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Anal. Chem. 1982, 54, 1806-1811 Kong, R. C.; Lee, M. L.; Tominaga, Y.; Pratap, R.; Iwao, M.; Castle, R. N. J. Chromatogr. Sci., in press. Lysyuk, L. S.; Koroi, A. N. Chromatographia 1977, IO, 712-719. Borwitzky, H.; Schomburg, G. J. Chromatogr. 1979, 170, 99-124. Janini, G.M.; Johnston, K.; Zieiinski, W. L., Jr. Anal. Chem. 1975, 47, 670-674. Janini, G. M.; Muschik, G. M.; Zieiinski, W. L., Jr. Anal. Chem. 1978, 48, 809-813. Zielinskl, W. L., Jr.; Scanian, R. A.; Miller, M. M. J. Chromatogr. 1981, 209, 87-95. Haky, J. E.; Muschik, G. M. J. Chromatogr. 1981, 214, 181-170. Laub, R. J.; Roberts, W. L. I n "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Proceedings of the Fourth International Symposium on Polynuclear Aromatic Hydrocarbons; Bjorseth, A., Dennis, A. J., Eds.; Batteile Press: Columbus, OH, 1980; pp 25-58. Laub, R. J.; Roberts, W. L.; Smith, C. A. HRC CC, J. High Resolut. Chromatogr Chromatogr. Commun 1980, 3 , 355-356. Laub, R. J.; Roberts, W. L.; Smith, C. A. I n "Polynuclear Aromatic Hydrocarbons: Chemical Analysis and Biological Fate"; Proceedings of the Fifth International Symposium on Polynuclear Aromatic Hydrocarbons; Cooke, M. W., Dennis, A. J., Eds.; Batteiie Press: Columbus, OH, 1981; pp 287-295. Finkeimann, H.; Laub, R. J.; Roberts, W. L.; Smith, C. A. I n Polynuclear Aromatic Hydrocarbons: Chemical Analysis and Biological Fate"; Proceedings of the Sixth International Symposium on Polynuclear Aromatic Hydrocarbons; Cooke, M. W., Dennis, A. J., Eds.;
.
.
Batteiie Press: Columbus, OH, 1982, in press. (14) Apfei, M. A.; Flnkelmann, H.; Laub, R. J.; Luhmann, B.-H.; Price, A,; Roberts, W. L.; Smith, C. A., to be submitted for publication in Makromol. Chem ., Rapld Commun . (15) Laub, R. J. Am. Lab. (Fairfleld, Conn.) 1981, 13 (3), 47-56. (16) Iwao, M.; Lee, M. L.; Castle, R. N. J. Heterocycl. Chem. 1980, 17, 1259-1264. (17) Tominaga, Y.; Lee, M. L.; Castle, R. N. J. Heterocycl. Chern. 1981, 18,967-972. (18) Wiiiey, C.; Iwao, M.; Castle, R. N.; Lee, M. L. Anal. Chem. 1981, 53, 400-407. (19) Laub, R. J.; Purneil, J. H.; Summers, D. M.; Williams, P. S. J. Chromatow. 1978, 155. 1-8. (20) Lser, D. W.; Wright, 8. W.; Lee, M. L. HRC CC J. High Resoluf. Chromatogr. Chromatogr. Commun. 1981, 4, 406-407. (21) Vilialobos, R.; Brace, R. 0.; Johns, T. I n "Gas Chromatography"; Noebeis, H. J., Wail, R. F.,Brenner, N., Eds.; Academic Press: New York, 1961; pp 39-51. (22) Viilaiobos, R.; Turner, C. C. I n "Gas Chromatography"; Fowler, L., Ed.; Academic Press: New York, 1963; pp 105-118.
RECEIVED for review April 21, 1982. Accepted June 21, 1982. This work was supported by the Department of Energy, Office of Health and Environmental Research, Contract No. DEAC02-79EV10237.
Fused Silica Capillary Gas Chromatography/Negative Chemical Ionization Mass Spectrometry for Determination of Catecholamines and Their 0-Methylated Metabolites Jeffrey T. Martin, Jack D. Barchas, and Kym F. Faull" Nancy Pritzker Laboratory of Behavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305
Determination of the pentafluoropropionyl derlvatlve of normetanephrine by GC/MS In the negative chemical ionlratlon mode yields a 200-fold and 350-fold Increase In sensitivity compared with determinatlon done In the posltlve chemical lonlratlon and electron impact modes, respectlvely. I n light of this flndlng, we have prepared two classes of derlvatlves for the catecholamlnes and their 0-methyl metabolites suitable for GCIMS appllcatlons In the NCI mode. The two derlvatlratlon schemes are discussed and the NCI mass spectral characterlstlcs of the correspondlng derlvatlves are compared. I n addltlon, the pentafluoropropionyl derivatives of the catecholamlnes and their 0-methylated metabolltes were found to have vastly improved chromatographic characteristics when fused slllca capillary columns were used compared to conventlonal packed columns. This Improvement, which Is attrlbuted to the reduced chemlcal reactlvlty of the fused slllca caplllary columns, offers advantages for trace level analysls of these compounds. Finally, a practical appllcatlon of this work Is Illustrated In the form of a sensltlve assay for normetanephrlne In human cerebrosplnal fluld.
In recent years, a number of workers have attempted to correlate levels of biogenic amines in various body fluids with specific psychiatric and neurologic disease states. For some of the most intensely studied disorders, this goal has remained elusive. This is in part because of the minute amounts of these compounds found in biological fluids and because any differences in the concentrations of these compounds between
the disease state and the control population may be quite subtle. These factors underscore the need for dependable and sensitive analytical procedures. The development of gas chromatography/mass spectrometry (GC/MS) and subsequently of selective ion monitoring techniques has provided a combination of sensitivity and selectivity unequaled by other methods which have found application in this area. The recent refinements of fused silica capillary gas chromatography ( I , 2) and electron capture negative chemical ionization mass spectrometry (NCI) (3) have led to further improvements in the specificity and sensitivity attainable with GC/MS. In order to be applicable to the GC/MS method, a molecule must be sufficiently nonpolar, volatile, and thermally stable to traverse the chromatographic column. Furthermore, to attain the enhanced sensitivity available in the electron capture NCI mode, the molecule must have a positive electron affinity. Few biological molecules meet these requirements in and of themselves. Accordingly, chemical derivatization is necessary. Derivatizing reactions conferring both volatility and electron capturing ability have found wide application in the area of gas chromatography with electron capture detection. Thus, a great deal of literature is already available regarding the preparation of derivatives which are potentially suitable for NCI GC/MS work. Perfluoroacylating reagents have perhaps found the most widespread utility as derivatizing reagents for electron capture gas chromatographic applications. These derivatives owe much of their prominence in this area to the volatility and electron capturing properties conferred by the perfluorocarbon group and the presence of the carbonyl group which synergistically helps to delocalize the captured electron and stabilize
0003-2700/82/0354-1806$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
the negatively charged ion ( 4 ) . In particular, the perfluoro acid anhydrides, trifluoroacetic anhydride (TFAA), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric anhydride (HFBA) have enjoyed considerable popularity because they facilitate rapid, complete, one-step derivatization reactions for a wide variety of organic molecules including the catecholamines and their metabolites (5-7). Other workers using electron capture detectors have noted markedly enhanced sensitivity with pentafluorophenyl derivatives, particularly pentafluorobenzamide derivatiives resulting from reaction of pentafluorobenzoyl chloride with an amine (8,9). In extending this application to electron capture NCI GC/MS, one group reported an (astonishingsensitivity increase with the pentafluorobenzyliniine-trimethylsilyl derivative of dopamine in which they could detect 25 fg of the derivatized compound (10). Fused silica capillary columns probably provide optimal overall performance for chromatographic resolution, chemical inertness, and sensitivity. The chemical inertness of these columns is attributable to the diminished number of surface silanol groups and the extremely low metal oxide content of the fused silica glass (1). Chemical inertmess is particularly valuable for the analysis of small amounts of derivatives which show a tendency for adsorption to chrom,atographic surfaces, like, for example, derivatives of the catecholamines. In light of these findings, we have studied the gas chromatographic and NCI mass spectral charalcteristicsof the PFP and tert-butyldimethylsilyl-pentafluorobenzyl(TBDMSPFB) derivatives of the catecholamines and their 0-methyl metabolites. In this paper we describe the results of this work and discuss in detail the analytical potential of electron capture NCI and fused silica capillary chromatography for the trace analysis of catecholamines and their 0-methylated metabolites. Furthermore, a practical application of this work is presented in the form of a sensitive and specific assay for normetanephrine in human cerebrospinal fluid. The results of this work give reason to believe that the combination of electron capture NCI CX/MS and fused silica capillary gas chromatography has applicability to other areas of analytical chemistry which require increased sensitivity and specificity.
EXPERIMENTAL SECTION Reagents. Pentafluoropropionic anhydride (PFPA) was obtained from Pierce Chemical Co. (Rockford, IL), pentafluorobenzoyl chloride, dopamine hydrochloride, norepinephrine hydrochloride, and epinephrine bitratrate were from Regis Chemical Co. (Morton Grove, IL),tlvt-butyldimethylchlorosilane/imidazole reagent was from Applied Science (State College, PA), metanephrine hydrochloride, normetanephrine hydrochloride, and 3-methoxytyraminehydrochloride were from Sigma Chemical Co. (St. Louis, MO). All other reagents and solvents were of the highest purity attainablle. Instrumentation. The work was carried out on a HewlettPackard 5985B GC/MS system. The mass spectrometer was equipped with a dual electron impact (EI)/chemical ionization (CI) source with the capability of analyzing positively and negatively charged ions. Thie gas chromatograph was equipped for packed and capillary columns. A covalently bonded fused silica capillary column (DB-5,30 m X 0.2 mm i.d., J and W Scientific, Rancho Cordova, CA) wa3 used by inserting the end of the column into the source of the mass spectrometer. Helium (1mL/min) was used as carrier gas, and methane was used as the CI reagent gas. The reagent gas was delivered to the source of the mass spectrometer coaxially with the capillary column. The packed column (2 m X 2 mm i.d.) was filled with 3% SP2250 on 80/100 mesh Supelcoport (Supeloo Inc., PA). When this column was used with the mass spectrometer in the E1 mode, helium (30 mL/min) was used as the carrier gas, much of which was removed from the column effluent via a single-stage glass jet separator. When the packed column was used with the mass spectrometer in the CI mode, methane was used as a carrier/reagent gas which was transferred directly to tho source of the masw spectrometer. With
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both the packed and capillary columns, the reagent gas pressure in the source of the mass spectrometer was adjusted to 1.4 and 1.0 torr in the positive and negative ion modes, respectively. At these pressures, the greatest sensitivity was found when tuning the mass spectrometer. The GC injector port and interface region between the gas chromatograph and mass spectrometer were maintained at 300 "C. Unless otherwise noted, the column oven was maintained at 100 "C for the first minute following injection and then raised to a maximum of 300 "C at a rate of 10 'C/min (packed column) or 30 'C/min (capillary column). When sensitivity comparisons were made between the EI, PCI, and NCI modes, the same electron multiplier voltages were used. Furthermore, the instrument was adjusted so that the mass spectral resolution was the same in the three modes. In the positive ion mode the mass spectrometer was tuned by using perfluorotri-n-butylamine for calibration, by focusing on the ions at m / z 69,219, and 502 in the E1 mode, and the ions at m/z 219, 414, and 614 in the CI mode. In each case, the parameters of the source were adjusted so that the ratios of the ions were approximately 100/75/3 and 10/40/6 for E1 and PCI, respectively. In the NCI mode the mass spectrometer was tuned by using the ions at m/z 182, from benzophenone, and 452 and 633 from perfluorotri-n-butylamine. In this mode the parameters of the source of the mass spectrometer were manipulated to maximize the signal at m / z 182 obtained from benzophenone. Derivatization. PFP Derivatives. The dried CSF extracts or HC1 salts of the pure compounds were heated with pentafluoropropionic anhydride (PFPA) (100 pL) for 15 min at 7 5 "C in a stoppered 15-mL conical test tube. The residual reagent was then evaporated under a stream of nitrogen and the remaining residue was taken up in heptane to the desired concentration. TBDMS-PFB Derivatives ( 8 , l l ) . Pure amine hydrochloride (100 pg-1 mg) or a mixture containing the amines was heated with the tert-butyldimethylchlorosilane/imidazolereagent (100 pL) (packaged as 1.0 mmol tert-butyldimethylchlorosilane and 2.5 mmol of imidazole in 1mL of dimethylformamide) at 75 "C for 30 min in a stoppered 15-mL conical test tube. The solvent was then evaporated under a stream of nitrogen and the residue was taken up in heptane (500 pL) and washed with water (500 pL). The heptane layer was then separated and the solvent was evaporated under a stream of nitrogen. The residue thus obtained was heated with ethyl acetate containing 0.2% triethylamine (100 pL) and perfluorobenzoyl chloride (25 pL) for 30 min at 75 "C in a stoppered test tube. Solvent and residual reagent were then evaporated under a stream of nitrogen and the remaining residue was taken up in heptane to the desired concentration. Preparation of Deuterated Normetanephrine. This was achieved by using a previously described procedure (12) in which normetanephrine hydrochloride (10 mg) was incubated for 1 month at room temperature with deuterium oxide (1mL) and freshly reduced platinum catalyst (derived from 33 mg of Pt02) in an evacuated sealed glass ampule. The contents of the ampule were then filtered, and the resulting yellow-brown filtrate was passed through a C-18 reverse-phase Sep-PAK cartridge (Waters Associates) which had been previously washed with methanol (20 mL) followed by water (15 mL). The cartridge was eluted with 5-mL volumes of water followed by 5-mL volumes of water containing increasing concentrations of methanol. Aliquots (10 pL) of each fraction were removed, taken to dryness under a stream of nitrogen and converted to the PFP derivative, and analyzed by GC/MS. Deuterated normetanephrine was located in the water fraction which was then lyophilized to dryness to give a white powder. Examination of the E1 (Figure 1)and CI (methane) mass spectra of the PFP derivative revealed a pentadeuterated product (normetanephrine-d&with two deuterium atoms on the aromatic ring and one and two deuterium atoms on the 0-and a-carbon atoms, respectively. The powder was redissolved in deuterium oxide (1 mL) and serial dilutions were made with additional deuterium oxide. The solutions were stored frozen at -10 "C. Selected ion monitoring, using the nondeuterated compound as internal standard, was employed to determine the concentration of deuterated normetanephrine in solution. Extraction of Human CSF Samples. To a 1.0-mL aliquot of CSF is added 100 pL of deuterium oxide containing deuterated normetanephrine (approximately 1ng). The samples were then
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
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AI PACKED COLUMN SP2250 2M x 2 mm id
NORMETANEPHRINE- PFP
,
c z
c
>
F
100
W
50
9 3
0 50 100
LXd 0
AUTHENTIC
60
120
160
520 m/e
Flgure 1. E1 mass spectra of the PFP derivatives of deuterated (top) and nondeuterated normetanephrine (bottom), and the proposed fragmentation pattern of the molecule.
acidified with 6 N HCl (10 yL) and extracted twice with ethyl acetate (4 mL each time) using centrifugation to separate the phases (3000g, 5 min). The organic phases are removed and subsequently used for the analysis of the acidic and alcoholic metabolites of the biogenic amines (13). The aqueous fraction was then lyophilized to dryness, and the residue redissolved in water (1mL) and brought to pH 12-13 by the addition of NaOH (10 N, 20 yL) before being passed through an anion exchange column (Biorad AG 1-X2, 200-400 mesh, acetate form, 5.2 cm X 0.7 cm, 2 mL bed volume). The column was then washed with dilute alkali (twice with 1.0 mL of water brought to pH 12-13 by addition of NaOH), water (three times with 5 mL), and acetic acid (1N, 0.8 mL). The column was eluted with additional acetic acid (1 N, 3 mL), the eluate lyophilized to dryness, and the resulting residue washed to the bottom of the tubes by addition of 80% methanol (100 yL) which was evaporated in a stream of nitrogen. The drying process was completed by addition of benzene (50 yL) which was also evaporated in a stream of nitrogen. The samples were then ready for chemical derivatization. Preparation of Standard Curves. With each set of samples, a standard curve was prepared for by addition of the internal standard (100 yL of deuterium oxide containing 1 ng of normetanephrine-d6)and known amounts of normetanephrine hydrochloride (0,250,500,1000, and 2500 pg) to samples of artificial CSF (containing millimolar concentrations of NaCl (140), KC1 (3.4), CaClz (1.3),MgC12.7Hz0(0.541, urea (0.22),NaH2P04(0.25), Na2HP04(0.25), glucose (3.33), and NaHC03 (3.57)). These standard samples were then treated in the manner already described for the human CSF samples.
RESULTS AND DISCUSSION Comparison between Fused Silica Capillary and Packed GC Columns. Representative chromatograms comparing the behavior of the PFP derivatives of the catecholamines and their 0-methylated metabolites in the E1 mode on a packed and fused silica capillary column are shown in Figure 2. The packed column showed a strong tendency to adsorb the derivatives. As previously reported (14, 15), this tendency was particularly dramatic with the derivatives of the catecholamines. With a new freshly conditioned packed column maximal sensitivity was only reached after the active sites on the column and transfer lines were saturated by injections of relatively large amounts of the derivatized standards (up to 250 ng) in heptane containing 1% PFPA. Furthermore, maximal sensitivity was only maintained if samples were dissolved in solvent containing 1% of the anhydride. Failure to include the anhydride resulted in an immediate loss of sensitivity of at least 100-fold. In addition, it was difficult to obtain a good blank with these columns as injection of solvent alone invariably produced small peaks at the retention times of the authent,ic compounds. These characteristics discouraged the use of these columns for trace level work. In contrast, fused silica capillary columns showed a minimal tendency for adsorption of the derivatives. With these columns it was not necessary to prime the system with large amounts of standards nor was it necessary to dissolve the
I
10 5
11
11 5
12
I
RETENTION TIME IN MINUTES
Comparison of selected ion traces obtained on packed (A) and fused silica capillary (B) columns of 100 pg of the PFP derivatives of the catecholamines, 3-methoxytyramine, and metanephrine. Chromatographic conditions were as described in the text except that in both cases an initial oven temperature of 100 O C was used, which 1 min after injection was increased at 10 'C/min. For analysis on the capillary column the sample was dissolved in heptane; for analysis on the packed column the sample was dissolved in heptane containing Flgure 2.
1 % PFPA.
derivatives in solvent containing the anhydride. By use of a new column the derivatives were detected in the eluate after the first injection which contained low nanogram quantities dissolved in heptane. Furthermore, the problem of memory effects was greatly diminished. Injection of solvent alone after the analysis of nanogram quantities of the derivatives invariably produced good blanks. An additional advantage of the fused silica columns was a reduced background signal in the mass spectrometer. These columns also produced sharper peaks with mean peak widths of the catecholamines and their metabolites reduced by a factor of 2 to 3 compared to the peak widths obtained from packed columns. These factors coupled with the reduced adsorption of the derivatives combine to significantly improve signal to noise ratios and reduce the smallest amount of any of the derivatives which could be detected with confidence. Mass Spectral Characteristics of the Derivatives. The NCI mass spectra of the PFP derivatives of dopamine, epinephrine, norepineprine, and their 0-methyl metabolites show marked variation in fragmentation pattern and relative intensities of the various fragments depending on the presence or absence of a @-hydroxyor 0-inethyl group in the parent compound (Figure 3). Whereas the NCI spectra of metanephrine, normetanephrine, epinephrine, and norepinephrine demonstrate dominant molecular ions, those of dopamine and 3-methoxytyramine show loss of H F and C2F,COH groups, respectively. The characteristic m/z values for the dominant species in the NCI mass spectra of each derivative provide ideal ions for specific analysis during selective ion monitoring. The NCI mass spectra of the TBDMS-PFB derivatives of the catecholamines and their 0-methyl metabolites are presented in Figure 4. For preparation of this class of derivatives the hydroxyl functionalities were reacted with TBDMS reagent (a silyating reagent which is nonreactive toward amines) prior to reaction with PFB, in order to avoid excessively bulky and nonvolatile derivatives. In this series, only metanephrine shows the molecular ion as the dominant species. The major ion for normetanephrine and norepinephrine results from cleavage between the nitrogen and
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
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Table I. Sensitivity Comparisons in the NCI Mode between the PFP and TBDMS-PFB Derivatives of the Catecholamines and Their 0-Methyl Metabolites sensitivity ratio a PFPJ TBDMSPFB
ions ( m / z ) TBDMSPFP PFB derivderivative ative dopamine norepinephrine epinephrine 3-methoxytyramine normetanephrine metanephrine
571 753
442 439
1.8
7 61 311
586 455
8.4 8.2
621
339
1.4
635
619
6.1
2.1
The sensitivity ratio was determined by derivatizing a known amount of each compound under optimal conditions and comparing the amount of each derivative necessary to produce an equal response during selected ion monitoring of the designated ions on a [used silica capillary column,
636 M - I
248 285 306
120 180 200 240 ZBI 120 JBO 400 440 680 520 550 BOO ea0 880 720 ,BO
mle
E
Dl 180
, 200
1-I 4
296 1
I p -
,
240
280
120
360
400
410
480
520
560
600
me
Figure 3.
NCI (methane) mass spectra of the PFP derivatives of the
catecholamines and their 0-methylated metabolites.
mie
Figure 4. NCI (methane) mass spectra of the TBDMS-PFB derivatives of the catecholamines andl their 0-methylated metabolites.
the a-carbon atom with concurrent transfer of a proton to the observed fragment. The dominant species for the derivatives
Flgure 5. PCI (methane) mass spectrum of normetanephrine-PFP.
of epinephrine and dopamine represent loss of 133 molecular weight units from the parent ion. In the absence of accurate mass measurements it would be difficult to unequivocally establish the structure of this fragment ion. However, loss of two tert-butyl groups and a fluorine atom is an attractive possibility. The NCI spectrum of the TBDMS-PFB derivative of 3-methoxytyramine shows loss of H F as the major fragment. The relative sensitivities of the PFP and the TBDMS-PFB derivatives of the catecholamines and their 0-methyl metabolites are compared in Table I. In each case, the values were arrived at by derivatizing a known amount of the amine by both methods under optimal conditions and then comparing the amount of each derivative necessary to get an equal response by selectively monitoring the dominant ionic species from the NCI spectrum of the corresponding derivative. As demonstrated, the PFP derivative yielded a more sensitive assay in every case, ranging from a 1.4-fold increase in sensitivity for normetanephrine to an 8.4-fold increase for epinephrine. This may be secondary to a higher yielding derivatization reaction or, more likely, to improved NCI mass spectral characteristics with the characteristic fragments carrying a greater proportion of the ion current. A representative PCI (methane) mass spectrum of the PFP derivative of normetanephrine is shown in Figure 5. Comparing this spectrum with the E1 and NCI spectra for this derivative (Figures 1 and 3) shows that the fragmentating pattern varies depending upon the mode of ion generation. In PCI, the dominant ion a t m / z 458 results from loss of pentafluoropropionic acid from the protonated molecular ion. In the E1 mode, the major fragment results from cleavage between the CY- and @-carbonatoms, with charge retention on the amino end of the molecule resulting in the observed fragment at m / z 176. The relative sensitivity of each mode was determined by comparing the amount of derivatized normetanephrine necessary to get equal responses when
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
monitoring the ions at m/z 176,458, and 621 in the EI, PCI, and NCI modes, respectively. By this method, the NCI mode was found to be 350 and 200 times more sensitive than the E1 and PCI modes, respectively. Furthermore, similar experiments with the PFP derivatives of the catecholamines, metanephrine and 3-methoxytyramine, suggested the NCI mode offered similar increases in sensitivity over the E1 and PCI modes. We have also studied the GC and mass spectral characteristics of the methyl ether-PFP derivatives of metanephrine and normetanephrine. These derivatives are formed by treating the parent amines with methanolic/HCl prior to reacting them with PFPA (7). This procedure results in conversion of the hydroxyl group on the @-carbonatom to a methyl ether, while the phenolic hydroxyl groups and the amines are converted to the pentafluoropropionyl forms. These derivatives are stable in solution at room temperature for several days and have good GC properties with longer retention times than the corresponding PFP derivatives. Their mass spectral characteristics are particularly suitable for selected ion monitoring in the E1 mode. The E1 mass spectra are extraordinarily simple, consisting of a single dominant fragment ion which results from cleavage of the bond between the a-and 8-carbon atoms with charge retention on the aromatic nucleus. However, our experience with these derivatives for quantitative work has demonstrated that caution must be exercised in the choice of internal standard. Ring-deuterated analogues of metanephrine and normetanephrine lose the label by back-exchange with protons during methanolic/HCl treatment, a phenomena which has also been observed with the ethyl ether-PFP derivatives of epinephrine and norepinephrine (16). This characteristic discouraged further use of these derivatives for quantitative work. Quantitative Analysis of NMN. In attempting to exploit the sensitivity and specificity increases available during NCI analysis of the PFP derivatives, we have developed a quantitative procedure for normetanephrine. The recovery of this metabolite during the sample workup was measured by addition of 2.5 ng of normetanephrine (HC1 salt) to 1.0 mL of artificial CSF. The samples were prepared as described and the peak intensities compared with those derived from the same amount of normetanephrine which had been derivatized directly. This approach yielded a mean percentage recovery of 86%. The quantitative assay was based on selected ion recordings in the NCI mode of the molecular anions a t m / z 621 (normetanephrine-do) and 626 (normetanephrine-d6). In the concentration range from 5 ng to 150 pg we have found this procedure produces linear standard curves. Selected ion traces obtained during the analysis of 10% of the final derivatized extract from 1.0-mL samples of human lumbar CSF show peaks in the corresponding ion traces for the undeuterated and pentadeuterated isomers of normetanephrine. The area of these peaks is estimated to average between 20- and 100-fold greater than the area of the smallest measurable peak. Inverse linear regression analysis has been used to calculate normetanephrine concentrations in CSF samples. The results show a wide concentration range between different subjects ranging from 55 pg to 7 ng (Table 11). The accuracy and reliability of the method are reflected in the variability between data obtained from replicate samples. Coefficients of variation of less than 20% were obtained in 62% of the cases examined. The combination of fused silica capillary gas chromatography with NCI mass spectrometry provides an analytical approach which is probably unequaled for combined specificity and sensitivity. In exploring the potential of this approach for the analysis of catecholamines and 0-methylated metabolites, we have compared the gas chromatographic and mass
Table 11. Concentrations of Normetanephrine in Human Lumbar CSF mean
subject 1 2 3 4 5 6
7
concn, ng/mL 1.21 1.23 1.01 0.06 0.14 1.20 2.27
subject 8
9 10 11 12 13
mean concn, ng/mL 2.42 6.83 3.82 2.70 3.09 2.65
a The figure for each subject represents the mean of measurements made on duplicate samples. The mean Concentration for the 1 3 subjects was 2.20 ng/mL (i'1.79, standard deviation), while the mean of the differences between the 1 3 pairs of duplicate measurements was 0.76 (i 1.1,standard deviation).
spectral characteristics of different classes of derivatives and have extended this work to include a quantitative procedure for the analysis of normetanephrine in small samples of human lumbar CSF. In addition to providing improved chromatographic resolution and greater sensitivity, the chemical inertness of the fused silica columns virtually eliminates the problem of memory effects. This problem plagues trace level work with these compounds on packed columns where special precautions need to be taken before reliable and reproducible results are obtained (see ref 17 for a discussion of this subject). Furthermore, the flexibility and durability of the fused silica capillary columns make them easy to use, while the low bleed rates result in low base lines and minimal contamination of the source of the mass spectrometer. Exploitation of the advantages of NCI GC/MS necessitates the use of derivatives suitable for electron capture detection. Of the derivatives compared, those produced by treatment with PFPA were found to be most suitable. They were simple to prepare in a one-step reaction and the excess reagent and reaction byproducts were easily removed by evaporation in a stream of nitrogen. Furthermore, the reaction yields derivatives of both primary and secondary amines and the resulting derivatives are sufficiently volatile to allow for their relatively rapid elution from the gas chromatograph column. Most importantly, these derivatives provided greatest sensitivity in the NCI mode which was 2 orders of magnitude better than that obtained in either the E1 or PCI modes. In addition, the derivative of each compound yielded characteristic and unique diagnostic ions of high m / z values ideal for selected ion monitoring. Although the pentafluorobenzyl Schiff base derivative of dopamine has been shown to give excellent sensitivity in the NCI mode (IO),this derivative is not applicable to the secondary amines epinephrine and metanephrine. The PFB derivatives of the catecholamines are ideally suited for electron capture GC detection (17). However, the presence of three or four PFB groups in each compound has the disadvantage of creating derivatives which have long retention times. At least four other GC/MS assays have been described for normetanephrine (18-21). However, the procedure described here has the advantage of increased sensitivity. Furthermore, we have developed this procedure in conjunction with assays for other biogenic amine metabolites (13) and y-aminobutyric acid (22). The organic acid extract not used here contains the 0-methylated-deaminated metabolites, while y-aminobutyric acid is copurified with normetanephrine. Separation of the eluate from the ion exchange column into two fractions, one for y-aminobutyric acid and the other for normetanephrine, allows the analysis of all compounds in the same CSF sample.
Anal. Chem. 1982. 54. 1811-1814
Our experience with this assay on a routine basis has shown that occasional samples fail to show acceptable agreement between replicates. This problem is related to the difficulty occasionally experiencedl in obtaining accurate measurements of peak area (or height) during selected ion monitoring in the NCI mode. This is attributable to an uneven base line which can occur during Chromatography of impure extracts. Further work is in progress to eliminate this prolblem by partial purification of the derivatieed samples (23). Further work is also in progress to expand tlhe quantitative assay to also include 3-methoxytyramine and metanephrine.
LITERATURE CITED
.
.
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RECEIVED for review April 29, 1982. Accepted June 22,1982. This work was carried out with financial support from an NIMH Program-Project Grant (MH 23861))an NIMH Mental Health Clinical Research Center Grant (MH 3084)) and an ONR Selected Research Opportunity Award (SRO001:N00014-79-C-0796).
Gas Chromatographic Determination of Pyrrolizidine Alkaloids in Goat’s Milk Max L. Deinzer,” Brian L. Arbogast, and Donald R. Buhler Environmental Health Scislnces Center, Depalrtment of Agrlcukural Chemistry, Oregon State University, Corvallis, Oregon 9733 1
Peter R. Cheeke Department of Animal Science, Oregon Stato University, Corvallis, Oregon 9733 1
A procedure was developed for the analysis of pyrrollrldine alkaloids (PAS) in milk. Milk samples were treated with base which converted the PAS to retronecine. Retronecine was recovered from the flltered sample by ion exchange chromatography, cleaned up by reversed-phase and porous polymer chromatography, and determined as the bls( heptafiuorobutyrate) by eleotron capture gas chromatography. Recoveries of 80-9096 were obtained from control milk samples fortifled with 0.2-1.0 ppm of the PA jacoblne. Sensitivity of the method was estimated to be 0.1 ppm. By use of this method, PAS have been determined in the milk from goats that were fed 1% of their body weights per day of dried flowering tops of Senedo lacobaea L . (tansy ragwort). The concentration range of alkaloids found in the milk was 0.33-0.81 ppm.
Pyrrolizidine alkaloids (PAS) are found in a wide variety of plant species of genera including Senecio, Crotalaria, Heliotropium, ErechtiEtes, Trichodesm.a, and Amsinckia.
These plants are widely distributed throughout the world and have been of particular concern to stockmen for at least 150 years because of their hepatotoxic effect in cattle and horses (172). In the Pacific Northwest, tansy ragwort (Senecio jacobaea L.),which contains the alkaloids jacobine, jacoline, senecionine, seneciphylline (Figure I), and others (3),has caused significant economic losses to ranchers and dairy farmers through livestock poisoning ( 4 ) . More recently, PAS have been detected in honey from bees pollinating tansy ragwort flowers (5, 6) and in milk from cattle that were deliberately exposed to ragwort (7,8). Since these alkaloids have been shown to be carcinogenic (9, IO), mutagenic ( I I ) , and teratogenic (12)in animals, there is concern for the consequences to the consumer of ingesting contaminated agricultural products. Large numbers of people have been poisoned through consumption of harvested cereal and grain crops contaminated with plants containing pyrrolizidine alkaloids (13,14). And recently the administration of infusions of Senecio longilobus as a cold remedy to two infants in the southwestern United States resulted in severe hepatic veno-occlusive disease that was fatal to one of them (15). Experimental animals fed diets containing
0003-2700/82/0354-1811$01.25/00 1982 American Chemlcal Soclety
