Anal. Chem. 1981, 53, 793-797 (12) Kimes, B. W.; Morris, D. R. Biochemistry 1973, 12, 442-449. (13) Long, K. R,; Long, R. C.; Goldstein, J. H. J. Magn. Reson. 1972. 8, 207-210. (14) Tabor, H. Biochemistry 1962, 1 , 496-501. (15) Welss, R. L.; Morris, D. R. Slochemistry 1973, 12, 435-441. (10) Weser, U.; Strobel, G.J.; Rupp, H.; Voeiter, W. Eur. J. Biochem. 1974, 50, 91-99. (17) Sarneski, J. E.; Reilley, C. N. Essays Anal. Chem. 1977, 35-49. (18) Sarneski, J. E.; Surprenant, H. L.;Reiiley. C. N. Spectrosc. Len. 1978, 9, 805-894. (19) van de Weijer, P.; Thijsse, H.; van der Meer, D. Org. Map. Reson. 1976, 8, 187-191. (20) van de Weijer, P.; van den Ham, D. M. W.; van der Meer. D. Org. Magn. Reson. 1977, 8 , 281-284. (21) Olson, A. R.; Koch, C, W.; Pimentel, G. C. “Introductory Quantitative Chemistry”; W. H. Freeman: San Franclsco, CA, 1950; Chapter 12.
793
(22) Sudmeier, J. L.; Reiiley. C. N. Anal. Chem. 1984, 36, 1098-1700. (23) Sarneski, J. E.; Surprenant, H. L.; Molen, F. K.; Reiiley, C. N. Anal. Chem. 1975, 47, 2116-2124. (24) Palmer, 8. N.; Powell, H. K. J. J . Chem. Soc., Dalton Trans. 1974, 2080-2088. (25) Hedwlg, G. R.; Powell, H. K. J. Anal. Chem. 1971, 43, 1206-1212. (20) Anichlnl, A.; Fabbrizzl, L.; Barbucci, R.; Mastroianni, A. J. Chem. Soc., Dalton Trans. 1977, 2224-2228. (27) Prue, J. E.; Schwarzenbach, G. HeEv. Chim. Acta 1050, 33,985-995. (28) Schwarzenbach, G. Helv. Chlm Acta 1950. 33, 974-985. (29) Hamann, S. D. Aust. J. Chem. 1970, 23, 1749-1705.
RECEIVED for review November 13,1980. Accepted January 23,1981. This research was supported in part by a grant (GM 10848) from the National Institutes of Health.
Determination of Flurazepam in Human Plasma by Gas Chromatography-Electron Capture Negative Chemical Ionization Mass Spectrometry B. J. Miwa” and W. A. Garland Department of Biochemistry and Drug Metabollsm, Hoffmann-La Roche Inc., Nutley, New Jersey 071 10
P. Blumenthal Department of Clinical Pharmacology, Hoffmann-La Roche Inc., Nutley, New Jersey 071 10
A specific and extraordlnarily sensitive GC/MS assay has been developed to quantitate flurazepam, a commonly prescribed hypnotic, In human plasma. The assay requires 2 mL of plasma and features the use of electron capture negative chemical ionization. A decadeuterated analogue of flurazepam is used as Internal standard. A 4 ft X 1 mm mlcropacked column contalning OV-17 on pPartkorb Is used for the GC analysis. A quadrupole mass spectrometer Is used to monitor in the GC effluent the M-• Ions of flurazepam and Its Internal standard. Methane is used as both GC carrier gas and negative chemical ionization reagent gas. The sendtlvlty of the assay Is 12 pg mL-‘ and at a concentration of 135 pg mL-’ the assay precision is 4 %. Three subjects who received a 30-mg oral dose of flurazepam showed peak flurazepam concentrations of 3.9, 2.3, and 0.4 ng mL-’ at 0.5, 1, and 1 h postdoslng, respectively. The ellminatlon half-llves for the three subjects were 1.9, 3.0, and 2.0 h, respectively.
Flurazepam (Dalmane, I, Table I) was introduced into general medical practice in 1970 as an orally active agent for the treatment of insomnia. Presently, flurazepam is the most widely prescribed drug for insomnia ( I ) and is the thirteenth most prescribed drug in the United States (2). Studies on the biotransformation and elimination of flurazepam in humans have shown that the compound is extensively metabolized and has a large apparent volume of distribution (3). Peak plasma concentrations of the intact drug rarely exceed several nanograms per milliter following the single or multiple dose administration of therapeutic amounts of flurazepam to humans (4,5).While de Silva and co-workers have reported spectrophotometric and EC/GC procedures which can sometimes measure the peak concentration of flurazepam following dosing (4,5), only the recently published RIA procedure of Glover et al. (6) can measure flurazepam 0003-2700/81/0353-0793$01.25/0
Table I. Chemical Structures and Their Designations
designation
over a sufficient period of time to determine the drug’s pharmacokinetic profile. In this paper, we report a sensitive and specific method for the quantitation of flurazepam which is the first reported “chemical” assay for flurazepam with sufficient sensitivity to measure useful concentrations of this widely used hypnotic in plasma. The assay uses electron capture negative chemical ionization mass spectrometry (EC/NCIMS) as the method of detection. NCIMS has been successfully used to analyze trace amounts of other medically used 1,4-benzodiazepin-2ones, i.e., desmethyldiazepam (7) and clonazepam (8),in biological matrices. The sensitivity of EC/NCI depends on the ability of the molecule in question to capture a thermal electron and form a stable molecular anion. In this regard, electron capture by the 1,4-benzodiazepin-2-onesis aided by the presence of two aromatic electron systems which can resonance stabilize the resulting anion (9). The highly adsorptive nature of flurazepam makes it a particularly difficult compound to quantitate. In addition to a deuterated internal standard (11), we also add a structural analogue, compound 111,to the plasma to increase extraction efficiencies and reduce adsorptive losses (IO). Another 0 1981 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 6, MAY 1981
structural analogue, compound IV, is used in the tuning procedure to eliminate "ghosting" produced when nanogram amounts of flurazepam are injected onto the GC column. EXPERIMENTAL SECTION Instrumentation. For selected ion monitoring measurements, a Finnigan 3200 quadrupole mass spectrometer was used in conjunction with a Finnigan PROMIM peak monitor. The ion tracings were recorded on a Rikadenki multichannel recorder. Mass spectra were obtained by use of a Finnigan Model 1015 quadrupole mass spectrometer and a Finnigan Model 6000 data system. Both mass spectrometers were coupled to Finnigan 9500 gas chromatographs. Each instrument was modified for negative ion detection using the method of Stafford et al. (11). The conversion dynode, obtained by removing a single dynode from a Finnigan discrete stage electron multiplier, was used with a Galileo Model 4770 continuous dynode electron multiplier. Voltage to the conversion dynode, usually +2.5 kV, was supplied by a Hewlett-Packard Model 6516A dc power supply. Two Hewlett-Packard Model 6209B dc power supplies provided voltages for the lens and the ion volume. The repeller and the trap were both electricallytied to the ion volume. The ion energy, lens, and electron energy were set to give the maximum signal intensity consistent with unit resolution and symmetrical peak shape. The voltage across the electron multiplier was -1.9 kV, and the filament emission was set to the lowest value consistent with maximum negative ion production. Magnets (0.0009 and 0.0007 T) were positioned on the vacuum housing above the conversion dynode and ion source, respectively. The positions of the magnets were adjusted until the m/z 452 ion intensity from perfluorotributylamine was at a maximum, Each PROMIM channel was operated with a 100-msdwell time, a 0.5-Hz frequency response, and a gain of lo8 V A-'. The Rikadenki recorder was operated at a chart speed of 2 cm min-'. For recording mass spectra, the Model 6000 data system was set to scan every 1.5 s from m / z 150 to m/z 450. The signal threshold was set at 1 bit. The GC column, 4 f t x 1 mm i.d., was made by glass blowers at Hoffmann-La Roche using glass purchased from Wilmad Glass Co., Buena, NJ. The column was packed by Whatman, Inc., Clifton, NJ, with OV-17 on ppartisorb. The column required no further conditioning. The temperatures of the injector, column oven, separator oven, and transfer line were 295, 290, 250, and 250 "C, respectively. Methane (3.21 atm, 15 mL min-') was used as GC carrier gas and CI reagent gas. Under these conditions the methane ion source pressure was 67-80 mPa, and the retention time of flurazepam was approximately 135 s. The ion source was operated without any external heating. The normal temperature of the Finnigan 3200 ion source when the filament is turned on and off repeatedly for short periods of time, i.e., less than 180 s, is in the range of 80-100 "C. The pressure in the ion source was estimated from published charts (12)and a thermocouple connected directly to the ion source was used to measure the temperature of the ion source. Materials. Glass-distilled benzene, dichloroethane, hexane, and acetone were obtained from Burdick and Jackson. The preparation of 1 M, pH 10 borate buffer has been described elsewhere (13). Methane (ultra high purity) was obtained from Matheson Gas Products. Unlabeled flurazepam.2HCl (I), comdihydropounds 111and IV, and 7-chloro-5-(2-fluorophenyl)-1,3 2H-1,4-benzodiazepin-2-one were obtained from W. E. Scott, Hoffmann-La Roche, Nutley, NJ. Extractions were performed in culture tubes with Teflon-lined screw caps (16 mL, Pyrex 8061), and conical centrifuge tubes (5 mL, Pyrex 8061) were used for the final evaporation step. Prior to use, all glassware was treated with a 1%aqueous solution of Prosil-28 (PCR Research Chemicals) and consecutively rinsed with water, dichloromethane, and methanol. Synthesis of 7-Chloro-1-[2 4diethyl- d5-amino)ethyll-5-(2fluoropheny1)-1,3-dihydro-2H-1,4-benzodiazepin-2-one Dihydrochloride (compound 11, flurazepam-dlo). A 500-pL sample of 2-aminoethanol (8.3 mmol, Aldrich Chemical Co.) and 5 mL of C2H3C2H0(82 mmol, 99% 'H4, Stohler Isotopes Inc.) were transferred into a 500-mL Parr hydrogenation flask containing 100 mL of D20 (Stohler Isotope Inc.) and 50 mg of Pd/C. The flask was attached to a Parr hydrogenation apparatus and
the solution was exposed, with shaking, to deuterium gas (99% 2Hz,1.4 atm, Matheson Gas Products) for 24 h (14), The solution was made alkaline with 1 M sodium hydroxide and was exhaustively extracted with ethyl ether. The combined ethyl ether extracts were dried over anhydrous magnesium sulfate. The residue remaining, after careful removal of the ethyl ether at room temperature under a stream of dry nitrogen gas, was dissolved in 500 p L of pyridine and was treated with 500 p L of thionyl chloride (Aldrich Chemical Co.), The solution was stirred for 3 h and then was cooled to 0 "C. Two milliliters of water wm added and the solution was titrated to approximate neutrality with 5 M sodium hydroxide. Five milliliters of 0.1 N sodium hydroxide, 5 mL of methylene chloride, 200 mg of tetrabutylammonium hydrogen sulfate (Aldrich Chemical Co.), and 287 mg of 7chlorc-5-(2-fluorophenyl)-l,3-dihydro-W-l,4-benzodiazepin-2-one (1mmol) were added, and the two-phase mixture was stirred for 18 h (15). The methylene chloride layer was isolated, and the residue remaining after removal of the CHC12was reconstituted in 200 pL of methylene chloride. Twenty 10-pL portions of this solution were analyzed by HPLC using the normal phase system of Strojny et al. (16). The column effluent from each analysis, at the retention time of authentic flurazepam, was collected. The effluent collections were combined and were treated with hydrogen chloride gas, and the solvents and excess gas were removed under a stream of nitrogen. The residue was then recrystallized from methanol-ethyl ether. The product (225 mg, 0.58 mmol) showed suitable mass spectral, 'H NMR, IR and UV properties. Positive and negative CI mass spectral analyses of the titled compound suggested the presence of less than 0.00015% unlabeled flurazepam. Solutions. One milligram (free base) per milliliter stock solutions of flurazepam, flurazepam-dlo, and compounds I11 and IV were prepared in methanol. For spiking plasma, the flurazepam stock solution was diluted with water to give working solutions A and B containing 1and 10 ng mL-' of flurazepam, respectively. The flurazepam-dlospiking solution was prepared by diluting this compound's stock solution with water to give solution C containing 100 ng mL-l of flurazepam-dlo. The compound I11 stock solution was diluted with 0.01 M HC1 to give solution D containing 4 pg mL-' of compound 111. The compound IV stock solution was diluted with hexane/acetone (9:l) to give solution E containing 50 ng pL-' of compound IV. External standard solutions were prepared by diluting the relevant stock solutions in hexane/acetone (91), These solutions each contained 125 ng mL-l of flurazepam-dlo and either 0,0.625, 1.25, 2.5, 12.5, or 25 ng mL-' of flurazepam, respectively. Assay Procedure. Two milliliters of either control plasma or the plasma from a subject who had received flurazepam was added to a culture tube containing 500 pL of solution D (2 pg of the compound 111). Tubes containing control plasma were spiked with either 0, 25, 50, or 100 p L of solution A corresponding to 0, 0.025,0.05, or 0.1 ng of flurazepam added respectively or 50, 100, 250, or 500 p L of solution B corresponding to 0.5, 1.0, 2.5, or 5.0 ng of flurazepam added, respectively. All plasma samples were then spiked with 50 pL of solution C (5 ng of flurazepam-dlo). The tubes were vortexed, capped, and placed on an Eberbach variable-speed reciprocating shaker for 2 h to ensure equilibration of all compounds. Prior to extraction, the tubes were stored overnight in a 4 "C refrigerator. One milliliter of 1 M, pH 10 borate buffer and 6 mL of benzene/dichloroethane (73) were added to each culture tube. The samples were extracted by gentle shaking on the Eberbach shaker for 30 min. The tubes were centrifuged (Damon Model CRU-500) at 10 "C for 10 min at 1500g. The organic phase was transferred, using Prosil-28 treated Pasteur pipets, to a 16-mL culture tube, and the solvent was evaporated at 50 "C under a gentle stream of nitrogen (N-Evap, Organomation Associates). One milliliter of 0.1 M HC1 was added to the residue and the tube was vortexed several times during a 15-min period. Six milliliters of benzene was added and the sample was extracted by shaking for 20 min followed by centrifugation for 10 min. The benzene layer was aspirated and 0.2 mL of 0.5 M NaOH, 0.5 mL of 1 M pH 10 borate buffer, sufficient NaCl crystals to saturate the solution, and 6 mL of benzene/dichloroethane (7:3) were added. The samples were shaken for 25 min and centrifuged for 10 min. The organic phase was transferred, with a Prosil-28 treated
ANALYTICAL CHEMISTRY, VOL. 53, NO. 6,MAY 1981 lCl0
387
1
I
4
A
FLURAZEPAM
IOoo 500
!
795
-1
I*
\
397
I
85
105
125
145
165
185
210
ION SOURCE TEMPERATURE, OC.
Figure 2. Effect of Ion source temperature on the absolute intenskies of the M-. ( m l z 387) and CI-. (mlz 35) ions in the EC/NCI mass spectrum of flurazepam.
1
twz
Figure 1. Methane EC/NCI mass spectra of (A) flurazepam, MW = 387,and (6) flurazepam-d,,, MW = 397,at an ion source temperature of approximately 100 "C. At this temperature virtually no other ions are present in the spectra. Pasteur pipet, to a 5-mL centrifuge tube, and the solvent was evaporated at 50 "C under a gentle stream of nitrogen. Prior to the injection of any sample, the GC column was primed by several injections of a concentrated ethyl acetate extract of control blood, followed by Silyl-8 (Pierce Chemical Co.) and several 1-pL injections of solution E. Aliquots of solution E were injected to determine the mass offset of compound Iv's M-. ion at m/z 369. By use of the same mass offset value, the mass marker was then used to set the mass spectrometer to monitor mlz 387 and mlz 397 corresponding to the M-. ions of flurazepam and flurazepam-dlo, respectively. At this point aliquots of the external standard solutions were injected to determine the performance of both the GC column and mass spectrometerand also the retention time of flurazepam. The residues from the sample extracts were redissolved in 40 j & of hexane/acetone (9:l) and 1-5 p L of these solutions were injected into the GC/MS with the GC divert valve open. Thirty-five seconds later the divert valve was closed to allow the GC effluent to enter into the ion source. The ion source filament was turned on 55 s after injection. Following collection of all data, the peak heights in the ion chromatograms were measured and the mle 387 to m/e 397 ion ratio was calculated. Ion ratio (y) vs. amount added ( x ) data from the calibration curve samples were analyzed by linear regression. The ion ratios were weighted by a factor of y-l. Constants from the least-squaresfitting were used to calculate the concentration of flurazepam in an experimental sample from the measured ion ratio.
RESULTS AND DISCUSSION The NCI mass spectra of flurazepam and flurazepam-dlo are shown in Figure 1. The spectra are quite simple consisting principally of each compound's M-. molecular ions. Both spectra show an ion of law abundance at a mass of M + 15. This ion is probably due to the addition of a methyl radical to flurazepam or flurazepam-dlo prior to ionization (17).
Experiments were performed t o determine the ion source pressure and temperature associated with the optimum intensity of flurazepam's M-. ion. Virtually no M-. ion was observed until the ion source pressure reached 53 mPa. The intensity of the M-. ion was at a maximum at 80 mPa and decreased to a value one-tenth of the maximum value a t 133 mPa. The effect of ion source temperature on the intensity of the M-. ion is shown in Figure 2. For this study, 25 ng of flurazepam was injected at several ion source temperatures, and the peak heights a t mlz 35 and mlz 387 were measured. As can be seen, the intensity of the M-. ion is at a maximum a t ion source temperatures less than 100 "C, the normal temperature range if the ion source is operated without any external heating. Consistent with a first-order decomposition mechanism, the intensity of the M-- ion falls in a logarithmic fashion for ion source temperatures between 125 and 185 "C. The principal fragment ion observed to increase over this temperature range is C1-a. These data suggest a dissociative electron capture mechanism for flurazepam for ion source temperatures greater than 100 "C and a nondissociative electron capture mechanism for flurazepam for ion source temperatures less than 100 "C (18). An ion chromatogram representing the plasma extract from the lowest concentration in the standard curve is shown in Figure 3. Five nanograms of flurazepam-dlo and 0.025 ng of flurazepam were added to 2 mL of control plasma, and the plasma was analyzed by using the procedure described in the Experimental Section. Five of the available 40 pL, approximately 3 pg, was injected. The relative sensitivities of PCI and NCI for the detection of flurazepam were compared. The ion source parameters for both the PCI and NCI mode of operation were optimized while observing the mlz 414 ion in the spectrum of perfluorotributylamine. Because the conversion dynode in the PCI mode acts as a signal multiplier, the electron multiplier was adjusted, during both tuning procedures, to produce the same noise level a t the same recorder setting. Six injections, 25 ng each, of flurazepam were made for each mode of ionization. The ion current at mlz 387 (M-.) was monitored for the determination of the NCI sensitivity and the ion current at mlz 388 (MH', the principal ion in the PCI mass spectrum of flurazepam) was monitored for determination of the PCI sensitivity. The peak heights from each set of six injections were measured, and the mean value was determined. From a comparison of
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 6, MAY 1981
Table 11. Seven 2-mI, Aliquots of Pooled Subject Plasma Spiked with 5 ng of Flwazepam-d,, a sample
amt, pg mL-'
1
135 133
2
amt, Pg mL-'
132 127 145
5 6 7
132 134 Mean f SD (CV)= 134 f 5.5 ve' mL-' (4%). 3
4
a
Figure 3. Selected ion current profile from the GC/EC/NCIMS analysis of an extract from a plasma sample containing 0.0125 ng mL-l of flurazepam and 2.5 ng mL-l flurazepam-d,,. I n viewing these ion current profiles it should be recalled that the recorder pens are slightly offset from one another. The arrows denote the retention time of authentic flurazepam.
the peak heights, the intensity of the M-. ions is estimated to be 50 times greater than that of the MH' ion for the detection of flurazepam. Selected ion current profiles showing the effect of the addition of the carrier substance, compound 111, on the recovery of flurazepam is shown in Figure 4. Although all 12 samples
were analyzed at the same time, only the samples which were spiked with 2 pg of compound I1 show reasonable recoveries of flurazepam and flurazeparn-dlo. Calibration curves from the analysis of plasma samples spiked with known amounts of flurazepam, flurazepam-dlo, and compound I1 all show a correlation coefficient of greater than 0.99. The ion ratios when no flurazepam is added are zero. The scatter of points in the calibration curve, especially a t subnanogram concentrations, is reduced when the spiked plasma samples are allowed to equilibrate several hours before extraction. Ion current profiles from the analysis of plasma extracts from a subject who had received a 30-mg oral dose of Dalmane are shown in Figure 5. The plasma concentrations a t 0, 5.5, 7.5,and 10.5 h were 0, 0.971,0.459,and 0.258 ng mL-l, respectively. The peak in the m / z 387 ion tracing which elutes after flurazepam is due to a residual lipid component from the plasma that was not eliminated during the acid wash. Plasma samples that did not receive an acid wash step showed a peak in the ion chromatogram tracings that was several orders of magnitude larger than the contaminant peak shown in Figure 5.
Flgure 4. Selected ion current profiles showing the effect of the addltion of carrier substance, compound 111, on the recoveries of flurazepam ( m l z 387) and flurazepam-d,, ( m l r 397). Selected ion current profiles 1-6 are from the analysls of 1 mL of plasma sample spiked with 1 ng of flurazepam, 5 ng of flurazepam-d,,, and 2 pg of compound 111. Selected ion current profiles 7-12 are from the analysis of 1 mL of plasma spiked only with 1 ng of flurazepam and 5 ng of flurazepam-d,,. All samples were analyzed at the same time. Each selected ion current profile is from the injection of 4 pL of the final 40 pL solution.
do x 5 d,OX 10,5 H R
7.5 H R
5.5 H R
OHR
+T
Flgure 5. Selected ion current profiles from the GC/EC/NCIMS analysis of plasma extracts from a subject who had received a 30-mg oral dose of Dalmane. I n viewing these ion current profiles, it should be recalled that the recorder pens are slightly offset from one another. Thus, the small peak in the do tracing in the 0-h sample which appears under the dlo profile actually elutes before authentic flurazepam.
ANALYTICAL CHEMISTRY, VOL. 53, NO. 6, MAY 1981
797
10050-
-1
-.
---v
2
l
C
a
10
12
2
4
e
a
i
0
1
2
HOURS
Flgure 6. Drug plasma levels from these patients administered an oral dose of 30 mg of Dalmane.
For determination of the assay precision a t subnanogram concentrations of flurazepam, several subject plasma samples were pooled and equilibrated for several hours on an Eberbach shaker. Seven 2-mL replicates of the pooled plasma were extracted and analyzed as described in the Experimental Section. The results are shown in Table 11. At a plasma concentration of approximately 135 pg/mI,, the precision was *4%. Flurazepam’s pharmacokinetic profile in three subjects, each of whom received a 30-mg oral dose of Dalmane, is shown in Figure 6. Peak flurazepam levels of 3.9,2.3, and 0.4 ng/mL were found at 0.5, 1,and 1h, respectively. The half-lives for each subject, determined by a least-squaresfit of the data from the 2-h sample to the last data point collected, were 1.9, 3.0, and 2.0 h, respectively. These values are in good agreement with the mean half-life of 2.3 h for flurazepam obtained in eight subjects by the RIA (6). Several technical problems were encountered while developing the assay. If more than 10 ng of either flurazepam or flurazepam-dIowas injected on the GC column to tune the mass spectrometer, severe compound “ghosting” a t the M-. ion of the injected compound could often be observed for several hours after the injection. Injections of a solution containing compound IV were used to tune the mass spectrometer, to avoid this problem. Solutions of compound IV, in conjunction with injections of Silyl-8 and the extract of control plasma, were also used to prime the GC column prior to its use. Without such priming, the sensitivity of the assay is only a thousandths of the sensitivity when the column is primed. The amount of priming required varies with the age and condition of the colurnn and the condition of the GC/MS interface. One variable in the assay is the position of the magnets above the ion source and conversion dynode. The function of the magnets is not known. The magnet above the ion source may serve to focus the thermal electrons in the ion source, while the magnet above the conversion dynode may serve to better focus negative ions on the conversion dynode or to better focus organic or inorganic positive ions emitted from the conversion dynode m t o the electron multiplier. The optimum position of either magnet can only be found by experiment. Although the magnets will often cause a large increase in absolute signal,,the gain in the signal to noise rates
is more modest, typically a factor of 3-5. Occasionally, the use of the magnets will yield no increase in the signal to noise ratio. Certain magnet positions must be avoided in order to maintain suitable ion peak shape and resolution. In conclusion, making use of the high electron capture affinity of flurazepam we have been able to develop a GC/ NCIMS assay for this widely prescribed hypnotic. The sensitivity limit of 12 pg/mL for this assay makes it approximately 8 times more sensitive than the RIA assay for flurazepam (6) and more than 100 times more sensitive than the EC/GC assay for the drug (5). Our assay for flurazepam gives support to the suggestion of Hunt e t al. that GC/electron capture NCIMS will be more sensitive than EC/GC for the analysis of pharmacologically important materials in biological matrices (19). LITERATURE CITED “Drug Store and Hospital Purchases In America”; IMS Corp.: Ambler, PA, 1976-1977; Category 67220, p 940. DeNuzzo, R. V. Medical Marketing Media, April 1980, p 18. Schwartz, M. A.; Postma, E. J . Pharm. Sci. 1970, 59, 1800-1806. de Silva, J. A. F.; Strojny, N. J . Pharm. Scl. 1971, 60, 1303-1314. de Silva, J. A. F.; Puglisl, C. V.; Brooks, M. A,; Hackman, M. R. J . Chromatogr. 1974, 99, 461-483. Glover, W.; Earley, J.; Delaney, M.; Dixon, R. J . Pharm. Sci. 1980, 69, 601-602. Garland, W. A.; Miwa, B. J. EPH, Envlron. Health Perspect. 1980, 36, 69-76. Garland, W. A.; Min, B. H. J . Chromatogr. 1979, 172, 279-286. de Silva, J. A. F. In “Antleplleptic Drugs: Quantitative Analysis and Interpretation”; Plppenger, C. E., Penry, J. K., Kutt, H., Eds.; Raven Press: New York, 1978; pp 111-137. Min, B. H.; Garland, W. A.; Khoo, K.-C.; Torres, G. S. Blomed. h&ss Spectrom. 1978, 5 , 692-698. Stafford, G.; Reeher, J.; Smith, R.; Story, M. In “Dynamic Mass Spectrometry”; Price, D., Todd, J. F. J., Eds.; Heyden: London, England, 1978; Vol. 5, pp 55-57. Munson, M. S. B.; Field, F. H. J . Am. Chem. SOC. 1986, 88, 2621-2630. de Silva, J. A. F.; Puglisl, C. V. Anal. Chem. 1970, 42, 1725-1735. Bowmann, R. E.; Stroud, H. H. J . Chem. SOC. 1950, 1342-1349. Vessman, J.; Johansson, M.; Magnusson, P.; Stromberg, S. Anal. Chem. 1977, 49, 1545-1549. Strojny, N.; Pugiisi, C. V.; de Silva, J. A. F. Anal. Lett. 1978, 1311(2), 135-160. McEwen, C. N.; Rudat, M. A. J . Am. SOC. 1979, 101, 6470-6472. Llndley, T. M. J . Anal. Toxicol. 1979, 3 , 18-20. Hunt, D. F.; Crow, F. W. Anal. Chem. 1978, 50, 1781-1784.
RECEIVED for review July 21,1980. Resubmitted January 22, 1981. Accepted January 22, 1981.
