Representative standards data for 'Li and l H are given in Table 11. The lH standards data were corrected for background protons which occur in the NMR insert and on the glass sample tubes. No background corrections were necessary for 7Li, 3H2, and %e. All background corrections were determined from blank runs for each nuclear species. The closeness to zero of the Y-intercepts indicates essentially no net bias in the experimental results. The day-to-day reproducibility of the pulsed NMR technique was determined by analyzing 1- to 2-year old LiT samples over a period of one month. Six determinations were made on each of six samples giving a relative standard deviation which varied from 6-7%. Part of this deviation is attributable to the unstable nature of LiT, although the results were fairly random over the period of observation. An investigation of the reproducibility of the results obtained from a %-ml proton standard (9.02 X 1020spins) over a period of one month yielded a relative standard deviation of 2%. A precision estimate for obtaining the data for the 1H standards was calculated by pooling the results of 36 duplicate runs, six for each standard. The standard deviation was 5% relative to the mean of the concentrations of the standards. The accuracy of the technique with respect to l H in liquid samples was estimated to be -0.24 X 1020 spins (or 3% relative) from the difference between the calculated concentration of a standard reference sample (9.02 X lozo 'H spins) and the mean (8.78 X 1020 l H spins) of five experimental values. Several sources of error are inherent in the pulsed NMR method. One of the largest errors arises from the "deadtime" introduced when the powerful rf pulse "rings down" for 10-20 psec with a consequential loss of FID signal. This loss accounts for less than 2% of FID signals greater than 2-msec duration. Certain FID signals ( > l o msec) show no apparent loss of signal, since the signal maintains zero slope for more than 50 psec after the overload period. The error in measuring the true nuclear magnetization is reduced by extrapolating the FID signal to the midpoint of the rf pulse (7). Variable errors are introduced in tuning the power amplifier, the preamplifier, and probe. These errors cannot be
eliminated, but they are minimized by running the standards and unknowns under the same instrument settings. Less controllable errors arise when adjusting the 90' pulse width and the rf reference phase, since pulse width is a function of rf power and the nuclear species being pulsed (IO). Furthermore, the rf reference phase is a function of the electronic circuitry and the physical characteristics of the sample. Finally, and no less important, is the error in the magnitude of the relative sen,sitivities between the nuclei of the standards and unknowns. A study (Table 11) was made of the experimental relative sensitivity between 7Li and l H to test the validity of using this technqiue for other nuclei standards. The theoretical ratio is 1.94 and the experimental value found in this work is 2.05 f 0.07. On the average, this would result in an experimental value which is 6% greater than the true value. The relative sensitivity of 19Fwith respect to 'H was also tested experimentally. A value of 0.926 f 0.001 was found. Compared to the theoretical vaiue of 0.941, an error of 1.6% is incurred here. The results of these two examples offer proof of the validity of analyses using other nuclei standards with the proper corrections applied.
LITERATURE CITED (1) P. C. Souers, T. A. Jolly, and C. F. Cline, J. Phys. Chem. Solids, 28, 1717-1 719 (1967). (2) P. C. Souers, T. S. Blake, P. M. Penpraze, and C. F. Cline, J. Phys. Chem. Solids. 30. 2649-2656 (1969). (3) T. C.Farrar and E. D. Becker, "Pulse and Fourier Transform NMR." Academic Press, New York, NY, 1971, pp 18-20, (4) Reference 3,p 29. (5) Reference 3,p 21. (6) Reference 3, pp 20-29. (7) D. Barnaal and I. J. Lowe, Phys. Rev. Lett., 11, 258 (1963);, (8) G. S.Rushbrooke, "Introduction to Statistical Mechanics, Oxford University Press, New York, NY, 1962, p 103. (9) J. A. Pople, W. G. Schneider, and H. J. Bernstein, "High-resolution Nuclear Magnetic Resonance," McGraw-Hill, New York, NY, 1959, p 39, Equation (3-79). (IO) Reference 3, pp 43-45.
RECEIVEDfor review August 2, 1974. Accepted December 11, 1974. Mound Laboratory is operated by Monsanto Research Corporation for the U S . Atomic Energy Commission under Contract No. AT-33-1-GEN-53.
Charge Exchange Mass Spectra of Morphine and Tropane AI kaloids Ian Jardine and Catherine Fenselau Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, MD 2 1205
One of the greatest strengths of mass spectrometry is the wealth of signals in a given spectrum. These provide considerable structural information when they can be interpreted, and make mass spectral fingerprinting a highly reliable method of compound identification. However, when the number of possible identities of an unknown is quite small, a few peaks may serve to characterize the sample, as in amino acid analyses and drug overdose or street drug analyses. In the latter forensic category, considerable emphasis has been placed on characterizing samples by their molecular ions alone and on the desirability of obtaining spectra simplified to contain primarily molecular ions ( I ) . 730 * ANALYTICAL CHEMISTRY, VOL. 47, NO. 4 , APRIL 1975
Chemical ionization ( 2 ) has been evaluated in this connection, both for analysis of samples introduced to the mass spectrometer through the gas chromatograph (3, 4 ) and introduced via the direct probe ( I , 5, 6). While chemical ionization does simplify the spectra of many drugs to essentially one peak at (M l),it does not give strong (M H ) + ions for many of the crucial compounds, including heroin and morphine (5, 6). The base peaks in the spectra of heroin and morphine correspond to (M H CH&OOH)+ and (M H - H20)+ fragment ions, respectively. These facile losses of acetic acid and water are characteristic of most acetates ( 5 ) and alcohols (7), respective-
+
+
+
+
Table I. 10% NO/N2 Mass Spectra of Selected Compounds Listed as Percentages of the Base Peakavb Peaks
EF
CIc
M'T
(M-,H)I
(%)
Compound
1
2
3
4
3
6
7
(%)
8
25 15 204 (9) 369 310 (30) 370 (28) 268 (23) 327 (20) 215 (18) 368 (9) 18 285 (4) 96 311 (5) 325 (5) 329 (5) 310 (19) 327 326 (35) 328 (24) 98 20 270 (3) 325 (5) 327 268 (98) 328 (41) 269 (28) 120 (15) 329 (9) 100 15 287 (5) 281 (5) 178 (5) 285 286 (23) 283 (16) 284 (11) 268 (9) 100 20 241 (2) 240 (4) 313 (5) 293 (7) 310 (12) 309 (19) 311 312 (26) 13 162 (5) 100 188 (5) 229 (5) 298 (6) 124 (7) 299 300 (20) 297 (7) 100 100 243 (4) 297 (4) 301 (6) 242 (6) 96 (7) 299 300 (30) 298 (7) 83 (9) 64 100 59 (13) 255 (10) 149 (IO) 150 (9) 257 258 (24) 256 (15) 100 100 57 (12) 199 (13) 85 (15) 83 (24) 282 (23) 47 (26) 283 284 (34) 22 100 69 (7) 172 (10) 43 (10) 246 (11) 70 (19) 7 1 (23) 247 248 (32) 96 (9) 26 100 94 (12) 105 (12) 83 (21) 82 (21) 303 304 (29) 182 (28) 15 16 289 124 (78) 290 (23) 125 (9) 43 (8) 60 (7) 71 (7). 73 (7) o The first eight peaks are listed in descending order of intensity with their abundances in parentheses. Peak 1 is 1Wh and is the molecular ion for each compound. r The relative intensities of the 51: and M + H A ions from the E1 and CI(Isobutane1 mass spectra, respectively, are also listed for comparison.
Heroin @-monoacetylmorphine 06-monoacetylmorphine Morphine Nalorphine Codeine Hydrocodone Levorphanol Levallorphan Meperidine Cocaine Atropine
nitric oxide are presented in Table I. The last two columns of Table I list the relative intensities of the M.+ and (M + H)+ ions taken from the E1 and isobutane CI spectra, respectively. Only the eight most intense ions are shown for the CE spectra since any other ions are of low abundance. The features of the charge exchange spectra are uniform throughout this set of compounds. That is, unlike the situation obtained using electron impact or chemical ionization, where the molecular ion species, M.+ or (M Hj+, do not consistently produce the base peak, the molecular ion produces the base peak in all of these spectra. They range from 14.6%, in heroin, to 56.9%, in nalorphine, of the total ion current not attributable to the reagent gas ions. The reliable occurrence of intense molecular ions in these compounds when ionized by charge exchange should be analytically useful and is not altogether unexpected from the theoretical point of view. Charge exchange can EXPERIMENTAL take place only in molecules or functional groups whose ionization potential is less than the recombination energy All mass spectra were measured on a DuPont 21-491 double foof the ionized reagent gas (9). The recombination energy of cusing mass spectrometer equipped with a dual EI/CI source and interfaced to a Varian 2700 gas chromatograph through a glass jet NO+ ion is between 8 and 9 eV (9); therefore the tertiary separator. When the GC was used, the carrier gas was helium a t 40 amine groups in the compounds studied here will be susml/min. The source has a separate introduction port for reagent ceptible to ionization since they have ionization potentials gas. E1 spectra of all the samples were recorded with a source temless than 8 eV (10).At the same time, charge exchange will perature of 220 "C, ionizing voltage of 80 eV and suitable probe not take place with hydroxy, acetoxy, or aromatic groups in temperature, generally 200-250 "C. CI and CE spectra of all the the molecules since their ionization potential (10) is greater samples were recorded using isobutane and a 10% mixture of nitric oxide in nitrogen reagent gases, respectively. The source was operthan the recombination energy of NO+ ion. Thus fragmenated a t a pressure of 0.5-1 Torr and a temperature of 200 "C. tation characteristic of these functional groups does not T h e sensitivity measurements on heroin were made hy injecting occur to the same extent as in E1 spectra. In fact, much of a measured quantity of a standard solut,ion into a capillary tube the fragmentation which is present in the CE spectra and and pumping off the solvent. With the mass spectrometer set for which closely parallels that of the E1 spectra, although of EI, isobutane CI, or nitric oxide CE operation and set to single ion much reduced intensity, is most probably resulting from monitor the appropriate ion for each method, the sample was inserted via the probe. The sample was rapidly heated by the probe charge exchange with N y + ions whose recombination enerheater to 350 "C and the molecular ion species was monitored on a gy is 15.3 eV, since a t the source pressure used in this work Bell and Howell UV recorder which was set at the slowest chart the intensity at mle 28 from Nz.+ ions is approximately speed of 0.1 inch/min and lowest frequency response of 3 Hz. The 20% of that at mle 30 from NO+ ions ( 1 1 ) . experiment was repeated a t least three times in each case t o ensure One further structural feature, which contributes to the reproducibility. uniformly high preservation of the molecular ions formed The isobutane (99.9% pure) and 10% mixture of nitric oxide in nitrogen were obtained from Union Carbide Corporation. All samfrom these compounds, must be pointed out. Charge exples were obtained either pure or as quaternary salts which were change ionization of open chain tertiary amines, methaextracted with chloroform from sodium bicarbonate solution as the done, methapyrilene, or quinine (12),for example, leads to free base. @-cleavage to the amine nitrogen, with subsequent fragRESULTS AND DISCUSSION mentation analogous to that occurring on electron impact. However, a-cleavage does not immediately bisect any of The spectra of heroin, 03-monoacetylmorphine! 06the compounds studied here and, hence, molecular ions are monoacetylmorphine, morphine, nalorphine, codeine, hyabundant. drocodone, levorphanol, levallorphan, meperidine, cocaine, Charge exchange also predominates in these molecules and atropine obtained by charge exchange ionization using
ly, under chemical ionization. Of the twelve pharmacologically active compounds studied here (Table I), most of which are commonly abused, (M 1j is the base peak in the isobutane chemical ionization spectrum of five. Interestingly, the relative size of (M H)+ and (M H HzO)+ ions in the chemical ionization spectrum of codeine has been found to vary according to the mixture of other compounds present in probe mixture samples (8). Five of the twelve compounds listed in Table I produce electron impact spectra in which Me+ ions form the base peak. We wish to report that charge exchange ionization using nitric oxide (9) uniformly leads to spectra in which molecular ions are the base peaks in this set of compounds, to point out the physical principles behind this, and to evaluate the relative sensitivity of this method of ionization.
+ +
+
+
ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 4, APRIL 1975
731
w
5
aw
I El, mie 369,Mng 2 Cl,m/e 370,5Gng 3 CE.m/e 369,IGGng 4 CE,m/e
369,50ng
a
Figure 1. Relative abundance of molecular ion species for heroin probe samples
From the scanned EI, isobutane CI, and CE spectra of heroin, the appropriate molecular ion species were found to carry 3.7%, 10.0%, and 14.6%of the total ion current not attributable to reagent gas ions, respectively. The ratio of the total io3 currents, obtained using the ratio of the molecular ion species combined with their percentage of the total ion currents, is approximately 20:5:1 for EI:CI:CE, respectively. Therefore, in terms of the total ion current generated for heroin, in the combination source and under the conditions of this study, E1 is more sensitive than isobutane CI which is more sensitive than nitric oxide CE. However, many analyses, for example, of seizure samples of opiates, do not usually require sensitivity below the microgram
'7 I CC€A#E
GCMSCEW)
Figure 2. GCMS CE(N0) mass spectrum of cocaine
over electrophilic addition of NO+, which has been observed in the spectra of simple ketones, esters, and carboxylic acids when pure nitric oxide is used as reagent gas ( 1 3 ) . (M NO)+ ions in these spectra are less than 3% relative intensity. It is noted, moreover, that (M H)+ ions are present in the CE spectra presented here. They are probably produced by proton transfer from ionized to neutral sample molecules when they collide in the confines of the chemical ionization source ( I d ) , but they may also result from interaction with hydrogenated impurities in the reagent gas. In fact, the (M H ) + ion is the second most abundant ion for most of the compounds and is only relegated from this position in those compounds where easy fragmentation is possible as, for example, with the loss of CH3COO- from heroin and 06-monoacetylmorphinein their 10% NO/N2 spectra. The differences in the monoacetylmorphine spectra are interesting and result from the fact that the major fragmentation occurs a t the 6 position where CH3COO. is lost more readily from 06-monoacetylmorphine than HO- from 03-monoacetylmorphine. Sensitivity measurements have usually been made by measuring the total ion current produced by a sample. However, this is difficult to measure under high pressure ionization conditions, where the ionizing gases contribute more ions to the total current than the sample. For this reason and because of our emphasis on characterization by molecular ions alone, we chose to measure the relative intensities of the molecular ions or (M H)+ ions from heroin produced by EI, CI, and CE ionization. Figure 1 shows the superimposed molecular ion profiles obtained when heroin was introduced via the probe and ionized by electron impact (50 ng), isobutane chemical ionization (50 ng), and nitric oxide charge exchange (50 ng and 100 ng). The ratio of the areas under the 50 ng peaks for EI, CI and CE is approximately 5.3:3.5:1,respectively. Therefore, in terms of the sensitivity of the methods relative to the molecular ion species of heroin, E1 is more sensitive than isobutane CI. which is more sensitive than CE.
+
+
+
+
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 4, APRIL 1975
range, and thus do not present critical problems in terms of absolute sensitivity. Although the spectra listed in Table I are probe samples, the NO/N2 reagent gas may be used for gas chromatography-mass spectrometry in the same way as methane or isobutane has been used, if, as in this case, the mass spectrometer source is fitted with a separate introduction port. For example, the GCMS CE(N0) mass spectrum of cocaine is presented in Figure 2. It was not expected that the helium carrier gas would have any effect on the CE spectra relative to those obtained from probe samples with the NO/N2 mixture, since spectra obtained with nitric oxide diluted with helium or argon have been found to be very similar to those obtained with nitric oxide diluted with nitrogen (11). This is found to be the case. In summary, therefore, nitric oxide diluted with nitrogen appears to be a useful reagent gas for select analytical applications for probe- or gas chromatography-mass spectrometry. It has been shown here to provide intense molecular ions and simplified mass spectra for a group of morphine and tropane alkaloids which have a common structural feature. That is, a tertiary amine function which, when cu-cleaved, will not immediately split the molecule. It is hoped that the method may be applied to any compound with this feature and, conversely, may be useful in structure elucidation when similar functionalities are being examined.
ACKNOWLEDGMENT The authors thank Solomon H. Snyder of this Department, A. Jacobsen of N.I.H., and A. Kemppainen of the Baltimore City Police Department for providing the s m ples used in this work.
LITERATURE CITED ( 1 ) R. Saferstein and J. Chao, J. Assoc. OFFic. Anal. Chem., 56, 1234
(1973). (2) F. H. Field, kcc. Chem. Res., 1, 42 (1968). (3) D. J. Jendon and A. K. Cho. Ann. Rev. Pharmacal., 13,371 ([973(/
(4) B. S. Finkle, R. L. Foltz, and D. M. Taylor, J. Chromafogr. Sci., 12, 304 (1974). (5) G. W. A. Milne, H. M. Fales, and T. Axenrod, Anal. Chem., 43, 1815 (1971). (6) J.-M. Chao, R. Saferstein, and J. Manura, Anal. Chem., 46, 296 (1974). (7) F. H. Field, J. Am. Chem. SOC.,92, 2672 (1970). (8)R. C. Dougherty, J. D. Roberts, L. Griffin, and H. M. Fales, Paper presented at the 22nd conference on Mass Spectrometry, Philadelphia, PA, May 1974. (9) N. Einolf and M. S. B. Munson, Int. J. Mass Spectrom. /on Phys., 9, 141 (1972). (10) J. L. Franklin, J. G. Diliard, H. M. Rosenstock, J. T. Herron, K . Draxl, and F. H. Field, NSRDS-NBS 26, Nat. Bur. Stand., 1969.
(11) B. L. Jelus, M. S. B. Munson, and C. Fenselau, Biomed. Mass Specfrom., 1, 96 (1974). (12) I. Jardine and C. Fenselau, J. Forensic Sci., in press. (13) D. F. Hunt and J. F. Ryan, J. Chem. Soc., Chem. Commun., 620 (1972). (14) H. M. Fales in "Mass Spectrometry, Techniques and Applications", G. W. A. Milne. Ed., Wiley-lnterscience, New York, NY. 1971, p 193.
RECEIVEDfor review August 29, 1974. Accepted January 8, 1975. This research was supported by USPHS Grants GM-16492, GM-21248, KO-4-6M-70417, and by the Andrew W. Mellon Foundation.
Preconcentration and X-Ray Fluorescence Determination of Copper, Nickel, and Zinc in Sea Water Donald E. Leyden' and Thomas A. Patterson Depadment of Chemistry, University of Georgia, Athens, GA 30602
James J. Alberts2 Department of Zoology, University of Georgia, Athens, GA 30602
Of the transition elements usually determined in sea water analyses, copper, nickel, and zinc are among the most frequently investigated elements (1-6). These and other elements must be preconcentrated from the sea water to be determined by most of the available analytical methods, or separated from interferences when methods of very low limits of detection are used. To satisfy these conditions, several preconcentration or separation techniques have been employed for the determination of trace elements in saline waters. Rona et al. ( I ) simply coprecipitated the metal hydroxides with ferric hydroxide followed by neutron activation analysis. Others found that procedure gave erratic results ( 4 ) . Riley and Taylor ( 2 ) ,Windom and Smith ( 5 ) ,and Chester and Stoner (6) used the chelating ion-exchange resin Chelex-100 to preconcentrate the metal ions, followed by atomic absorption (2, 5 ) or spectrophotometric (6) determination of the metals eluted from the resin. Spencer and Brewer ( 3 )extracted the metal ions directly into methyl isobutyl ketone using ammonium pyrrolidine dithiocarbamate and aspirated this solution directly into a flame for atomic absorption. Slowey and Hood ( 4 ) first treated the sample with peroxydisulfuric acid to decompose the organic material, then extracted the metals into chloroform with diethyl dithiocarbamate. The extract was evaporated to' dryness and the metals were determined by neutron activation analysis. The separation of the desired elements from the sample matrix is necessary when neutron activation is employed because of interference from constituents such as W J , *"a, and All of the above mentioned preconcentration techniques suffer from one or more of the following disadvantages: liquid extractions involving large volume ratios; several chemical manipulations prior to analysis; small throughput and/or long analysis time; incomplete preconcentration due to loading of Chelex-100 with calcium or magnesium ions; inability to determine some elements due to interferences. Of the preconcentration techniques used, ion-exchange is one of the most practical for ship-board sampling. As much sample as desired may be passed through a column and retained for analysis in the land-based laboratory (6). CheAuthor to whom reprint requests should be sent. Present address, Argonne National Laboratory, Argonne, IL.
lex-100 has been successfully applied in this manner (2, 5, 6). A successful ion-exchange material should have a high distribution ratio for the elements of interest in oxidation states present in the sample so that recovery of those ions is quantitative. Chelex-100 has a potentially serious disadvantage since the resin has an affinity for alkaline earth metals. For example, the distribution coefficient of calcium on the resin is almost the same as that of zinc (7). Should sufficient sea water be added to a column of the resin, it is possible that the more abundant calcium ions would displace the less abundant ions such as zinc. A second disadvantage is that the resin contains an immobilized anionic group which must be provided a counter cation, which is primarily sodium after treatment with sea water. If neutron activation analysis is to be used, this sodium must be exchanged for a cation such as ammonium ion. A resin more specific for the ions of the transition metals is preferred. Recently Siggia and coworkers (8) reported a comprehensive study of a series of polyamine-polyurea resins. A resin prepared from tetraethylenepentamine (TEPA) and toluene diisocyanate showed particular promise. These resins are exceedingly simple to prepare and have a much greater affinity for transition metal ions than alkaline earths. This report describes a study of the recovery of several ions from simulated sea water using the TEPA resin described by Siggia. The use of X-ray fluorescence for the direct determination of copper, nickel, and zinc on the resin is described. Determination by X-ray fluorescence and atomic absorption of these elements in sea water samples is reported.
EXPERIMENTAL Apparatus. The X-ray determinations were performed using a Philips PW-1410 X-ray spectrograph. A LIF-200 analyzing crystal was used for all elements studied. Both a flow proportional detector using P-10 gas (Selox, Inc.) and a scintillation detector were used. The 28 angle for each peak was located by counting a t angles on either side of the tabulated 28 valve until a maximum signal was located. The K a emission line of each element was used. The optimum detector voltage and gain was selected using a Philips pha2100 pulse height analyzer scope. The instrument used is equipped with a sine potentiometer circuit so that the energy window selected is compensated for goniometer settings and rapid sequential determination of several elements may be performed. A molybdenum ANALYTICAL CHEMISTRY, VOL. 47, NO. 4, APRIL 1975
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