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Separation of benzodiazepines by electrospray ionization ion mobility spectrometry–mass spectrometry
Analytica Chimica Acta 457 (2002) 235–245
Separation of benzodiazepines by electrospray ionization ion mobility spectrometry–mass spectrometry Laura M. Matz, Herbert H. Hill Jr.∗ Chemistry Department, Washington State University, P.O. Box 644630, Pullman, WA 99164-4630, USA Received 2 July 2001; received in revised form 11 December 2001; accepted 2 January 2002
Abstract Benzodiazepines are a commonly abused class of drugs; requiring analytical techniques that can separate and detect the drugs in a rapid time period. In this paper, the two-dimensional separation of five benzodiazepines was shown by electrospray ionization (ESI) ion mobility spectrometry (IMS)–mass spectrometry (MS). In this study, both the two dimensions of separation (m/z and mobility) and the high resolution of our IMS instrument enabled confident identification of each of the five benzodiazepines studied. This was a significant improvement over previous IMS studies that could not separate many of the analytes due to low instrumental resolution. The benzodiazepines that contain a hydroxyl group in their molecular structure (lorazepam and oxazepam) were found to form both the protonated molecular ion and dehydration product as predominant ions. Experiments to isolate the parametric reasons for the dehydration ion formation showed that it was not the result of corona discharge processes or the potential applied to the needle. However, the potential difference between the needle and first drift ring did influence both the relative intensity ratios of the two ions and the ion sensitivity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Electrospray; Ion mobility spectrometry; Benzodiazepines
1. Introduction Benzodiazepines are a widely abused class of drugs used as minor tranquilizers, hypnotics, and muscle relaxants [1]. Because benzodiazepines are often seen in forensic and clinical cases, many of these compounds are included in routine drug tests. The increasing number of these samples has created a need for higher sample throughput, requiring faster sample preparation and analysis. Currently, gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) are the most com∗ Corresponding author. Tel.: +1-509-335-5648; fax: +1-509-335-8867. E-mail address: [email protected] (H.H. Hill Jr.).
mon methods employed for benzodiazepine detection [1–3]. Also, thin layer chromatography and micellar electrokinetic capillary chromatography (MECC) are employed [1]. However, chromatographic separations are typically lengthy and require extensive sample preparation prior to analysis. In this paper we present the direct and rapid separation of benzodiazepines by electrospray ionization (ESI) ion mobility spectrometry (IMS)–mass spectrometry (MS). Until recently, employing IMS–MS for complex mixture analysis was limited due to the low instrumental resolution [4]. In the past several years, IMS instruments have evolved as higher resolution instruments [5–10], obtaining resolution values exceeding LC and GC and approaching capillary electrophoresis values [10]. With these new instruments,
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the separation of several complex mixtures have been shown including chemical warfare agents [11], explosives [12], and drugs [13]. Previously, Lawrence evaluated the separation of some benzodiazepines by IMS using a radioactive 63 Ni foil as the ionization source [14]. However, the low resolution of the IMS used in these studies led to incomplete resolution of several peaks. Although, there were some problems with the separation at the time, the work of Lawrence showed that IMS could be a rapid way (separation times in milliseconds to seconds) to detect and perhaps, separate benzodiazepines [14]. In this study, we evaluate the separation of benzodiazepines by high-resolution ESI-IMS–MS. ESI may be an ideal ionization source as a precursor to analysis of benzodiazepines in biological samples. However, ESI and 63 Ni foil are very different ionization sources and a part of this study was to evaluate any similarities and differences between the two techniques (comparison between the study published by Lawrence [14]). Furthermore, the advantage of high resolution separations was examined. The goal of this study was two-fold: (1) to investigate the differences between ions formed in 63 Ni, ESI-IMS and ESI-MS; and (2) to evaluate ESI-IMS–MS as an analytical technique for benzodiazepine separation and detection.
2. Experimental 2.1. Reagents and chemicals All solvents used in the study were HPLC grade (methanol, water, acetic acid) and were obtained from J.T. Baker (Phillipsburgh, NJ, USA). All five benzodiazepines were obtained from Radian International (Austin, TX, USA) as 1 mg/ml standards in methanol. All solutions were diluted with the electrospray solvent (47.5% water/47.5% methanol with 5% acetic acid). 2.2. IMS instrumentation Two ESI sources were employed in this study: the water-cooled ESI source described previously [15] and a nitrogen gas cooled ESI source [16]. The water-cooled ESI source was employed for all experiments except where specified. Also, both ESI needles
were maintained at 12.5 kV, except when indicated in the figures. The ESI solution was composed of 47.5% water/47.5% methanol with 5% acetic acid and was pumped by a Brownlee Labs (Santa Clara, CA, USA) dual syringe-pump at a flow rate of 5 l/min. For all studies, a high-resolution IMS–MS instrument was employed. This instrument was built at Washington State University (Pullman, WA, USA) and has been previously described [8] (modifications to original design are described in [10]). A voltage of 9.5 kV was applied to the drift tube (7.9 kV at the ion gate). The drift gas (nitrogen) flow rate was operated at 1 l/min for all experiments. Both the desolvation (7.5 cm) and drift region (22.5 cm) of the IMS tube were maintained at 250 ◦ C. Atmospheric pressure in Pullman, WA, USA is typically between 690 and 700 Torr. The IMS was a stacked ring design with alternating alumina and stainless steel rings. The MS instrument was a 150-QC quadrupole (ABB Extrel, Pittsburgh, PA, USA) with a mass range of 0–4000 u. The lenses between the IMS and the quadrupole were as follows: +8.0 V (40 m pinhole), −42.8, −116.3, −34.5, −17.3, and −50.9 V. The first MS chamber was maintained at a pressure of 2.0 × 10−4 Torr and the second chamber was maintained at a pressure of 2.0 × 10−5 Torr. The MS and IMS data acquisition systems have been improved from previous studies [8]. First, the MS software (Merlin, ABB Extrel) enabled the quadrupole to be slowly scanned (i.e. 1 u/50 s) over a specified m/z range. The IMS gate control and data acquisition used the Labview software (National Instruments, Houston, TX, USA) which was modified so that each ion mobility spectra was sequentially saved and spectra could be obtained for up to one hour. Similar to the previous IMS software [15], the scan time, gate open pulse time (pulse width), and number of averaged spectra could be varied. Due to the additional data processing incurred by the Labview software, there was a 40% time overhead added to the time required for acquisition of each IMS spectra. The IMS controlling electronics were the same as described in previous studies [15] (amplifier and gating electronics). The instrument was operated in one of three ways (specified for each figure), non-selective ion monitoring (NSIM), selective ion monitoring (SIM), and two-dimensional IMS–MS spectral acquisition (2D-IMS–MS). For all three cases, the ion mobility
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instrument was continually gated to obtain mobilities for the ESI ions. The difference in the three operational modes was the operation of the QMS. In NSIM, the QMS direct current (dc) voltage was turned off and all ions above a specified mass were transferred through the MS. The NSIM mode is similar to a total ion chromatogram in chromatography–mass spectrometry. In SIM, one specific m/z value was transmitted through the MS and detected. In the third mode of operation (2D-IMS–MS), the quadrupole was scanned so ions in a 1 u m/z window were transmitted per ion mobility spectrum. For example, if the desired mass range was 130–150 u and each completely averaged IMS spectra took 30 s, then the quadrupole would initially transmit the m/z value 130 u and increase this value by 1 u every 30 s (i.e. continual SIM IMS spectra with increasing m/z). In this way, both the mass and ion mobility information were obtained simultaneously. 2.3. Calculations The reduced mobility values reported were calculated based on the following equation [17]: 2 273 L P Ko = (1) Vtd T 760 where L is the drift region length (22.5 cm), V the drift voltage (7900 V), td the ion drift time, T the effective temperature in the drift region (523 K), and P is the pressure in the drift region (∼690–700 Torr in Pullman, WA, USA). Detection limits were de-
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fined to be the concentrations or quantities providing a signal-to-noise ratio of 3. 3. Results and discussion 3.1. ESI-IMS spectra of benzodiazepines The structures of the five benzodiazepines are shown in Fig. 1. All five compounds contain nitrogen atoms in their structure and oxazepam and lorazepam contain a hydroxyl moiety as well. In Table 1, the Ko values, mass identities and literature Ko values are listed. Initial inspection of the table showed that both lorazepam and oxazepam formed two predominant ions; a loss of water (due to the hydroxyl moiety) and the protonated molecular ion. Since an ion’s drift time is inversely related to the Ko values (see Eq. (1)), slower drifting ions have smaller Ko values. The literature values reported in Table 1 were obtained with air as the drift gas. Therefore, the Ko values (drift gas, nitrogen) reported in this study will not be the same, but the trends were similar. Furthermore due to the similar size and polarizability of nitrogen and air, Ko values will be similar in values as seen in Table 1, where the values differ by 7–8%. For both compounds that produced two ions (lorazepam and oxazepam), the (M–H2 O)H+ drifted faster than the MH+ which was expected due to the decreasing mass. Comparison of both the literature and reported Ko values showed that the largest (fastest drifting ion) Ko value was one of the higher mass ions
Table 1 List of benzodiazepine ions, Ko values, and literature Ko values Compound
MW
Ko (cm2 V−1 s−1 )
Ion formula
Ion mass (u)
Ko (cm2 V−1 s−1 ) [14]
Diazepam
284.8
1.123
MH+
286, 288
1.21
Detection limits (ppba , pmol) 52.8, 0.463
Oxazepam
286.7
1.138 1.184
MH+
(M–H2 O)H+
288, 290 270, 272
1.23 1.28
162, 1.41
Chlordiazepoxide
299.7
1.091
MH+
300, 302
1.18
253, 2.11
Lorazepam
321
1.085 1.118
MH+
(M–H2 O)H+
322, 324 304, 306
1.19 1.22
305, 2.38
Bromazepam
316
1.144
MH+
317, 319
1.24
200, 1.58
Literature Ko values were obtained with air as the drift gas and were obtained with 63 Ni foil as the ionization source [14]. Therefore, the mass identities of the literature values differed slightly from the ions observed in this study. a Detection limits in picomoles were defined based on 30 s acquisition time and 5 l/min sample flow rate.
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Fig. 1. Structure of five benzodiazepines that were electrosprayed, separated and detected by ESI-IMS–MS.
(the protonated ion of bromazepam). Although, the ion’s mobility is a function of mass, it is also a function of its size and interaction with the drift gas. These results are similar to the previous study by Lawrence (63 Ni ionization sources) which showed there was not a correlation between the ion mobility values and ion mass [14]. Inspection of Table 1 showed that the general trends for Ko values were similar for both the literature values and the measured values. In Fig. 2, the non-selective IMS–MS spectra for the five benzodiazepines are shown. As discussed in Table 1, the compounds without the hydroxyl moiety produced one predominant ion (MH+ , labeled 1) and both compounds with the hydroxyl moiety produced two ions (1 and (M–H2 O)H+ , labeled 2). As expected due to the nitrogen atoms in the molecular structures, all five compounds produced strong signals at the
20 ppm concentration range. For the five analytes, minor mobility peaks were observed and mass identified as sodium and potassium adducts. The smaller side peaks following the protonated ions in Fig. 2 correlated with these two ions. 3.2. Investigation of dehydration products Since ESI is a soft ionization process, dehydration ions observed in the IMS spectra were not expected. In MS studies employing ESI as the ionization source, the protonated ions were the only ions observed [3] and multiple stages of MS were employed to invoke fragmentation. In the second MS stage, dehydration ions were observed similar to the dehydration ions discussed above. Sometimes corona discharge ionization (a gas-phase ionization process) can occur
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Fig. 2. Non-selective ESI-IMS–MS spectra of five benzodiazepines: (1) MH+ and (2) (M–H2 O)H+ . Graph shows that benzodiazepines which contained a hydroxyl group also formed both a protonated and dehydration ion. Spectra were the result of 1000 averages. All mobility peaks were also mass identified and each analyte was present at a concentration of 20 ppm.
simultaneously with ESI conditions unless specific measures are taken to eliminate these processes. Chen et al. found that the water-cooled ESI source required a piece of plastic tubing surrounding the metal needle to eliminate any corona discharge [18]. The metal needle used in this study was insulated with a piece of polyethylene tubing, although it melted after an hour of operation. To insure that the insulating tube was positioned properly, experiments were performed to isolate any corona discharge processes occurring. A gas-phase corona discharge ionization process may have been the reason for the appearance of the dehydration ions. In Fig. 3, the NSIM IMS spectrum of oxazepam is shown for both the water-cooled ESI source with metal needle (a) and a nitrogen cooled ESI source (b) with a fused-silica needle. Since corona
discharge is a gas-phase process occurring due to the breakdown of gas molecules surrounding the needle [19], employing a fused silica needle would minimize corona discharge. If the dehydration ions were due to corona discharge, the dehydration ions should not be seen with the fused-silica needle. However, as seen in Fig. 3, both ions were found in the IMS spectra. The fused silica needle actually produced more dehydration ion than the water-cooled needle. Therefore, based on these studies, it was concluded that corona discharge processes were not causing the dehydration ions. In ESI, a potential is applied between the needle and a counterelectrode (for IMS, this was the first ring in the desolvation region). The potential difference between the needle and the ring was varied in order to
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Fig. 3. Comparison of oxazepam IMS spectra with (a) water-cooled ESI needle and (b) nitrogen-cooled ESI spectra. Graph shows that dehydration ion was formed regardless of ESI needle.
determine its effect on ion signal intensity and on the ion identity. Since altering the drift region voltage would affect the electric field and the ion transmission efficiency, the voltage on the ESI needle was varied and the drift region voltage (8.0 kV) was held constant. In Fig. 4, the signal intensity of three ions for oxazepam; MH+ , (M–H2 O)H+ , and (M + Na)+ were evaluated as a function of potential difference between the needle and first drift ring. In Fig. 4a, the ion intensity for the protonated molecular ion of oxazepam was plotted as a function of potential difference. The graph shows that the highest signal intensity was obtained for a difference of 3.5–4.0 kV and was significantly lower above and below these potentials. This is consistent with the range of potentials observed for ESI conditions reported previously [19].
Typically in ESI-MS, the ESI voltage at the ESI-MS interface can be increased to cause collision induced dissociation (CID) for fragmentation of the parent ion. However, this is not true for ESI-IMS since the instrument is operated at atmospheric pressures and the voltages utilized are not sufficient to cause CID. Manipulation of the lens voltages at the IMS–MS interface could be utilized to invoke fragmentation [20], but the daughter and parent ions would then have the same mobility values. As previously shown in Fig. 2, the protonated and dehydration ions have different drift times, indicating that the ions were formed prior to the mobility separation. A more plausible solution is that the ions form the dehydration ions due to stability of the structures at the elevated temperatures, similar to the reasons for
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Fig. 4. Evaluation of ESI potential on (a) analyte sensitivity and (b) ion fragmentation ratios. Intensity values were obtained for oxazepam. Graph shows that the potential difference between the needle and drift ring alters both the sensitivity and fragmentation.
formation of multiple ions with the radioactive ionization source. One interesting feature was that the highest MH+ : (M–H2 O)H+ ratios correlated with the highest signal intensities found in the upper graph. As the potential
difference was increased past 4 kV, the MH+ : (M + Na)+ was greater than for the dehydration ratio. The results of these experiments showed that the potential difference had little influence on minimizing the dehydration product and was highly correlated with the
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Fig. 5. Reproducibility of ion ratios for oxazepam at three different concentrations: (a) 1 ppm; (b) 10 ppm; and (c) 100 ppm.
potential difference that provided the greatest oxazepam intensity. In Fig. 5, the non-selective ESI-IMS spectrum of oxazepam was shown at three concentrations: (a) 1 ppm; (b) 10 ppm; and (c) 100 ppm. Based on inspection of the relative signal intensities in the graph, it was clear that the ratios were not consistent. In Fig. 5b, the MH+ ion intensity was approximately 50% of the dehydration product. In contrast, the 100 ppm (Fig. 5c) spectra showed that the protonated molecular ion was 75% of the dehydration product. It was clear from these studies that there needs to be a greater understanding of the ion formation process in order to quantify the ions observed for both lorazepam and oxazepam. Mass-selected mobility spectra for the protonated ion of each benzodiazepine were attained and the detection limits were estimated. The detection limits are
presented in Table 1 both in concentration units of ppb and in the quantity injected (picomoles). Although typical concentrations in biological samples could be present at low ppb, an order of magnitude below the detection limits, the minimal quantity required for a complete analysis would enable pre-concentration steps to provide adequate concentrations for IMS–MS analysis. Furthermore, it is expected upon optimization of the instrumental parameters, the detection limits could be further reduced. 3.3. Two-dimensional separation of five benzodiazepines In Fig. 6, a two-dimensional contour plot of m/z values and drift times was plotted for a mixture of the five benzodiazepines. As seen in Table 1, diazepam
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Fig. 6. Two-dimensional ESI-IMS–MS spectra of five benzodiazepines. Graph shows that six benzodiazepines were separated and identified due to the two dimensions of separation (ion mobility and m/z value).
and oxazepam only differ by one mass unit and due to the isotope patterns of Cl atoms, the mass spectra would be expected to overlap significantly. However, the contour plot in Fig. 6 shows that the two analytes can be easily identified due to the differences in mobilities (seen on the graph as drift times). Furthermore, Fig. 6 showed that all five analytes could be easily distinguished by evaluation of the m/z drift time values. In the previous work performed by Lawrence [14], the separation of diazepam and bromazepam (difference in Ko values was 0.3 cm2 V−1 s−1 ) was not achieved with conventional spectral techniques. However, inspection of Fig. 6 shows that bromazepam and diazepam do not overlap in drift times and would be easily separated by IMS alone. This example shows the improvements in instrumental resolution that have been made in the past 10 years. In IMS, separation efficiencies are typically reported as resolving power and are calculated based on the following equation: td Rp = (2) wh where Rp is resolving power, td the drift time, and wh is the ion pulse duration measured at half of the
maximal intensity [21]. Typical Rp values are around 30 for commercial instruments and up to 150 (for singly charged ions) for high-resolution instruments [8]. In order to compare the separation efficiencies between other separation techniques and ion mobility, the experimentally determined resolving power (Eq. (2)) can be converted to theoretical plates (N) by the following equation: N = 5.55(Rp )2
(3)
Recently, our laboratory developed the equations to describe the separation efficiency in terms of theoretical plates and resolution similar to the equations predominantly used in chromatography [10]. Eq. (4) shows the fundamental resolution equation derived for IMS: √ N α−1 R= (4) 4 α where R is resolution and α is the selectivity factor defined as Ko1 /Ko2 . By determining the Ko values for two compounds and the number theoretical plates achievable with the instrument, an estimation of the resolution between two ions can be attained. When discussing the separation efficiencies and resolution
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of two compounds, only the mobility separation was evaluated (separation along the y-axis in Fig. 6). In ion mobility, the resolving power can be improved by increasing the voltage, decreasing the temperature, decreasing the initial width of the ion injection (labeled pulse width) by decreasing the time the electronic gate is opened and improving the electric field homogeneity [10]. In the early reports by Lawrence, some of these variables were discussed (pulse width and voltage) as ways to improve the resolving power [14]. However, it was noted that decreasing the pulse width comes with a subsequent decrease in sensitivity which is typically unacceptable. Recently our lab reported substantial improvements in resolving power due to increases in the voltage with an accompanied increase in the drift tube length, and improvements in the drift tube design. With these design enhancements, resolving powers now reach 80–125 on a regular basis (with a 0.2 ms pulse width) and resolving power values up to 220 have been noted for multiply charged ions [10]. In the work performed by Lawrence, an instrumental resolving power of 39 (calculated based on reported peak resolution) was demonstrated [14]. Based on this resolving power, diazepam and bromazepam could not be distinguished and second derivative techniques were employed to identify the two ions. In the current study, the average resolving power (based on a pulse width of 0.2 ms) obtained was 90 (45 000 theoretical plates). The resolving power has been more than doubled from the previous work. Based on this average resolving power, the protonated ions of bromazepam and diazepam were separated with a resolution of 1.0, adequate for separation as described in chromatographic terms. The protonated ion of diazepam and the dehydrated ion of lorazepam had a Ko value difference of 0.005 cm2 V−1 s−1 . Based on this Ko difference (and the calculated α value), a resolving power of 570 (1.8 million plates) would be required to baseline resolve these two ions (resolution of 1.5).
4. Conclusions High-resolution IMS–MS coupled with ESI has been shown to be a potential technique to separate and detect benzodiazepines in liquid samples. For the benzodiazepines that contain hydroxyl moieties
in the molecular structure, two predominant ions are observed; both the protonated molecular ion and dehydration product. However, even with the formation of two ions, the technique is sensitive for all five benzodiazepines. In addition, the two-dimensional separation of a mixture of the five benzodiazepines showed that all five could be easily separated, demonstrating the potential of IMS–MS for high throughput drug analysis.
Acknowledgements This work was supported by the National Institutes of Health (Grant 8RO3DA1192302) and the National Science Foundation (Grant CHE9870850). The authors would like to thank the National Institutes of Drug Abuse for the scholarship funding for Laura M. Matz. References [1] O.H. Drummer, J. Chromatogr. B 713 (1998) 201. [2] V. Cirimele, P. Kintz, B. Ludes, J. Chromatogr. B 700 (1997) 119. [3] S.J. McClean, E.J. O’Kane, W.J. Smyth, Electrophoresis 21 (2000) 1381. [4] G.A. Eiceman, Z. Karpas, Ion Mobility Spectrometry, CRC Press, Boca Raton, FL, 1994. [5] J.L. Brokenshire, in: Proceedings of the FACSS Meeting, Anaheim, CA, USA, October 1991. [6] J.W. Leonhardt, W. Rohrbeck, H. Bensch, in: Proceedings of the Fourth International IMS Workshop, Cambridge, UK, 1995. [7] P.H. Dugourd, R.R. Hudgins, D.E. Clemmer, M.F. Jarrold, Rev. Sci. Instrum. 68 (1997) 1122. [8] C. Wu, W.F. Siems, G.R. Asbury, H.H. Hill Jr., Anal. Chem. 70 (1998) 4929. [9] C.A. Srebalus, J. Li, W.S. Marshall, D.E. Clemmer, Anal. Chem. 71 (1999) 3918. [10] G.R. Asbury, H.H. Hill Jr., J. Microcol. Sep. 12 (2000) 172. [11] G.R. Asbury, C. Wu, W.F. Siems, H.H. Hill Jr., Anal. Chim. Acta 404 (2000) 273. [12] G.R. Asbury, J. Klasmeier, H.H. Hill Jr., Talanta 50 (2000) 1291. [13] C. Wu, W.F. Siems, H.H. Hill Jr., Anal. Chem. 72 (2000) 396. [14] A.H. Lawrence, Anal. Chem. 61 (1989) 343. [15] D.P. Wittmer, Y.H. Chen, B.K. Luckenbill, H.H. Hill Jr., Anal. Chem. 66 (1994) 2348. [16] P.S. Tornatore, Design enhancements for high-flow, high-resolution stand alone ion mobility spectrometry, Masters Thesis, Washington State University, May 2000.
L.M. Matz, H.H. Hill Jr. / Analytica Chimica Acta 457 (2002) 235–245 [17] H.H. Hill Jr., W.F. Siems, R.H. St. Louis, D.G. McMinn, Anal. Chem. 62 (1990) 1201A. [18] Y.H. Chen, H.H. Hill Jr., D.P. Wittmer, Int. J. Mass Spectrom. Ion Proc. 154 (1996) 1. [19] C.B. Shumate, H.H. Hill Jr., Anal. Chem. 61 (1989) 601.
245
[20] W.E. Steiner, B.H. Clowers, K. Fuhrer, M. Gonin, L.M. Matz, W.F. Siems, A.J. Schultz, H.H. Hill Jr., Rapid Commun. Mass Spectrom. 15 (2001) 2221–2226. [21] W.F. Siems, C. Wu, E.E. Tarver, H.H. Hill Jr., P.R. Larsen, D.G. McMinn, Anal. Chem. 66 (1994) 4195.
Separation of benzodiazepines by electrospray ionization ion mobility spectrometry–mass spectrometry Laura M. Matz, Herbert H. Hill Jr.∗ Chemistry Department, Washington State University, P.O. Box 644630, Pullman, WA 99164-4630, USA Received 2 July 2001; received in revised form 11 December 2001; accepted 2 January 2002
Abstract Benzodiazepines are a commonly abused class of drugs; requiring analytical techniques that can separate and detect the drugs in a rapid time period. In this paper, the two-dimensional separation of five benzodiazepines was shown by electrospray ionization (ESI) ion mobility spectrometry (IMS)–mass spectrometry (MS). In this study, both the two dimensions of separation (m/z and mobility) and the high resolution of our IMS instrument enabled confident identification of each of the five benzodiazepines studied. This was a significant improvement over previous IMS studies that could not separate many of the analytes due to low instrumental resolution. The benzodiazepines that contain a hydroxyl group in their molecular structure (lorazepam and oxazepam) were found to form both the protonated molecular ion and dehydration product as predominant ions. Experiments to isolate the parametric reasons for the dehydration ion formation showed that it was not the result of corona discharge processes or the potential applied to the needle. However, the potential difference between the needle and first drift ring did influence both the relative intensity ratios of the two ions and the ion sensitivity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Electrospray; Ion mobility spectrometry; Benzodiazepines
1. Introduction Benzodiazepines are a widely abused class of drugs used as minor tranquilizers, hypnotics, and muscle relaxants [1]. Because benzodiazepines are often seen in forensic and clinical cases, many of these compounds are included in routine drug tests. The increasing number of these samples has created a need for higher sample throughput, requiring faster sample preparation and analysis. Currently, gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) are the most com∗ Corresponding author. Tel.: +1-509-335-5648; fax: +1-509-335-8867. E-mail address: [email protected] (H.H. Hill Jr.).
mon methods employed for benzodiazepine detection [1–3]. Also, thin layer chromatography and micellar electrokinetic capillary chromatography (MECC) are employed [1]. However, chromatographic separations are typically lengthy and require extensive sample preparation prior to analysis. In this paper we present the direct and rapid separation of benzodiazepines by electrospray ionization (ESI) ion mobility spectrometry (IMS)–mass spectrometry (MS). Until recently, employing IMS–MS for complex mixture analysis was limited due to the low instrumental resolution [4]. In the past several years, IMS instruments have evolved as higher resolution instruments [5–10], obtaining resolution values exceeding LC and GC and approaching capillary electrophoresis values [10]. With these new instruments,
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the separation of several complex mixtures have been shown including chemical warfare agents [11], explosives [12], and drugs [13]. Previously, Lawrence evaluated the separation of some benzodiazepines by IMS using a radioactive 63 Ni foil as the ionization source [14]. However, the low resolution of the IMS used in these studies led to incomplete resolution of several peaks. Although, there were some problems with the separation at the time, the work of Lawrence showed that IMS could be a rapid way (separation times in milliseconds to seconds) to detect and perhaps, separate benzodiazepines [14]. In this study, we evaluate the separation of benzodiazepines by high-resolution ESI-IMS–MS. ESI may be an ideal ionization source as a precursor to analysis of benzodiazepines in biological samples. However, ESI and 63 Ni foil are very different ionization sources and a part of this study was to evaluate any similarities and differences between the two techniques (comparison between the study published by Lawrence [14]). Furthermore, the advantage of high resolution separations was examined. The goal of this study was two-fold: (1) to investigate the differences between ions formed in 63 Ni, ESI-IMS and ESI-MS; and (2) to evaluate ESI-IMS–MS as an analytical technique for benzodiazepine separation and detection.
2. Experimental 2.1. Reagents and chemicals All solvents used in the study were HPLC grade (methanol, water, acetic acid) and were obtained from J.T. Baker (Phillipsburgh, NJ, USA). All five benzodiazepines were obtained from Radian International (Austin, TX, USA) as 1 mg/ml standards in methanol. All solutions were diluted with the electrospray solvent (47.5% water/47.5% methanol with 5% acetic acid). 2.2. IMS instrumentation Two ESI sources were employed in this study: the water-cooled ESI source described previously [15] and a nitrogen gas cooled ESI source [16]. The water-cooled ESI source was employed for all experiments except where specified. Also, both ESI needles
were maintained at 12.5 kV, except when indicated in the figures. The ESI solution was composed of 47.5% water/47.5% methanol with 5% acetic acid and was pumped by a Brownlee Labs (Santa Clara, CA, USA) dual syringe-pump at a flow rate of 5 l/min. For all studies, a high-resolution IMS–MS instrument was employed. This instrument was built at Washington State University (Pullman, WA, USA) and has been previously described [8] (modifications to original design are described in [10]). A voltage of 9.5 kV was applied to the drift tube (7.9 kV at the ion gate). The drift gas (nitrogen) flow rate was operated at 1 l/min for all experiments. Both the desolvation (7.5 cm) and drift region (22.5 cm) of the IMS tube were maintained at 250 ◦ C. Atmospheric pressure in Pullman, WA, USA is typically between 690 and 700 Torr. The IMS was a stacked ring design with alternating alumina and stainless steel rings. The MS instrument was a 150-QC quadrupole (ABB Extrel, Pittsburgh, PA, USA) with a mass range of 0–4000 u. The lenses between the IMS and the quadrupole were as follows: +8.0 V (40 m pinhole), −42.8, −116.3, −34.5, −17.3, and −50.9 V. The first MS chamber was maintained at a pressure of 2.0 × 10−4 Torr and the second chamber was maintained at a pressure of 2.0 × 10−5 Torr. The MS and IMS data acquisition systems have been improved from previous studies [8]. First, the MS software (Merlin, ABB Extrel) enabled the quadrupole to be slowly scanned (i.e. 1 u/50 s) over a specified m/z range. The IMS gate control and data acquisition used the Labview software (National Instruments, Houston, TX, USA) which was modified so that each ion mobility spectra was sequentially saved and spectra could be obtained for up to one hour. Similar to the previous IMS software [15], the scan time, gate open pulse time (pulse width), and number of averaged spectra could be varied. Due to the additional data processing incurred by the Labview software, there was a 40% time overhead added to the time required for acquisition of each IMS spectra. The IMS controlling electronics were the same as described in previous studies [15] (amplifier and gating electronics). The instrument was operated in one of three ways (specified for each figure), non-selective ion monitoring (NSIM), selective ion monitoring (SIM), and two-dimensional IMS–MS spectral acquisition (2D-IMS–MS). For all three cases, the ion mobility
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instrument was continually gated to obtain mobilities for the ESI ions. The difference in the three operational modes was the operation of the QMS. In NSIM, the QMS direct current (dc) voltage was turned off and all ions above a specified mass were transferred through the MS. The NSIM mode is similar to a total ion chromatogram in chromatography–mass spectrometry. In SIM, one specific m/z value was transmitted through the MS and detected. In the third mode of operation (2D-IMS–MS), the quadrupole was scanned so ions in a 1 u m/z window were transmitted per ion mobility spectrum. For example, if the desired mass range was 130–150 u and each completely averaged IMS spectra took 30 s, then the quadrupole would initially transmit the m/z value 130 u and increase this value by 1 u every 30 s (i.e. continual SIM IMS spectra with increasing m/z). In this way, both the mass and ion mobility information were obtained simultaneously. 2.3. Calculations The reduced mobility values reported were calculated based on the following equation [17]: 2 273 L P Ko = (1) Vtd T 760 where L is the drift region length (22.5 cm), V the drift voltage (7900 V), td the ion drift time, T the effective temperature in the drift region (523 K), and P is the pressure in the drift region (∼690–700 Torr in Pullman, WA, USA). Detection limits were de-
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fined to be the concentrations or quantities providing a signal-to-noise ratio of 3. 3. Results and discussion 3.1. ESI-IMS spectra of benzodiazepines The structures of the five benzodiazepines are shown in Fig. 1. All five compounds contain nitrogen atoms in their structure and oxazepam and lorazepam contain a hydroxyl moiety as well. In Table 1, the Ko values, mass identities and literature Ko values are listed. Initial inspection of the table showed that both lorazepam and oxazepam formed two predominant ions; a loss of water (due to the hydroxyl moiety) and the protonated molecular ion. Since an ion’s drift time is inversely related to the Ko values (see Eq. (1)), slower drifting ions have smaller Ko values. The literature values reported in Table 1 were obtained with air as the drift gas. Therefore, the Ko values (drift gas, nitrogen) reported in this study will not be the same, but the trends were similar. Furthermore due to the similar size and polarizability of nitrogen and air, Ko values will be similar in values as seen in Table 1, where the values differ by 7–8%. For both compounds that produced two ions (lorazepam and oxazepam), the (M–H2 O)H+ drifted faster than the MH+ which was expected due to the decreasing mass. Comparison of both the literature and reported Ko values showed that the largest (fastest drifting ion) Ko value was one of the higher mass ions
Table 1 List of benzodiazepine ions, Ko values, and literature Ko values Compound
MW
Ko (cm2 V−1 s−1 )
Ion formula
Ion mass (u)
Ko (cm2 V−1 s−1 ) [14]
Diazepam
284.8
1.123
MH+
286, 288
1.21
Detection limits (ppba , pmol) 52.8, 0.463
Oxazepam
286.7
1.138 1.184
MH+
(M–H2 O)H+
288, 290 270, 272
1.23 1.28
162, 1.41
Chlordiazepoxide
299.7
1.091
MH+
300, 302
1.18
253, 2.11
Lorazepam
321
1.085 1.118
MH+
(M–H2 O)H+
322, 324 304, 306
1.19 1.22
305, 2.38
Bromazepam
316
1.144
MH+
317, 319
1.24
200, 1.58
Literature Ko values were obtained with air as the drift gas and were obtained with 63 Ni foil as the ionization source [14]. Therefore, the mass identities of the literature values differed slightly from the ions observed in this study. a Detection limits in picomoles were defined based on 30 s acquisition time and 5 l/min sample flow rate.
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Fig. 1. Structure of five benzodiazepines that were electrosprayed, separated and detected by ESI-IMS–MS.
(the protonated ion of bromazepam). Although, the ion’s mobility is a function of mass, it is also a function of its size and interaction with the drift gas. These results are similar to the previous study by Lawrence (63 Ni ionization sources) which showed there was not a correlation between the ion mobility values and ion mass [14]. Inspection of Table 1 showed that the general trends for Ko values were similar for both the literature values and the measured values. In Fig. 2, the non-selective IMS–MS spectra for the five benzodiazepines are shown. As discussed in Table 1, the compounds without the hydroxyl moiety produced one predominant ion (MH+ , labeled 1) and both compounds with the hydroxyl moiety produced two ions (1 and (M–H2 O)H+ , labeled 2). As expected due to the nitrogen atoms in the molecular structures, all five compounds produced strong signals at the
20 ppm concentration range. For the five analytes, minor mobility peaks were observed and mass identified as sodium and potassium adducts. The smaller side peaks following the protonated ions in Fig. 2 correlated with these two ions. 3.2. Investigation of dehydration products Since ESI is a soft ionization process, dehydration ions observed in the IMS spectra were not expected. In MS studies employing ESI as the ionization source, the protonated ions were the only ions observed [3] and multiple stages of MS were employed to invoke fragmentation. In the second MS stage, dehydration ions were observed similar to the dehydration ions discussed above. Sometimes corona discharge ionization (a gas-phase ionization process) can occur
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Fig. 2. Non-selective ESI-IMS–MS spectra of five benzodiazepines: (1) MH+ and (2) (M–H2 O)H+ . Graph shows that benzodiazepines which contained a hydroxyl group also formed both a protonated and dehydration ion. Spectra were the result of 1000 averages. All mobility peaks were also mass identified and each analyte was present at a concentration of 20 ppm.
simultaneously with ESI conditions unless specific measures are taken to eliminate these processes. Chen et al. found that the water-cooled ESI source required a piece of plastic tubing surrounding the metal needle to eliminate any corona discharge [18]. The metal needle used in this study was insulated with a piece of polyethylene tubing, although it melted after an hour of operation. To insure that the insulating tube was positioned properly, experiments were performed to isolate any corona discharge processes occurring. A gas-phase corona discharge ionization process may have been the reason for the appearance of the dehydration ions. In Fig. 3, the NSIM IMS spectrum of oxazepam is shown for both the water-cooled ESI source with metal needle (a) and a nitrogen cooled ESI source (b) with a fused-silica needle. Since corona
discharge is a gas-phase process occurring due to the breakdown of gas molecules surrounding the needle [19], employing a fused silica needle would minimize corona discharge. If the dehydration ions were due to corona discharge, the dehydration ions should not be seen with the fused-silica needle. However, as seen in Fig. 3, both ions were found in the IMS spectra. The fused silica needle actually produced more dehydration ion than the water-cooled needle. Therefore, based on these studies, it was concluded that corona discharge processes were not causing the dehydration ions. In ESI, a potential is applied between the needle and a counterelectrode (for IMS, this was the first ring in the desolvation region). The potential difference between the needle and the ring was varied in order to
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Fig. 3. Comparison of oxazepam IMS spectra with (a) water-cooled ESI needle and (b) nitrogen-cooled ESI spectra. Graph shows that dehydration ion was formed regardless of ESI needle.
determine its effect on ion signal intensity and on the ion identity. Since altering the drift region voltage would affect the electric field and the ion transmission efficiency, the voltage on the ESI needle was varied and the drift region voltage (8.0 kV) was held constant. In Fig. 4, the signal intensity of three ions for oxazepam; MH+ , (M–H2 O)H+ , and (M + Na)+ were evaluated as a function of potential difference between the needle and first drift ring. In Fig. 4a, the ion intensity for the protonated molecular ion of oxazepam was plotted as a function of potential difference. The graph shows that the highest signal intensity was obtained for a difference of 3.5–4.0 kV and was significantly lower above and below these potentials. This is consistent with the range of potentials observed for ESI conditions reported previously [19].
Typically in ESI-MS, the ESI voltage at the ESI-MS interface can be increased to cause collision induced dissociation (CID) for fragmentation of the parent ion. However, this is not true for ESI-IMS since the instrument is operated at atmospheric pressures and the voltages utilized are not sufficient to cause CID. Manipulation of the lens voltages at the IMS–MS interface could be utilized to invoke fragmentation [20], but the daughter and parent ions would then have the same mobility values. As previously shown in Fig. 2, the protonated and dehydration ions have different drift times, indicating that the ions were formed prior to the mobility separation. A more plausible solution is that the ions form the dehydration ions due to stability of the structures at the elevated temperatures, similar to the reasons for
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Fig. 4. Evaluation of ESI potential on (a) analyte sensitivity and (b) ion fragmentation ratios. Intensity values were obtained for oxazepam. Graph shows that the potential difference between the needle and drift ring alters both the sensitivity and fragmentation.
formation of multiple ions with the radioactive ionization source. One interesting feature was that the highest MH+ : (M–H2 O)H+ ratios correlated with the highest signal intensities found in the upper graph. As the potential
difference was increased past 4 kV, the MH+ : (M + Na)+ was greater than for the dehydration ratio. The results of these experiments showed that the potential difference had little influence on minimizing the dehydration product and was highly correlated with the
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Fig. 5. Reproducibility of ion ratios for oxazepam at three different concentrations: (a) 1 ppm; (b) 10 ppm; and (c) 100 ppm.
potential difference that provided the greatest oxazepam intensity. In Fig. 5, the non-selective ESI-IMS spectrum of oxazepam was shown at three concentrations: (a) 1 ppm; (b) 10 ppm; and (c) 100 ppm. Based on inspection of the relative signal intensities in the graph, it was clear that the ratios were not consistent. In Fig. 5b, the MH+ ion intensity was approximately 50% of the dehydration product. In contrast, the 100 ppm (Fig. 5c) spectra showed that the protonated molecular ion was 75% of the dehydration product. It was clear from these studies that there needs to be a greater understanding of the ion formation process in order to quantify the ions observed for both lorazepam and oxazepam. Mass-selected mobility spectra for the protonated ion of each benzodiazepine were attained and the detection limits were estimated. The detection limits are
presented in Table 1 both in concentration units of ppb and in the quantity injected (picomoles). Although typical concentrations in biological samples could be present at low ppb, an order of magnitude below the detection limits, the minimal quantity required for a complete analysis would enable pre-concentration steps to provide adequate concentrations for IMS–MS analysis. Furthermore, it is expected upon optimization of the instrumental parameters, the detection limits could be further reduced. 3.3. Two-dimensional separation of five benzodiazepines In Fig. 6, a two-dimensional contour plot of m/z values and drift times was plotted for a mixture of the five benzodiazepines. As seen in Table 1, diazepam
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Fig. 6. Two-dimensional ESI-IMS–MS spectra of five benzodiazepines. Graph shows that six benzodiazepines were separated and identified due to the two dimensions of separation (ion mobility and m/z value).
and oxazepam only differ by one mass unit and due to the isotope patterns of Cl atoms, the mass spectra would be expected to overlap significantly. However, the contour plot in Fig. 6 shows that the two analytes can be easily identified due to the differences in mobilities (seen on the graph as drift times). Furthermore, Fig. 6 showed that all five analytes could be easily distinguished by evaluation of the m/z drift time values. In the previous work performed by Lawrence [14], the separation of diazepam and bromazepam (difference in Ko values was 0.3 cm2 V−1 s−1 ) was not achieved with conventional spectral techniques. However, inspection of Fig. 6 shows that bromazepam and diazepam do not overlap in drift times and would be easily separated by IMS alone. This example shows the improvements in instrumental resolution that have been made in the past 10 years. In IMS, separation efficiencies are typically reported as resolving power and are calculated based on the following equation: td Rp = (2) wh where Rp is resolving power, td the drift time, and wh is the ion pulse duration measured at half of the
maximal intensity [21]. Typical Rp values are around 30 for commercial instruments and up to 150 (for singly charged ions) for high-resolution instruments [8]. In order to compare the separation efficiencies between other separation techniques and ion mobility, the experimentally determined resolving power (Eq. (2)) can be converted to theoretical plates (N) by the following equation: N = 5.55(Rp )2
(3)
Recently, our laboratory developed the equations to describe the separation efficiency in terms of theoretical plates and resolution similar to the equations predominantly used in chromatography [10]. Eq. (4) shows the fundamental resolution equation derived for IMS: √ N α−1 R= (4) 4 α where R is resolution and α is the selectivity factor defined as Ko1 /Ko2 . By determining the Ko values for two compounds and the number theoretical plates achievable with the instrument, an estimation of the resolution between two ions can be attained. When discussing the separation efficiencies and resolution
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of two compounds, only the mobility separation was evaluated (separation along the y-axis in Fig. 6). In ion mobility, the resolving power can be improved by increasing the voltage, decreasing the temperature, decreasing the initial width of the ion injection (labeled pulse width) by decreasing the time the electronic gate is opened and improving the electric field homogeneity [10]. In the early reports by Lawrence, some of these variables were discussed (pulse width and voltage) as ways to improve the resolving power [14]. However, it was noted that decreasing the pulse width comes with a subsequent decrease in sensitivity which is typically unacceptable. Recently our lab reported substantial improvements in resolving power due to increases in the voltage with an accompanied increase in the drift tube length, and improvements in the drift tube design. With these design enhancements, resolving powers now reach 80–125 on a regular basis (with a 0.2 ms pulse width) and resolving power values up to 220 have been noted for multiply charged ions [10]. In the work performed by Lawrence, an instrumental resolving power of 39 (calculated based on reported peak resolution) was demonstrated [14]. Based on this resolving power, diazepam and bromazepam could not be distinguished and second derivative techniques were employed to identify the two ions. In the current study, the average resolving power (based on a pulse width of 0.2 ms) obtained was 90 (45 000 theoretical plates). The resolving power has been more than doubled from the previous work. Based on this average resolving power, the protonated ions of bromazepam and diazepam were separated with a resolution of 1.0, adequate for separation as described in chromatographic terms. The protonated ion of diazepam and the dehydrated ion of lorazepam had a Ko value difference of 0.005 cm2 V−1 s−1 . Based on this Ko difference (and the calculated α value), a resolving power of 570 (1.8 million plates) would be required to baseline resolve these two ions (resolution of 1.5).
4. Conclusions High-resolution IMS–MS coupled with ESI has been shown to be a potential technique to separate and detect benzodiazepines in liquid samples. For the benzodiazepines that contain hydroxyl moieties
in the molecular structure, two predominant ions are observed; both the protonated molecular ion and dehydration product. However, even with the formation of two ions, the technique is sensitive for all five benzodiazepines. In addition, the two-dimensional separation of a mixture of the five benzodiazepines showed that all five could be easily separated, demonstrating the potential of IMS–MS for high throughput drug analysis.
Acknowledgements This work was supported by the National Institutes of Health (Grant 8RO3DA1192302) and the National Science Foundation (Grant CHE9870850). The authors would like to thank the National Institutes of Drug Abuse for the scholarship funding for Laura M. Matz. References [1] O.H. Drummer, J. Chromatogr. B 713 (1998) 201. [2] V. Cirimele, P. Kintz, B. Ludes, J. Chromatogr. B 700 (1997) 119. [3] S.J. McClean, E.J. O’Kane, W.J. Smyth, Electrophoresis 21 (2000) 1381. [4] G.A. Eiceman, Z. Karpas, Ion Mobility Spectrometry, CRC Press, Boca Raton, FL, 1994. [5] J.L. Brokenshire, in: Proceedings of the FACSS Meeting, Anaheim, CA, USA, October 1991. [6] J.W. Leonhardt, W. Rohrbeck, H. Bensch, in: Proceedings of the Fourth International IMS Workshop, Cambridge, UK, 1995. [7] P.H. Dugourd, R.R. Hudgins, D.E. Clemmer, M.F. Jarrold, Rev. Sci. Instrum. 68 (1997) 1122. [8] C. Wu, W.F. Siems, G.R. Asbury, H.H. Hill Jr., Anal. Chem. 70 (1998) 4929. [9] C.A. Srebalus, J. Li, W.S. Marshall, D.E. Clemmer, Anal. Chem. 71 (1999) 3918. [10] G.R. Asbury, H.H. Hill Jr., J. Microcol. Sep. 12 (2000) 172. [11] G.R. Asbury, C. Wu, W.F. Siems, H.H. Hill Jr., Anal. Chim. Acta 404 (2000) 273. [12] G.R. Asbury, J. Klasmeier, H.H. Hill Jr., Talanta 50 (2000) 1291. [13] C. Wu, W.F. Siems, H.H. Hill Jr., Anal. Chem. 72 (2000) 396. [14] A.H. Lawrence, Anal. Chem. 61 (1989) 343. [15] D.P. Wittmer, Y.H. Chen, B.K. Luckenbill, H.H. Hill Jr., Anal. Chem. 66 (1994) 2348. [16] P.S. Tornatore, Design enhancements for high-flow, high-resolution stand alone ion mobility spectrometry, Masters Thesis, Washington State University, May 2000.
L.M. Matz, H.H. Hill Jr. / Analytica Chimica Acta 457 (2002) 235–245 [17] H.H. Hill Jr., W.F. Siems, R.H. St. Louis, D.G. McMinn, Anal. Chem. 62 (1990) 1201A. [18] Y.H. Chen, H.H. Hill Jr., D.P. Wittmer, Int. J. Mass Spectrom. Ion Proc. 154 (1996) 1. [19] C.B. Shumate, H.H. Hill Jr., Anal. Chem. 61 (1989) 601.
245
[20] W.E. Steiner, B.H. Clowers, K. Fuhrer, M. Gonin, L.M. Matz, W.F. Siems, A.J. Schultz, H.H. Hill Jr., Rapid Commun. Mass Spectrom. 15 (2001) 2221–2226. [21] W.F. Siems, C. Wu, E.E. Tarver, H.H. Hill Jr., P.R. Larsen, D.G. McMinn, Anal. Chem. 66 (1994) 4195.