2240
Anal. Chem. 1988, 60, 2240-2243
Comparison of Ion Mobility Constants of Selected Drugs after Capillary Gas Chromatography and Capillary Supercritical Fluid Chromatography R. L. Eatherton,' M. A. Morrissey, and H. H. Hill*
Department of Chemistry, Washington State University, Pullman, Washington 99164-4630
Several test compounds were introduced into a unidirectional flow Ion mobillty spectrometer by both gas chromatography (GC) and supercritical fluld chromatography (SFC). The test samples were drugs from three general classes, opiates, benrodiazoplnones,and steroids. Drm spectra were collected for each compound by Fourier transform ion moblllty spectrometry. The drlft veioclty data for each compound was corrected for temperature and pressure dlfferences by conversion to reduced mobllity values, K,. Agreement of K O values for each method confkmed that the mobility of product ions In the unldirectlonai flow ion mobility spectrometer is independent of the introduction method. I n addition, drift tknes of the reactant ions In the spectrometer were compared for conditions of the presence of a contaminating CO, flow and the absence of any CO, flow. I t was found that there were no differences In the mobliitles of the reactant ions caused by CO, contamination, Indicating that the identities of the reactant ions were not affected by CO, flow.
The ion mobility detector (IMD), first introduced in 1982 (1) and based on principles from ion mobility spectrometry,
has been found to function as a multipurpose, sensitive detection method for capillary gas chromatography (CGC) and capillary supercritical fluid chromatography (CSFC) (2). Ion mobility monitoring as a chromatographic detection method has several clear advantages: (1)Because it is an atmospheric pressure ionization process followed by atmospheric pressure ion separation, no complicated or expensive vacuum equipment is required. (2) Quantitative and semiqualitative data can be obtained from compounds present at trace levels. (3) When operating as a continuous chromatographic detector, tunable selectivity is available throughout a range in which the detector can be essentially specific for a single compound or function as a universal detector with a nonselective response (2). A study of SFC/IMD was conducted to investigate the potential of using CO, as the drift gas rather than the standard drift gas, nitrogen (3). The use of COz as a drift gas would ensure that the composition of the drift gas would remain constant as the flow of CO, into the IMD changes during SFC pressure programming. It was found that ion mobility spectra in CO, produced patterns similar to those found in Nz, but the mobilities of the individual ions were considerably lower than those obtained with nitrogen. With lower mobilities, drift times were longer and peak broadening was greater. The sensitivity of the IMD was significantly reduced due to increased diffusional broadening when COz was used as the ion mobility drift gas. Thus, it seems reasonable that although classical ion mobility spectra can be obtained when CO, is used as the drift gas, for maximum sensitivity nitrogen is far superior to CO,. Author t o whom correspondence should be addressed. Current address: International Clinical Laboratories. S\5' 2203 Airport b'ay S , Seattle, \+'A 98131. a
I
Using nitrogen as a drift gas after CSFC in which COz is used as the mobile phase could present a serious problem. In opposing flow ion mobility spectrometers of the type used for the experiments reported in ref 3, COz flows typical of that which would be introduced from CSFC caused drift times to vary (4). During a pressure programmed SFC separation, the volume flow rate of the COz introduced into the detector changed, causing ion mobilities to vary and making IMD detection of CSFC effluents impossible. These results showed that varying amounts of C 0 2 introduced into a bidirectional flow IMD swept by nitrogen drift gas would compromise the integrity of the mobility data. In the unidirectional flow design neutrals are rapdily swept from the drift region by the drift gas and CO, flow from the chromatograph into the spectrometer should not change the mobilities of the compounds of interest. In this work the ion mobility integrity of a unidirectional flow detector design similar to that described for gas chromatographic detection was investigated. Mobilities of several test compounds were compared after introduction by CGC, where no COz was introduced into the IMD, and after CSFC, where gaseous COz was introduced into the detector at a rate of approximately 2 mL/min. Nitrogen was used as the mobility drift gas.
EXPERIMENTAL SECTION Instrumentation. The CSFC/IMD instrument used in this study was assembled in our laboratory from a variety of offthe-shelf instruments plus several components that were constructed at the Washington State University Technical Services facility. A schematic of the complete instrument similar to that used in this study is shown in ref 2. Primary components of the SFC portion of the instrument were the following: A 24.7-L COz reservoir (Instrument grade Liquid Air Corp, Tacoma, WA), a 250-mL pressure controlled syringe pump (Lee Scientific, Salt Lake City, UT), a 60-nL four-port injector (Valco Instruments. Co., Inc., Houston, TX), a precolumn splitter, and a column oven (HP 5830A GC Avondale, PA). The primary components of the IMD portion of the instrument consisted of an ion drift tube (W.S.U. Technical Services), a scanning square wave generator (W.S.U. Technical Services), control electronics for the entrance and exit gates (W.S.U. technical Services), an electrometer (Keithly 417 Picoammeter, Keithly Instruments, Inc., Cleveland, OH) a fast ADC storage scope (RC Electronics Inc., Santa Barbara, CA), a dot printer (Epson America, Inc., Torrence, CA), and a strip chart recorder (Western Scientific Associates, Danville, CA). The ion drift tube was simply a series of insulated stacked rings that were maintained at decreasing potential such that a constant electric field was established in the tube. The drift tube was filled with nitrogen through which the gas phase ions migrated in the electric field. In this work the ionization source of the ion mobility spectrometer consisted of a 63Niradioactive foil that produced about 1.5 nA of current. Background ions from the source underwent ion/ molecule reactions with organic compounds contained in the column effluent to produce product ions and the chromatographic response. Briefly, there are three methods of operation of an ion mobility instrument by which spectra can be obtained: A signal averaging method, a scanning second gate method, and a Fourier transform
0003-2700/88/0360-2240$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
Table I. Comparative Operating Conditions CGC
CSFC
column inside diameter, mm length, m stationary phase coating thickness, pm mobile phase column temperature, O C injection temperature, "C transfer line temp, O C column pressure, atm
fused silica 0.25 30 DB-5 0.25 He 250-280 300 300 1
fused silica 0.1 16 SPB-1 0.25 COZ 100 25 150 200-300
IMD pressure, Torr IMD temperature, O C IMD electric field, V/cm drift length, cm drift gas drift gas flow rate, mL/min
699 300 240 7.2
703 150 240 7.2
NZ
NZ
600
600
method. The Fourier transform mode of operation was used for the spectra reported in this paper. In this mode of operation both the entrance and exit gates were operated simultaneously. The frequency at which they were opened and closed was scanned from 20 to 5020 Hz. As ions of differing mobilities came in and out of phase with the scanning gate frequencies, a mobility interferogram was created. Time domain spectra were reclaimed through a fast Fourier transform operation. For the CGC/IMD portion of this work, the IMD instrument was identical with the instrument described above. The only difference was insertion of the capillary gas chromatographic column into the ionization source rather than the restrictor of the SFC system. The capillary gas chromatograph used for this work was a Model 560 Tracor chromatograph equipped with a split/splitless capillary column injector (Tracor Instruments, Austin, TX). Operating Conditions. In general, the IMD, the CSFC, and the CGC were operated under typical conditions. Table I provides a summary of the operating parameters used in this work. With the exception of temperature, conditions for the IMD were the same for both SFC and GC. The temperature of the ion mobility spectrometer when used with CGC sample introduction was 250 OC, while when the spectrometer was interfaced to SFC, the drift tube temperature was only 150 OC. The gas chromatographiccolumn was a DB-5 coated fused silica capillary column 30 m long with a 0.25 mm i.d. (J&W Scientific, Inc., Rancho Cordova, CA). The injector for the CGC was operated in the split mode with a split ratio of 50:l. Column temperatures ranged from 250 to 280 "C, depending on the compounds used. The carrier gas was helium at a flow rate of 0.78 mL/min. The portion of the column that was used as a transfer line from the chromatograph to the detector was maintained at 210 OC. The column used for the CSFC portion of the work was a 16 m x 0.1 mm i.d. fused silica capillary column coated with a cross-linked methyl-silicone stationary phase (SPB-1,Supelco Inc., Bellefonte, PA). Samples were chromatographed isobaricdy and isothermally at 180 atm and 100 OC. This corresponded to a flow rate of roughly 2 mL/min of C02 gas into the detector. Chemicals. Test compounds selected for this study were a series of drugs for which there had been no previous reports of separation by SFC or reported detection by IMS. The nine compounds investigated fell into three groups: steroids, opiates, and benzodiazopinones. The three test steroids chosen were estrone, progesterone, and testosterone with molecular weights ranging from 270 to 314. Codeine, hydromorphone, and morphine made up the opiate group with molecular weight range from 285 to 299. The benzodiazopinone group had a molecular weight range from 281 to 387 and consisted of diazepam, flurazepam, and nitrazepam. These particular drugs were chosen because they could be chromatographed by both gas and supercritical fluid chromatography. Procedures. For the experiments using gas chromatography, two of the compounds were derivatized to increase their vapor pressure sufficiently so they would elute from the column. Trimethylsilyl derivatives of these compounds were made fol-
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lowing standard procedures. The drug to be derivatized was dissolved in 500 pL silylation grade pyridine (Pierce Chemical Co., Rockford, IL). After dissolution, 500 p L of BSA, N,O-bis(trimethylsily1)acetamide (Supelco, Inc., Bellefonte, PA), was added and the mixture heated to 70 OC for 20 min. After silylation, the reagent was evaporated under a nitrogen stream at room temperature. The derivative was redissolved in dichloromethane for gas chromatography. Final concentrations of the test compounds were as follows: Diazepam, 1.6 mg/mL; flurazepam, 0.9 mg/mL; nitrazepam, 2.1 mg/mL; morphine, 2 mg/mL; codeine, 2 mg/mL; testosterone, 1.6 mg/mL; progesterone, 1.0 mg/mL; estrone, 1.0 mg/mL. For the GC experiments 1 pL of the test solution was injected into the GC followed by a 501 vapor split. For SFC the test compounds,except cholesterol,were prepared as 10 mg/mL solutions in methanol. Cholesterol was prepared as a 10 mg/mL solution in toluene. Sixty nanoliters of the solutions was injected into the column via a four-port valve followed by a 5:l supercritical fluid split. A series of 3.63-5 ion mobility scans was obtained for each compound as it eluted from the column. The resulting ion mobility spectra were compared to ensure that the product ion peaks were stable from scan to scan and that the most intense product ion peak was selected for use in comparison between the two chromatographic introduction methods. Calculations. After ion mobility spectra had been obtained and selected for all of the test compounds by using both chromatographic methods, reduced mobility values were calculated for each of the product ion peaks. In ion mobility spectrometry, the separation of ions in the spectrometer is based on differences in the average velocities of the ions. Under low electric field conditions, which are achieved by using the typical operating conditions reported here, the velocity (u) of an ion is proportional to the electric field (E). This proportionality constant (eq 2) is called the ion mobility constant (K)and can be expresed in terms of the ion d r i i length (4,the potential drop across the drift length (V), and the time the ion takes to traverse the drift length (t). K = u/E = d2/Vt Of course, the time it takes an ion to traverse the drift region is also dependent on the temperature, pressure, and composition of the drift gas. If the same ion is used as a probe and we make corrections for temperature and pressure, then any change in mobility constants from one set of experiments to another would indicate a change in composition of the drift gas. The mobility constant can be corrected to standard temperature ( T ) and pressure (P) by using the following equation: KO = K(273/7')(P/760) This corrected value is known as the reduced mobility constant.
RESULTS AND DISCUSSION When 63Niis used as an ionization source for ion mobility spectrometry, a standing current of reactant ions is produced. In earlier work performed with a bidirectional flow ion mobility spectrometer (3),the small quantities of COPintroduced into the spectrometer from the supercritical fluid chromatograph altered the drift times of the reactant ions. Figure 1 shows ion mobility spectra of both positive and negative reactant ions with and without the introduction of C 0 2 from the supercritical chromatograph. The lower two tracings in the figure were obtained when no C02 was entering the spectrometer and are representative of typical reactant ion spectra. Tracing c shows the positive ions while tracing d shows the negative ions. The upper two spectra were obtained when the capillary supercritical fluid chromatograph was interfaced to the spectrometer. Again, the spectrum on the left is the positive ion spectrum and the spectrum on the right is the negative ion spectrum. As is clear from visual comparison of these spectra, the introduction of CSFC quantities of COz did not alter the the reactant ion spectra. Table I1 provides more detailed information about these spectra, listing the reduced mobility constants for both the COz contaminated and noncontaminated situations. The excellent agreement of these values with each other and with
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988 a.
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