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Selective Detection of Chlorine-Containing Compounds by Gas Chromatography/Chemical Reaction Interface Mass Spectrometry Hengchang Song and Fred P. Abramson' Department of Pharmacology, The George Washington University, Washington, D.C. 20037
ChemicalreactionInterfacemass spectrometry (CRIMS) was studled as a gas chromatographic detection technique for c h b r l ~ a BothS~andHBrwwetested ~ c ~ as reactant gases. With SO2, a detectlon limit of 50 pg of dlrzepam and a ilnear r a g . of 4 orders of magnitude were achieved, and the experimentaldata were reproducible. With HBr, the detection limit was 10 ng of diazepam and the linear dynamk range was only 2 orders of magnitude. The pordblo phamacokgicaiappllcatknof CRIMS was s t w udng urine spiked with dlrzepam and several of its metabolites, and the resuits show CRIMS to be a dmpk but potentially powerful method In drug metaboilsm studies.
INTRODUCTION Chlorine-containing compounds are widely used in industrial, agricultural and medical fields. Selective detection of these compounds is of great importance especially in the pharmacological and environmental areas. The electron capture detector' and Hall electrolytic conductivity detector2 are very sensitive to chlorine-containing compounds. Ionspecific electrodes3 and neutron activation4 methods also have been employed to measure the total organic chloride in environmental samples. However, none of these methods has flexibility comparable to a GUMS system. Chemical reaction interface mass spectrometry (CRIMS)S is a technique that can be used both as a selective and a general detector for chromatographic methods. Ita applications to the selective detection of isotopes such as l3C, 14C, l5N, and 2H6-9 and heteroatomslOJ1 in organic compounds have been reported, and the technique has proved to be useful in metabolism studies. Recently, Morre and Moini12reported the selective detection of chlorine- and bromine-containing compounds using CRIMS and found useful applications in environmental studies. In this investigation, we evaluated two selective CRIMS detection strategies for C1-containing (1)Miller, D.;Grimsrud, E. Anal. Chem. 1979,51,851-859. (2)Nulton, C. P.; Haile, C. L.; Redford, D. P. Anal. Chem. 1984,56, 598-599. (3)McCahill,M.P.; Conroy, L. E.; Maier, W. J. Enuiron.Sci. Technol. 1980.14. 201-203. (4)L&de,G.;Gether, J.; Josefsson,B. Bull.Enuiron. Contam. Toxicol. 1976,13,656-661. (5)Chace, D.H.; Abramson, F. P. Anal. Chem. 1989.62, 2724-2730. (6)Markey, S.P.; Abramson, F. P. Anal. Chem. 1982,54,2375-2376. (7)Chace, D.H.; Abramson, F. P. Biomed. Enuiron. Mass Spectrom. 1990,19,117-122. (8)Chace, D. H.; Abramson, F. P. J. Chromatogr. 1990,527,l-10. (9)Chace, D.H.;Abramson, F. P. In Synthesis and Applications of IsotoDicallv Labelled Compounds, 1988; Baillie, T. A., Jones, J.R., Eds.; ElseGer: Amsterdam, 1989;p 253. (10)Abramson,F.P.; Markey, S. P. Biomed.Enuiron. Mass Spectrom. 1986,13,411-415. (11)Moini, M.; Chace, D. H.; Abramson, F. P. J. Am. SOC.Mass Spectrom. 1991,2,250-255. (12)Morre, J. T.;Moini, M. Biol. Mass Spectrom. 1992,21,693-699. ~
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compounds as first reported by Morre and Moini12for their potential applicability to drug metabolism research.
EXPERIMENTAL SECTION Apparatus. The GC/CRIMS system we used was described previously.ll A Varian Model 3300gas chromatograph equipped with a Varian 1075splitless injector was used with a 30-m-long, 0.25-mm-i.d., 0.25-pm f i b thickness DB-5capillarycolumn (J&W Scientific,Rancho Cordova,CA). A microwave-poweredreaction chamber was installed in the GC oven. The device consists of a44n.-long, l/&-o.d., l/&n.-i.d. alumina tube and amicrowave cavity which was powered by a 100-W, 2450-MHz Opthos (Rockville,MD) Model MPG-4 microwave supply. The transfer line from the reaction chamber to the MS ion source was 15 in. of deactivated, uncoated wide-bore (0.53-mm) fused-silica tubing (Hewlett-Packard,Avondale, PA). The reactant gas was introduced into the reaction chamberby a stainless steel Swagelok "T"between the chromatographiccolumn and reaction chamber, and the flow of the reactant gas was adjusted by a GranvillePhillips (Boulder, CO) Series 203 variable leak. The Extrel C50/ 400quadrupole maas spectrometer (Pittsburgh,PA) was operated using a Teknivent (Maryland Heights, MO) Vector/One data acquisition interface, the accompanying software, and an IBM PC-AT computer. The GC carrier gaswas ultrapure grade helium (Air Products, Allentown, PA) with a flow rate of about 1mL/ min. Two kinds of reactant gases, SO2 and HBr (bothMatheson, East Rutherford, NJ) were used in the research. To minimize air trapped in these liquid-filled tanks, the manufacturer had prepurged the SO2 with He and had bled half of the HBr tank out. All the gas lines were preevacuated prior to use to remove air and contaminants. Materials and Methods. Diazepam and desmethyldiazepam were obtained from Hoffman-La Roche Inc. (Nutley, NJ), oxazepamand lorazepamwere obtained from Alltech (Deerfield, IL), and&-DDT (l,l-bis(4chlorophenyl)-2,2,2-trichloroethane) was from Aldrich (Milwaukee,WI). All the chemicalswere used without further purification. Solvents (toluene and methanol) were HPLC grade from Fisher Scientific (Fair Lawn, NJ). For the linearity and dynamic range studies, a series of diazepam solutions from 10pg/pL to lo00 ng/pL with a constant DDT concentration of 7.24 ng/pL were prepared in toluene. A urine sample was prepared for the selectivity study. A 10-mL aliquot of urine which was obtained from a healthy adult human volunteer was spiked with 5.4 pg of diazepam, two diazepam metabolites,8.4pg of desmethyldiazepamand 6.3 pg of oxazepam, and 8.3 pg of lorazepam, another benzodiazepine that contains two chlorine atoms. The sample extraction procedure was reported previously.s A 10-cm-long,3-mm4.d.column was packed with XAD-2resin (Mallinckrodt,St.Louis,MO) and then washed with (1)30 mL of methanol, (2) 30 mL of 0.2 M bicarbonate acid buffer, and (3) 30 mL of distilled water. The spiked urine was added to the column, rinsed with 30 mL of distilled water, and then extracted in the followingorder by (1)20 mL of 3:1 methylene chloride:2-propanol and (2) 20 mL of methanol. The extracts were combined and dried at 50 O C under a stream of nitrogen. The dried sample was derivatized by adding 250 pL of N-methylN-(trimethylsily1)trifluoroacetamide (MSTFA) and 250 pL of dry acetonitrile and then heated at 100"C for 30 min. The same procedure was used to prepare the urine sample for the HBr selectivity study except the concentrations for all the spiked analytes were 5-fold higher than those for the SO2 study. 0 1993 American Chemical Society
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The injector temperature was 250 "C. For the sensitivity and linearity studies, the column temperature was programmed from 80 to 235 "C at a rate of 40 W m i n after a 2-min delay; for urine sample analysis, the column temperature was initially set at 60 OC for 2 min and then programmed to 110 "C at a rate of 30 OC/min and 110-235 "C at a rate of 4 "C/min. All samples were injected in the splitless mode. The mass spectrometerwas set in the selected ion monitoring (SIM)mode. When SO2 was the reactant gas, masses 36 (H35Cl), 44 (Con), and 76 (CSz) were detected. Previous research in this lab showed that CSz, a reduction product of sulfur, appeared only when the concentrationof an analyte was so high that the SOn signal declined significantly, which indicated the oxidative capacity of SO2 flowing into the CRI was overwhelmed. As long as the m/z 76 signal is no bigger than a few hundreds of 1% of the m/z 44 signal, one is assured that the SOz-scavenged CRI chemistry is functioning appropriately. When HBr was the reactant gas, masses 16 (CHI), 28 (CO), and 36 (H35Cl)were monitored.
RESULTS AND DISCUSSION Chemistry. In the reaction chamber, the microwave plasma decomposes all the entering molecules, including the reactant gas and chromatographic column effluents. The decomposed products react rapidly, forming simpler molecules. By using a mass-selective detector to monitor these molecules, we can obtain qualitative and quantitative information about the GC effluents. With SO2 as the reactant gas, H W l was the most abundant of all chlorine-containing products. H W l was the second highest peak showing a relative height of 1:3 to H35Cl, representing the natural abundance of the 37Clisotope. All other chlorine-containing products were less than 1%.This is comparable to Morre and Moini.12 Therefore, rnlz 36 (H35Cl) was chosen as the channel to monitor chlorinecontaining compounds and rnlz 44 (COZ) was used as a general detection channel for organic compounds. Subsequent work on authentic biological samples showedthat very large matrix peaks give small mlz 36 signals. This may be due to H234S. Using the H W l channel at rnlz 38 was completely selective and is recommended for work on very complex problems. During the experiment, the SIM chromatogram of rnlz 36 tailed much more severely than the SIM chromatogram of rnlz 44 (Figure 1). When the partial pressure of SO2 was increased, the tailing of rnlz 36 was reduced and the peak shape improved (Figure 2). In the figure the increase of SO2 is indicated by the MS ion source housing pressure, because the absolute amount of SO2 in the reaction chamber is not determined directly in our system. A possible explanation for the tailing effect is that HC1is a very polar compound and is adsorbed on the wall(@ of the reaction chamber andlor transfer line. The adsorptionldesorption process of HCl produces the peak tail of the m/z 36 SIM chromatogram. However, S02, the reactant gas, can also be adsorbed, and increasing SO2 competes a t the adsorption sites with HC1, thus reducingthe tailing of HC1and improvingthe peak shape. Morre and Moini also found improved peak shape with higher reactant gas pressure in their system.'2 A sensitivity decrease (ca. 40%) was observed at the highest SO2 pressure, presumably resulting from a change in ion source efficiency. A key reason why HBr was used as a reactant gas for C1 detection was that we expected a product of Br-Cl to be produced. This product is formed from Br-containing compounds when HCl is the reactant gas (unpublished observations). The Br-Cl molecule is much less polar than HC1; thus the peak tailing of C1-containing compounds could be reduced. Contrary to our expectation, the experimental results indicated that the amount of Br-Cl produced in the reaction chamber was too small to be detected. Even with an overload sample injection, the m/z 116 (79Br-37C1and 81Br-
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Figure 1. Ion current chromatograms of COz(mlz44)and H3%I (m/z 36) for DDT. The Ion source houslng pressure was 2 X Torr. Intensity
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35Cl) signal was difficult to detect. Among the chlorinecontaining products from the reaction, HCl (mlz 36,38) was the most abun&mt, and no other chlorine-containingproduct ever produced a signal that was greater than 10% of the H35Cl
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1093 Intensity
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(mlz36) peak, except HW1. Therefore, HClagain was chosen as the channel to monitor C1-containing compounds. rnlz 28, presumably CO, was the most sensitive channel for carbon and was chosen to generally detect all organic compounds.13 A similar tailing effect of the HC1 product peak was observed. When the amount of HBr reactant gas was increased, the tailing effect was decreased. However, the pressure of HBr in the reaction chamber was so high that it was difficult to ignite the plasma, or when ignited, the plasma was unstable. A deposit of carbon on the wall of the reaction chamber was observed. Later experiments showed that as the carbon deposit became heavier, the experimental results were less reproducible. Sensitivity and Dynamic Range. Diazepam was chosen as the compound to study the detection sensitivity and dynamic range for C1-containing compounds. A number of diazepam solutions were prepared with p,p'-DDT as the reference (7.24 pg1mL). With SOz as the reactant gas, the rnlz 36 SIM showed excellent reproducibility and a broad linear dynamic range. Figure 3 indicates that the linear dynamic range was at least 4 orders of magnitude. In order to study reproducibility, the dynamic range experiments were repeated on three different days, and the results were in good agreement with each other. A linear regression analysis showed a correlation coefficient (R2)of 0.9945. With an integration time of 300 ms/mass, the detection limit for diazepam was 50 pg with a signal to noise ratio >3, which translates into a chlorinedetection limit below 1 pgls. This level of performance is 1 or more orders of magnitude better than recently published work using microwave-induced plasma (MIP) with optical detection methods14-16 and comparableto MIP with mass spectrometric detection.17 When HBr was used as the reactant gas, the linear dynamic range and sensitivity were much worse than with SOZ. With mlz 36 as the monitoring channel, the linear dynamic range was only about 2 orders of magnitude, and the relative peak areas of diazepam and DDT were less reproducible. At the high concentration end, the peak shape of diazepam was significantly deformed, although the integration of the peak areas was still proportional to the concentration. The correlation coefficient (R2) was only0.8845. For an integration time of 300 ms/mass, the sensitivity limit was 10 ng of diazepam with a signal to noise ratio greater than 3. One important factor that causes the poor sensitivity of HBr as (13)Heppner, R. A. Anal. Chem. 1983,55,2170-2174. (14)Hooker, D. B.; Dezwaan, J. J. Pharrn. Biomed. Anal. 1989,7 , 1591-1597. (15)Quimby, B.D.;Sullivan, J. J. Anal. Chern. 1990,62,1027-1034. (16)Abdillahi, M.M.J. Chrornatogr. Sci. 1990,28,613-616. (17)Creed, J.T.;Davidson, T. M.; Shen, W.; Caruso, J. A. J.Anal. At. Spectrorn. 1990,5, 109-113.
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Figure 4. Chromatograms of urlne spiked with four benzodiazeplnes usingSO2as the reactant gas. mlz36 Isthe selective chkrkre detection channel; mlz 44 Is the carbon detectlon channel. The basellne for mlr 36 was orlglnallyat 1300 In Intensity. These benzodiazepines are (a)desmethyldlazepam, (b)oxazepam, (c)dkepam, and (d) lorazepam.
a reactant gas is the high background of HC1, as there was always a trace amount of HC1 in the HBr gas. Selectivity and Applications to Drug Metabolism Studies. In order to determine the selectivity of rnlz 36 to chlorine-containing compounds and to explore the possible application of this method to drug metabolism studies, we prepared a urine sample which was spiked with four chlorinecontaining drugs: diazepam,desmethyldiazepam,oxazepam, and lorazepam. For the SO2 experiments the concentrations of these drugs in urine were 540, 840,630, and 830 ngImL, respectively. After extraction and derivatization, 1pL of the sample was injected into the gas chromatograph in the splitless mode. Figure 4 is the chromatogram of the sample. The result indicated that mlz 36 is an excellent channel for chlorinecontaining compounds. While the carbon channel (mlz 44) showed a complex chromatogram,the selective detector (mlz 36) responded only to a few minor peaks, four of which were the spiked chlorine-containing drugs. Except for a peak of unknown origin eluting between 11 and 12 minutes, this detection was completely selective for the drug molecules. One general assumption of CRIMS is that once a molecule enters the reaction chamber, it is decomposed into atoms. Since the reactant gas is in great excess, the atoms of the sample are quantitatively converted into certain molecules according to the reactant gas type; thus the physicochemical characteristics of the sample molecule such as size and structure have no effect on the detection response, only the elemental compositionand the concentration matter. In our experiment, the carbon to chlorine ratios of the drugs were correctly reflected by the relative integration areas of rnlz 44 to mlz 36. When diazepam was used as the standard (=16.0), the observed carbon to chlorine ratios for derivatized desmethyldiazepam, oxazepam, and lorazepam were 18.5 (RSD 7 % , n = 31, 21.8 (RS 2 % , n = 31, and 10.4 (RS5%, n = 31,
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while the theoretical data are 18, 21, and 10.5, respectively. These experimental results agree well with structure, in particular the presence of two chlorines in lorazepam. This result clearly shows that the responses of the selective C1 and C channels are only proportional to the amount of the selected element or isotope in the sample, regardless of its molecular structure. In other words, CRIMS as a detection technique is not only selective but also structure-independent. Structure independence is one important advantage of CRIMS. For other chromatographic detection methods, quantitation of different compounds always requires different calibration curves, since each structure makes the response of detection different. This is a particular difficulty when analytical standards are not available, as in the case of new drug metabolites. However, with CRIMS as the detection method, such standards might not be necessary. If the C1 response of one compound is obtained, the concentrations of other compounds can be calculated, provided C1 is not removed from the structure by metabolism, the extraction efficiencies are comparable, and the adsorption of sample in the injector and column is neglectable. The same experiment was done with HBr as the reactant gas except with 5-fold higher concentrations of spiked drugs. The result indicatesthat mlz 36 was also completely selective; however, the baseline of this channel was not very stable, and the quantitation of chlorine-containing compounds was difficult. Comparison of 902 and HBr as the Reactant Gases. Clearly, SO2 has major advantages over HBr as a reactant gas. When SO2 was used, the producta in the reaction chamber were gases. In contrast, a carbon deposit was observed on the wall of the reaction chamber when HBr was used. This was the first time a carbon deposit was observed in 10 years of CRIMS development and indicates that HBr is not an effective reactant gas. The linear dynamic range for C1 detection with SO2 was at least 4 orders of magnitude, while it was only 2 orders of magnitude with HBr. The sensitivity
was at least 100 times higher with SO2 as the reactant gas, and the experimental results were much more reproducible than with HBr. The experiments for the drug metabolism studies showed that if SO2 was used, the chromatogram of m/z 36 SIM was completely selective and clean and, if HBr was used, although it was selective, the poor sensitivity and stability made it difficult to pursue further investigation. The optimum pressure for both reactant gases was much higher than required for isotope analyses of C, N, or H.5-9 A similar elevation of reactant gas pressure was also needed for sulfurselective detection with HC1 reactant gas.” We believe this arises from the action of the reactant gas to modify reactive sites and scavenge water, rather than the actual reaction in the plasma.
CONCLUSIONS The results indicate that CRIMS with SO2, but not HBr, combines high sensitivity, high selectivity, a wide linear range, and structure independence for chlorine detection. The possible pharmacological application of CRIMS was studied with diazepam and several of its metabolites as the probes, and the results showed CRIMS to be a simple but powerful method in the study of drug metabolism. The presence of a metabolicallystable atom of chlorine in the parent drug means that it is intrinsically “labeled” and this “label” could be used to define metabolic patterns just as a stable or radioisotopic label can.
ACKNOWLEDGMENT This research was supported by USPHS Grant GM36143. We thank Dr. Mehdi Moini for much helpful discussion and advice. RECEIVEDfor review August 24, November 16, 1992.
1992.
Accepted
