecule formation properties [3]. Generally, the CI mechanisms involve bimolecular processes during analyte ions generation. The occurrence of bimolecular reactions requires a sufficiently large number of ion-molecule collisions during the dwell time of the reagent species in the ion source. This is generally achieved by largely increasing the partial pressure of the CI reagent gas in the ionization chamber. In any CI plasma, both positive and negative ions are present, e.g., [M+H]+ and [M−H]– ions (albeit in different amounts), and it is just the polarity of the

L. Guandalini et al. / International Journal of Mass Spectrometry 388 (2015) 34–39

acceleration voltage which determines the extraction of positive or negative ions from the ion source [4]. The positive-ion chemical ionization (PICI) is a multi-step process that involves different reactive ionic species. In the first step, a reagent “R” is ionized by interaction with the electrons emitted by the filament, allowing the formation of [R]+. ions. In the following step, [R]+• interacts with the R neutral molecules, present in the ionization source in large excess, leading to the formation of other reacting species, such as [R+H]+ . In the last step, the analyte reacts with ionic species present in reagent plasma. Common reactions occurring in the last step of the PICI process are [5]: (A) Protontransfer : [RH]+ + M → [MH]+ + R (B) Hydrideabstraction : [R]+ + M → [M−H]+ + RH (C) Association : [R]+ + M → [MR]+ (D) Chargetransfer : [R]+ + M → [M]+ + R Most of the common reagents show the proton transfer as the major reaction, leading to the formation of the protonated analyte ions. On the other hand, the energy involved in the CI ionization mechanisms can be enough to activate the fragmentation processes of the [M+H]+ species with consequent increase of the complexity of the CI mass spectrum. Traditional CI reagents are gases in standard conditions (i.e. methane, isobutane, ammonia) endowed with some evident drawbacks: the need of high pressure cylinders and pressure regulation devices, risk of explosions with flammable gases (i.e. methane, isobutane), limited choice of a suitable reagent, high management costs, etc. Many of these problems could be overcome employing vapors from liquid reagents. In fact, in this case the choice of a suitable reagent is greatly enhanced since there are many compounds available with respect to gases, and the chance to find a suitable reagent is higher. In addition, the limited amounts needed avoid the toxicity problems or safety risks. In the present work, the ionization features of the “in situ”(ionization) ion trap mass spectrometer (in situ IT-MS) were evaluated. This instrumental configuration enables the trapping of ions for a relatively long time (tens of milliseconds) thus increasing tremendously the probability of interaction with neutral molecules present in the surroundings [1]. Therefore, it is possible to obtain a CI reaction plasma with only ppm concentrations of reagent gas, so that also liquid reagents (with rather low vapor pressure) can be employed as such. Gas chromatography coupled with mass spectrometry (GC–MS) and CI have been widely used for decades to determine small quantities of drugs in multiple matrices. This is the case of gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), the two “prodrugs” of gamma-hydroxybutyric acid (GHB). They are drugs of abuse often associated with drug-facilitated sexual assault (DFSA) [6]. After administration, GBL and 1,4-BD are enzymatically converted to GHB in many tissues, with comparable biological effects and risks [7–10]. GHB and GBL have been recently subjected to legal constraints in many countries, while 1,4-BD is not included in any prohibited substances list and is commercially available as industrial solvent [11]. Qualitative and quantitative determination of GHB and GBL in various matrices has been carried out employing GC-PICI-MS, often using methane as CI reagent [12]. In many published methods, the procedure takes advantage of the quantitative conversion of GHB into GBL by dehydration; such reaction can occur both at high temperatures and in acidic conditions [13–16]. Recently, a method for quantitative determination of GHB, GBL and 1,4-BD in dietary supplements using GC-PICI-MS was published [17]. In this research, full

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conversion of GHB to GBL was achieved in the gas chromatograph injection port and a comparison between the EI and PICI mass spectra of GBL was made. In order to preserve the information derived from molecular ions, with the intrinsic improvement in both specificity and sensitivity, CI was selected for the quantitative analysis and acetonitrile was chosen as CI reagent. The aim of this work is the evaluation of the analytical performances, employing different liquid CI reagents, using the GC-PICI-MS method for the determination of GBL and 1,4-BD as case study. The influence of parameters, such as the nature of the liquid reagent (different proton affinity) and reaction time (different yield of CIreaction), on the analytical results was studied. The choice of the suitable CI reagent is of primary importance on the analytical results such as quality of spectra or ionization yield. In fact, the thermodynamics of the reagent-analyte interaction determines the amount of the energy deposited, and consequently, the degree of fragmentation of the analyte along with all the related information regarding the molecular ions species (e.g. [M+H]+ , [M−H]+ , etc.). Moreover, the choice of the reagent gas can be finely tuned and could greatly affect specificity, with the possibility to modulate the ionization of the analyte with respect to the matrix interferences [18]. For instance, if only the proton transfer mechanism is considered, the yield of reaction largely depends on the different proton affinities of analyte and CI reagent [5]. On the other hand, different reagent species could be present in the reagent plasma leading to different ionization mechanisms. Therefore, many solvents commonly used in laboratory can be used as CI reagent to afford a wide range of proton affinities [19] and many other potential ionization species generated in the in situ IT-MS. In the present work, the effect of four liquid reagents, acetonitrile, methanol, acetone and diethylamine, was investigated. This selection was motivated by both their boiling points (82, 65, 56 and 55 ◦ C, respectively), so as to obtain a suitable vapor pressure for CI, and the range of proton affinities, that were similar to those of the analytes (Table 1). Diethylamine was chosen as a high-end reference for high proton affinity with respect to the analytes. The effect of different reaction times between the analytes and ionic reagent species was also investigated. 2. Materials and methods 2.1. Chemicals GBL was purchased from Merck (Milan, Italy). 1,4-BD, acetonitrile, methanol, acetone and diethylamine were all supplied by Sigma–Aldrich (Milan, Italy). 2.2. Instruments The GC-PICI-MS analysis was performed by a Varian CP-3800 gas-chromatograph coupled with a Saturn-2200 ion trap mass spectrometer (Palo Alto, CA, USA) equipped with multiple CI reagent module. Chromatographic separations were performed by a Zebron ZB-WAX capillary column (30 m × 0.25 mm i.d., 0.25 ␮M

Table 1 Proton affinities of GBL, 1,4-BD and the CI liquid reagents data given from E.P.L. Hunter and Lias [19]. Compound

Proton affinity (kJ mol−1 )

GBL 1,4-BD Methanol Acetonitrile Acetone Diethylamine

840.0 915.6 754.3 779.2 812.0 952.4

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Table 2 PICI experimental conditions for all gas reagents tested. Liquid reagent

Isolation window (m/z)

Ejection amplitude (V)

Scan range (m/z)

Methanol Acetonitrile Acetone Diethylamine

20–55 25–65 35–68 50–80

9.0 9.0 12.0 9.0

65–250 65–250 65–250 80–250

d.f., Phenomenex Bologna, Italy) with the following oven temperature program: 50 ◦ C for 1 min, then 30 ◦ C/min up to 100 ◦ C and then 20 ◦ C/min up to 200 ◦ C, hold time 2.33 min. A split/splitless injector was used at 250 ◦ C, in splitless mode for 1 min, and 1 ␮L of sample was injected. Helium, at a constant flow rate of 1.2 mL/min, was used as the carrier gas. The PICI conditions were optimized for every gas reagent tested using a multiple CI reagent module. This apparatus allows the introduction of the vapor into the CI chamber, through a circuit connected from individual glass reservoir, filled with the reagent, and regulated by a proper needle valve. The flow of each reagent vapor was regulated so that an ionization time of 100 ␮s produced an ion current of about 10,000 counts at filament current of 10 ␮A. In order to maintain these conditions, the multiple CI reagent case was kept at 25 ◦ C ± 3 ◦ C with a thermostatic device, since solvent evaporation or ambient temperature variations would have affected the vapor pressure. The ion trap ionization mode selected was CI Auto, and maximum ionization time and target TIC were set at 2 ms and 5000 counts respectively, while the specific parameters used for each reagent gas tested are listed in Table 2. Different isolation width was defined for each reagent so as to collect the reactive ions species and eliminate undesired reactive ions, such as the ubiquitous m/z 19 corresponding to [H3 O]+ , whose presence may have invalidated the properties of reactive vapors from the various solvents used. Therefore, the analytical scan range was adjusted for each solvent to allow the acquisition of only sample ionic species, avoiding those from the reagent gas. Each liquid reagent tested was evaluated using five different reaction times (10, 20, 40, 80, 120 ms), analyzing samples containing GBL and 1,4-BD at concentrations near to the detection limit. 2.3. Preparation of standard solutions Stock solutions of the analytical standards (GBL and 1,4-BD) were prepared in acetonitrile at 10.0 mg/mL and stored in the dark at 4 ◦ C for no longer than 2 months. Working solutions were freshly prepared for each experiment by diluting stock solutions of GBL and 1,4-BD in acetonitrile up to a concentration of 10 ␮g/mL (working solution 1) and 1 ␮g/mL (working solution 2). Six concentration levels for each calibration curve were prepared by adding proper volumes of working solutions 1 (for the higher concentration levels) or working solutions 2 (for low concentration levels) of GBL and 1,4-BD. The mixtures obtained were dried under a gentle nitrogen stream and dissolved in 1 mL of acetonitrile. Following this procedure, the final concentrations of GBL and 1,4-BD in the standard solutions were: 25, 50, 100, 250, 500, 1000 ng/mL. The obtained standard solutions were analyzed in triplicate by GC-PICI-MS method described above; this procedure was repeated for each combination of CI reagent and reaction times (four CI reagents at five different reaction times). GBL and 1,4-BD at the chromatographic conditions employed showed retention times of about 5.9 and 7.3 min, respectively, and

were fully separated from the solvent peak so as to rule out any contribution of the dilution solvent to the chemical ionization of the molecules (Supplemental Material Figure S1).

3. Results and discussion 3.1. Analysis of GC-MS-PICI spectra The mass spectra of GBL and 1,4-BD were obtained analyzing the standard solution at 1000 ng/mL with the GC-PICI-MS method using the CI reagents reported above. The spectra of GBL obtained employing methanol or acetonitrile or acetone were similar to each other and very simple, since only the signal relative to [M+H]+ species at m/z 87 was present (Supplemental Material Figures S2-4). Conversely, the PICI spectra of 1,4-BD resulted to be very different depending on the CI reagent employed. The spectrum obtained with methanol (Fig. 1A) showed the signal at m/z 73 as base peak assigned to [M+H-H2 O]+ species, and a group of signals at 10% relative abundance corresponding to [M+H]+ at m/z 91 and [M−H]+ at m/z 89 ions. The [M+H]+ ions were derived from proton transfer reaction while [M−H]+ species could be provided both hydride abstraction ionization mechanism and hydrogen loss from [M+H]+ . The abundance of [M+H-H2 O]+ species can be explained through the properties of 1,4-BD which dehydrates quickly after protonation. The corresponding spectrum obtained with acetonitrile (Fig. 1B) showed the same base peak at m/z 73, while the [M+H]+ species was increased to about 20%, and [M−H]+ ion was not detected. The spectrum obtained with acetone (Fig. 1C) showed the m/z 91 ion as base peak while the signals at m/z 73 and m/z 89 were present at 78% and 10% relative intensity, respectively. The different behaviors of 1,4-BD in these conditions can be explained by the difference in proton affinity between reagent gas and analyte, i.e. about 103 kJ mol−1 , less than the differences with the other gas reagents considered (about 136 and 161 kJ mol−1 for methanol and acetonitrile respectively). Therefore, a lower excess energy was transferred during the ionization process and consequently a preservation of [M+H]+ species was observed. The analysis carried out with diethylamine did not produce relevant results, in accordance with its higher proton affinity with respect to both analytes, and the chromatograms obtained did not show any signals related to the analytes (Supplemental Material Figure S5). The obtained results confirmed that proton transfer was the major process among the reactions occurring in the CI conditions tested, in full accordance with previous observations of “in situ”(ionization) IT-CI-MS system [1]. The [M−H]+ species, originating as explained above, are present at about 10% in the 1,4-BD spectra when methanol or acetone was used as reagent gas but not with acetonitrile. The hydride abstraction or hydrogen loss mechanisms should be preferably avoided or negligible, especially when an isotopic dilution method with deuterated internal standards is used. In this case, hydride abstraction or hydrogen loss processes may generate competitive reactions leading to possible mistakes, e.g. impure deuterated internal standards (ISTD) containing traces of partially deuterated species, generating signals with the same m/z of the analyte. Moreover, the formation of several kinds of ions, in the range of m/z of interest, splits the analyte signal decreasing the detection performances. Therefore, for the determination of 1,4-BD acetonitrile appears to be a good compromise, giving 20% of [M+H]+ and a negligible hydride abstraction or hydrogen loss mechanisms.

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Fig. 1. PICI spectra of 1,4-BD obtained with the following reagent gases: (A) methanol, (B) acetonitrile and (C) acetone.

3.2. Comparison among the different experimental conditions on the method sensitivity and detectability The method performances as a function of the experimental conditions were evaluated through the determination of the slope of calibration function and limit of detection (LOD). The slope of the regression line is important, as it determines the sensitivity of the calibration function, that is, the rate at which the signal changes with analyte concentration. The higher the slope values, the easier it is to distinguish between concentrations which are close to each other. The LOD is the smallest signal that is distinguishable from the background (baseline) noise. Various criteria have been applied in quantifying this limit, but a generally accepted rule is that the signal must be at least three times greater than the background noise. When the LOD values were checked as Signal-to-Noise (S/N) evaluation approach, its value is strongly influenced by the stability and reproducibility of the background noise. It often happens that high S/N ratio can be obtained likely resulting from only low baseline noise, without any information about the signal reliability. Therefore, to obtain reliable LOD values, the variations of the analyte signals in the low concentration range must be considered, employing the standard deviation of response and slope approach [20]. The standard solutions, prepared as described in Section 2.3, were analyzed in triplicate by GC-PICI-MS method; the peak areas relative to [M+H]+ ion species of GBL and 1,4-BD were plotted versus the nominal concentrations, and the best fitting to the linear regression was calculated for each analyte. Following this procedure, for each combination of CI reagent-reaction times, 15 linear regressions were obtained for each analyte (Supplemental Material Table S1-2). LOD and slope of each regression line were calculated and evaluated in order to rationalize the effect of the different experimental parameters.

The standard deviation of response and slope approach uses the standard error of curve (SE-curve) or the standard deviation of y-intercepts (SE-y) as sigma value (). In the regression line parameters, SE-curve represents the variability around the regression curve throughout the whole range of the curve. Therefore, it may occur that the variability of the larger concentrations overpowers the variability of the lower ones and gives an unrealistically high value of the LOD. Hence, for evaluation of the actual LOD values, the use of the SE-y limits the range of the regression curve (25–250 and 100–1000 ng/mL for GBL and 1,4-BD respectively) which is considered more appropriate [21]. LOD values were calculated by the standard deviation of response and slope approach using SE-y of regression lines as following: LOD =

3.3 ∗  slope

 = SE-y

The calculated slopes for each CI reagent for both analytes were plotted versus reaction times and are reported in Figs. 2 and 3. The values of slope increased with reaction times for any reagent employed (Figs. 2 and 3). In accordance to the proton affinities, the GBL slopes obtained when methanol was used as reagent gas were higher with respect to those with acetonitrile and acetone. This trend is evident with GBL, where [M+H]+ is the only ion species obtained, in opposition of 1,4-BD, where the signal is split between [M+H]+ , the dehydrated ion [M+H-H2 O]+ and other minor ionic species. In particular, when [M+H]+ signal was evaluated as quantification ion, the proton affinity rule is not followed. In this case, the slopes obtained from acetone were higher respect than obtained from acetonitrile and the differences with methanol slopes were decreased (Figs. 2 and 3). Otherwise, when [M+H-H2O]+ species was selected as quantification ion, it follows again the proton affinity rule with high difference between the slopes obtained from different reagents (Supplemental Material Figure S6). The use of fragmented ions as quantitative signal in CI could be an opportunity

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L. Guandalini et al. / International Journal of Mass Spectrometry 388 (2015) 34–39

Fig. 2. Plot of slopes calibration curves vs reaction times (ms) obtained from [M+H]+ species of GBL using different CI reagents.

Fig. 3. Plot of slopes calibration curves vs reaction times (ms) obtained from [M+H]+ species of 1,4-BD using different CI reagents.

but must be evaluated and checked, especially when an isotopic dilution method is applied. However, in this study, it was preferred to consider the [M+H]+ species as target, because it preserved all information about analyte structure and conformation. The analysis of slopes vs reaction times plots would suggest that methanol and long reaction times are the best choices to improve the ionic signal and consequently the sensitivity of determination, although the contribution of background noise has not been taken into account. It is interesting to observe that these linear functions do not pass through the origin of axes. This evidence suggests that it is possible to decrease the reaction time until a threshold value below which the analyte ions formed are not enough to overcome the detection limit. Interestingly, different information can be drawn from the analysis of LOD values (Tables 3 and 4).

The lowest absolute values of LOD were shown when methanol or acetone was used as gas reagent for GBL and 1,4-BD respectively, but actually the reaction times or different gas reagent did not affect significantly the LOD values (i.e. larger than ± 2 SD) for both analytes. Table 3 LOD’s values (ng/mL) obtained for GBL using different gas reagent and reaction times. Reaction time (ms)

10 20 40 80 120

Reagent gas Methanol (ng/mL)

Acetonitrile (ng/mL)

Acetone (ng/mL)

30 20 20 20 20

50 60 20 30 70

20 20 30 50 30

L. Guandalini et al. / International Journal of Mass Spectrometry 388 (2015) 34–39 Table 4 LOD’s values (ng/mL) obtained for 1,4-BD using different gas reagent and reaction times. Reaction time (ms)

10 20 40 80 120

Reagent gas Methanol (ng/mL)

Acetonitrile (ng/mL)

Acetone (ng/mL)

410 500 280 250 380

170 420 330 400 410

300 110 90 170 180

4. Conclusions The information derived from the experiments reported above suggests that longer reaction times, during chemical ionization process, improve the ionic signal and consequently the sensitivity of the method. Therefore, the reaction time value should be limited only for ensuring a suitable sampling frequency (dwell time or duty cycle), necessary to define the chromatographic peak properly. Methanol resulted as the most efficient reagent gas with respect to acetone and acetonitrile for the determination of studied analytes. In any case, considering the 1,4-BD, it should be noted that methanol favors the hydride abstraction, during ionization process, or hydrogen loss from [M+H]+ species and these phenomena can be considered as a possible source of uncertainty or errors. The same considerations can be made regarding acetone as reagent gas that, in spite of a lesser sensitivity, shows low LODs, primally for 1,4-BD. Hence, acetonitrile represents a good compromise between sensitivity and reliability of signal for both analytes, also because it does not occur with hydride abstraction or hydrogen loss from [M+H]+ species. This case study indicates that a comprehensive approach through the evaluation of LOD and sensitivity is useful for the choice of the most appropriate CI reagent in GC PICI-MS analysis. Acknowledgements The authors wish to thank R. Di Loreto and S. Federighi for their support in the revision version of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijms.2015.07.018 References [1] S. Bouchonnet, D. Libong, M. Sablier, Low-pressure chemical ionization in ion trap mass spectrometry, Eur. J. Mass Spectrom. 10 (2004) 509–521.

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