Instrumentation
Radiogas Chromatography/ Mass Spectrometry Iain M. Campbell Department of Biological Sciences University of Pittsburgh, GSPH 130 DeSoto Street Pittsburgh, Pa. 15261
In many areas of research there is a need to analyze complex mixtures of nonpolymeric substances for the identity, level of occurrence, and sta ble and/or radioisotope content of each substance. This necessity is most often encountered where in vivo iso tope incorporation experiments are run; for instance, in studies of: • Drug or toxicant metabolism • Cell metabolite biosynthesis or degradation • How drugs or toxicants interact/ interfere with cell metabolism • How nutritional factors or in duced mutations influence small mol ecule metabolism • How small molecule metabolism correlates with development and mor phogenesis. In these assays, comprehensiveness, speed, and sensitivity are usually im portant. Of all the analytical methods avail able, radiogas chromatography/mass spectrometry (RGC/MS) is the only one wherein the whole analysis can be conducted with a single instrument. T h e gas chromatographic module sep arates the mixture into its compo nents; the gas flow counter assays ra dioactivity in each; and the mass spec trometer, functions as a specific and/ or nonspecific mass detector, mea sures stable isotope enrichment and provides structure definition of each component. Particularly if some data collection and processing are available (see later), an RGC/MS can handle mixtures containing many hundreds of components in the space of a few hours, yielding accurate mass and iso tope content data for each duly identi fied component in the mixture. Despite their considerable poten tial, RGC/MS-based methods have 1012 A ·
not been exploited extensively since Hobbs (/) announced the construction of the first R G C / M S unit in 1970. T h e purpose of this article is to summarize how an RGC/MS unit is best con structed and employed, and to provide examples of how effective the technol ogy can be in monitoring in vivo iso tope incorporation experiments.
Construction of an RGC/MS Unit To the best of our knowledge, there are four RGC/MS units currently op erating in the United States (1-4). Their construction involved the cou pling of a commercial or custom-made gas flow radioactivity counter to a commercial gas chromatograph/mass spectrometer (GC/MS). The MS mod ule of the GC/MS can be of the mag netic sector or of the quadrupole type. Theoretically, there are two ways in which the coupling can be per formed; the counter can be placed in parallel or in series with the mass spectrometer. Commercially available gas flow counters are exclusively of the combustion type and convert organic materials to CO2 and H 2 0 prior to proportional counting (see basic layout on the left-hand side of Figure 1). Parallel coupling of this type of counter to a GC/MS is essential. Fig ure 1 shows how coupling is achieved. A splitter is inserted between the ef fluent end of the GC column and the carrier gas separator. Although a fixed-ratio splitter will serve, a vari able-ratio splitter is better. The addi tional gas stream that the splitter has created is led by means of a heated line from the oven of the GC to the combustion furnace of the counter. T h e path length to the counter should be held to a minimum. The delay shown in Figure 1 as being inserted
ANALYTICAL CHEMISTRY, VOL. 5 1 , NO. 9, AUGUST 1979
between the splitter and the entrance to the carrier gas separator serves to equalize path lengths to the ion source of the mass spectrometer and to the counter tube. The delay can be elimi nated if a data system is available to correlate mass and radioactivity traces following collection. T h e alternative and potentially more efficient series-coupled RGC/ MS design has not yet been imple mented. It involves placing a counting tube between the GC and MS mod ules. The tube must run at elevated temperatures and deal with organic substances without prior combustion. Such counting tubes exist (5, 6'). Since in this design, counter quench gas en ters the MS, the series-coupled RGC/ MS will work best in the chemical ion ization mode with the counter quench gas doubling as the chemical ioniza tion reagent gas. Whatever the design of the RGC/ MS, provision must be made to dis pose of the radioactive exhaust gases. With 14C in the parallel-coupled sys tem, the bulk of the isotope can be trapped as carbonate following issue from the counter tube. The labeled solid residue can then be disposed. T h e remainder exits the RGC/MS unit in the fore pump exhaust gases and should be vented to an authorized fume hood. Were the series-coupled principle to be implemented, all the radioactivity would exit through the pumping system. Use of sealed pumps and venting them through an autho rized fume hood, with or without prior chemical scrubbing (CO2 —- CO :t 2 ~ = solid), would be essential. I f ' Ή is used, radioactive hydrogen (obtained by reduction of the H^O of combus tion) emerges from pumps and count ing tube vents. These exit points 0003-2700/79/0351-1012 A $01.00/0 © 1979 A m e r i c a n Chemical Society
GC Injector
Current Status and Future Prospects
GC Column
Heated Line
should be exhausted to a fume hood. Although this is not the place to discuss the fine points of RGC/MS setup and maintenance, potential builders of an RGC/MS might benefit from two observations. Firstly, it is essential to have a multieomponent mass and radioactivity standard that can be used routinely to check out and calibrate the RGC/MS unit. Using a bootstrapping approach, gas flows can be varied so that the sensitivities of the mass spectrometer as a mass detector and of the gas flow counter are maximized. Thereafter, the standard can be used to ensure that maximal performance is being maintained. We use a mixture of n-alkenes and rc-alcohols (C12-C19, the "ene-ol" standard) in which three of the alcohols are radiolabeled (2). T h e second observation concerns unit computerization. While computerization is not essential for effective operation, it is our experience that as the full potential of the unit is realized, data collection and processing become the rate-limiting steps. Some six years of software development have already been completed in our laboratory which potential users of an R G C / M S need not duplicate. Our programs RADGLC and RADSIM are available in FORTRAN IV at no cost. Details of RADGLC have appeared in the literature (7); RADSIM is an extension of RADGLC to include up to three channels of selected ion monitoring. Performance That Can Be Expected from a Typical RGC/MS Unit Potential users of RGC/MS technology can be convinced readily that: • A standard packed gas chromato-
Combustion Furnace (CuO)
Hydrogen Inlet Reduction Furnace (Fe)
MS Carrier Gas Separator
MS Ion Source
Quench Gas Inlet
Counting Tube
MS Analyzer
Radioactivity Trap
To Fume Hood Figure 1. Block diagram of combustion mode gas flow counter in parallel with a GC/MS. Two left-hand panels show components of counter ANALYTICAL CHEMISTRY, VOL. 5 1 , NO. 9, AUGUST 1979 ·
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graphic column with suitable temper ature programming will provide at least partial resolution of the compo nents of a 100+ component mixture. • A mass spectrometer total ion current detector will, for a given com ponent, give a reproducible signal pro portional over a wide range to the mass of that component in the gas stream: precision ±10% or better. • With selected ion current moni toring, partially resolved GC peaks can be resolved fully, and reproducible mass quantitation of selected GC ef fluent components at the picomolar level can be made: precision ±10% or better. • In the vast majority of cases, the combination of the GC retention time with the mass spectrum of any com pound defines the structure of the compound uniquely. • Mass spectral ion intensities yield stable isotope enrichments. These facts are the operating princi ples of GC/MS. The additional oper ating principle needed in dealing with RGC/MS is that a radioactivity flow counter is reproducible, is efficient, and within the frame of reference of an experiment that is being analyzed exclusively by RGC/MS, is effective in measuring radioactivity. T h e reproducibility and efficiency issues are easily addressed. Provided gas flow, quench gas/carrier gas com position ratio, and high voltage are kept constant (factors that all should be under control in a quality commer cial or custom gas flow counter), preci sion should be no worse than ±10% above the natural statistical variance
in measuring a random event. Ef ficiencies of modern gas flow propor tional counters can be made better than 80% for 14 C and than 50% for ;i H. Further justification of the efficiency of gas flow counters is available in the review by Matucha and Smolkova (8) and in its extensive bibliography, and in the recent theoretical paper of Kiricsi et al. (9). The issue of gas flow counter effec tiveness requires more extensive dis cussion. Critics compare the gas flow counter to the scintillation counter and note that the residence time of a sample in a gas flow counter is usually 1 min or less whereas it can be 20 min or more in a scintillation counter. It is obvious that the flow counter will never be able to compete favorably with a scintillation counter under cir cumstances where the latter instru ment is forced, for instance, to collect 20-min counts from a 100-mg sample to obtain acceptable counting statis tics. It should be equally obvious that in vivo incorporation experimental protocols that were optimized to pro cure the 100 mg of sample in pure form for scintillation counting may not be optimal for an RGC/MS exper iment. Different experimental designs are appropriate to each counter. An example makes this latter point more forcefully. Consider a situation where there is need to assay for mass and radioactivity, 20 compounds in a biological extract. Assume that all 20 occur at a level of 10 _ 2 % biomass weight and that all 20 have been la beled to a specific activity defined by the ratio: total dose of isotope fed/bio-
Table 1. Comparison of Effectiveness of Scintillation and Gas Flow Counters in Assaying Various Scales of Isotope Incorporation Experiment Scale 1
2
Biomass weight used in 50 g experiment Component level (10~ 2 % 5 mg biomass weight) Component specific 2000 activity (calculated as dpm/mg 8 10 dpm/biomass weight)
5g 500 Mg
3
4
500 mg
50 mg
50 Mg
5 Mg
5
β
0.5 mg
5 mg
50 ng
0.5 Mg
20 000 200 2000 20 000 dpm/mg dpm/Mg dpm/Mg dpm/Mg
200 dpm/ng
Counting statisticsa as a function of amount counted for each of the six scales: Amount counted
200 μς 20 Mg 2M9 0.2 M9 20 ng
Counting statistics (upper figure corresponds to a 20-mln count; lower to % mln) 1 2 3 4 5 β
2%/14% 7%/44% 22%/140% Both high Both high
0.7%/4% 2%/14% 7%/44% 22%/high Both high
0.7%/4% 2%/14% 7%/44% 22%/high
0.7%/4% 2%/14% 7%/44%
0.7%/4% 2%/14%
* Obtained as described by Cooper (29).
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0.7%/4%
-
mass weight. Table I shows for a series of biomass weights, the absolute level of occurrence of each component, its specific activity, and the probable error (95% confidence) of counting 200-, 20-, 2-, 0.2-, and 0.02-Mg samples for periods of 20 and 1/2 min. The longer interval is taken as being char acteristic of the scintillation counter, the shorter of the gas flow counter. Entries are omitted if the biomass would not provide sufficient material to count the components at the stated level. In all experiments a dose of 10 8 dpm is administered. Table I indicates t h a t the scintillation counter can be made to give good statistics at all bio mass levels. The flow counter can also be made to give acceptable counting statistics at all but the 50-g biomass level (at the 5-g level, for instance, counting 200 Mg of each compound). It must be remembered, however, t h a t factors other than counting sta tistics enter into judging the relative effectiveness of scintillation and flow counters. It is important to ask what is involved in going from the stage of a substance being a component in a mixture to the stage of having a known amount of a pure, structurally defined material in the counting chamber. With the RGC/MS ap proach, this is not a problem. The GC module can operate on the crude ex tract or on a very grossly performed fractionation thereof; the counter will efficiently accept, count, and relate these counts to each separated compo nent; the total ion current monitor will record the mass precisely over the range 200 mg —• 20 ng and below; and the MS module will record a struc ture-definitive mass spectrum to pro vide positive identification and to act as a purity check (in the selection ion monitoring mode, the MS could also act as a very selective and sensitive detector). Except for the 50-g biomass sample (which incidently presents the machine with a vast excess of sample), the 20-component analysis could be accomplished in a few hours. More over, it would be easy to repeat the analysis 10-50 times in succession. For methods not based on RGC/ MS, the same happy outlook might not prevail. Classical, nonintegrated methods based on column or layer chromatography, crystallization to constant specific activity, and gravi metric assay of samples prior to count ing would be hard pressed to cope with all but the 50-g biomass sample. The needed precision and accuracy could be obtained, but it would take time. The above example shows that while a gas flow counter may not be as effective as a scintillation counter can be made to be in measuring low specific activities, experiments can be
Figure 2. Mass spectra (70 eV) of dimethyl ester of authentic 4-(2'-carboxyphenyl)4-oxobutyrate (spectrum 1) and of a component of /. balsamina tissue (spectrum 2) Insert shows radiogas chromatographic trace of mixture in which natural oxobutyrate was found (arrows indicate radioactivity and mass)
designed easily where this situation does not occur. The secret is to use small samples of biomass in incorpora tion experiments. Moreover, as part of an RGC/MS assembly, any limited accuracy in counting that the gas flow counter might have is outweighed by the overall speed and efficiency of op eration, by the enhanced precision in mass measurement, and by the reli ability of purity assay that the inte grated unit can bring to bear on the problem. Two additional points have to be made regarding the effectiveness of RGC/MS in tackling biological prob lems. Because it relies on gas-phase separation of mixture components, the technique is applicable only to cell constituents that are volatile in their own right or can be so rendered by derivatization. Since elevated temper atures are involved, heat labile compo
nents are also excluded. These two limitations are not as disadvantageous as might first appear. The vast majori ty of the primary and secondary me tabolites of cells (Krebs cycle acids, glycolytic intermediates, amino acid precursors, nucleosides, fungal prod ucts, etc.) can be tackled, as can most drugs and toxicants currently under study. Moreover, as gas chromato graphic technology continues to devel op, a greater spectrum of biologically relevant molecules will come within the scope of RGC/MS analysis. E x a m p l e s of the Use of an R G C / M S Unit
In the previous sections we have shown t h a t the conversion of a G C / M S unit to an RGC/MS unit is simple and straightforward; in this third section we provide evidence of
the effectiveness of the RGC/MS tech nology in solving biological problems. Details will be given of the biomass amount used in the experiments, of the isotope dose fed, and of the dura tion of the incubation. These data will provide readers a clearer picture of the experimental frame of reference ap propriate to RGC/MS-monitored ex periments. Search for Structurally Un known Components of Metabolic Pathways. In his seminal work on RGC/MS, Hobbs (1) reported the identification of phenylenediamine and an hydroxylated nitroaniline as metabolites of 1 4 C-p-nitroaniline in the rat. Trimethylsilylation was used to derivatize the urine constituents prior to analysis. In another study re ported by Hobbs (10), tritiated sub strates were used to examine the me tabolism of indan-5-ol and indanyl carbenicillin in rats arid dogs. Animals were dosed at the rate of 28 mg/kg with :! H-indan-5-ol (sp. act. 256 MCi/mg) and of 50 mg/kg with ;! H-indanyl carbenicillin (sp. act. 2.67 MCi/mg). Samples of urine, feces, and, in some cases, bile were taken over 144 h. T h e bulk of the radioactivity was found in the urine. RGC/MS analysis of urine extracts indicated that in the rat, the indan-5-yl unit was excreted intact following conjugation with glu curonic or sulfonic acids. In the dog, extensive oxidation to indanediols and hydroxy indanones was demonstrated. Braun et al. (3), by way of illustra tion in their paper, showed figures produced by feeding 14 C-styrene to rats; conversion of this substrate to benzoic, phenylglyoxylic, mandelic, and hippuric acids was obtained. Braun et al. also indicate that, as yet unpublished, work had been done on the metabolism of "alkyl halides, halogenated aromatic hydrocarbons, pesti cides and experimental drugs." T h e bulk of our own work on this topic has been done with anabolic rather than catabolic reactions. Figure 2 shows the first data we obtained with a (noncomputerized) RGC/MS unit (11). It established that 4-(2'-carboxyphenyl)-4-oxobutyrate (I) was an intermediate in the biosynthesis of lawsone (II). The data were obtained by feeding 1 4 C-glutamate (250 μϋί), a primary precursor of lawsone (see ref. 12 for review), to Impatiens bal samina cuttings (4 g wet weight). At the close of the incubation (5 h), a total organic soluble extract was made and was methylated with diazomethane. Following sizing of this derivatized extract by gel permeation chromatography, fractions were as sayed by RGC/MS. All the radiola beled cell constituents detected by the counter were cataloged by the mass spectrometer. Figure 2 shows the spec-
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trum of one constituent together with the spectrum of an authentic sample of dimethyl 4-(2'-carboxyphenyl)-4oxobutyrate. Even from the primitive mass and radioactivity traces shown in Figure 2, it was possible to estimate t h a t the incorporation value for the process 1 4 C-glutamate -»· butyrate I was 1.7 X 10- :i %. A particularly valuable aspect of the application of RGC/MS technology to a single component metabolism study emerged from the lawsone work. In looking for intermediates in the anabolic pathway, intermediates in the catabolic pathway were found gratuitously, to wit 3-(2'-carboxyphenyl)-3oxomalonate (III), o-acetylbenzoate (IV) and phthalate (V). It is also easy with the RGC/MS method to determine that isotope from the precursor has entered and is being metabolized by the cells. Although often disregarded, this is an important control parameter. In the particular experiment mentioned above, radiolabeled peaks associated with, inter alia, glutamate, 2-ketoglutarate, citrate, malate, squalene, and the Cie and Ci 8 fatty acids, were encountered. Using similar methods, the biosynthetic pathway to the mycotoxin mycophenolic (VI) acid has been examined. In addition to establishing the presence in the tissue of three antici-
Figure 3. Traces of mass and radioactivity produced from methylated total extract of P. brevicompactum tissue Peaks associated with citrate (1), C 16 (2) and C, 8 (3) fatty acids, phthalide (VIII) (4), and mycophenolic acid (5) are shown. Phthalide radioactivity peak (4) corresponded to 10 900 dpm
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pated intermediates (VII, VIII, IX), a new intermediate was encountered and characterized partially (X) (2). In Figure 3 the peaks of mass and ra dioactivity associated, inter alia, with intermediate VIII are seen. The sam ple that gave rise to Figure 3 was 10% of a methylated unfractionated total extract of the fungal tissue (490 mg). The incubation had been with 1-14Cacetate (25 /nCi) for 3 h. R G C / M S in Drug Mode of Action Studies. Elucidating how a drug, toxi cant, or environmental pollutant in terferes with cellular biochemistry is a major concern in many laboratories. RGC/MS can make a significant con tribution to these studies since it has the power to overview large sections of metabolism. We have been using RGC/MS technology to study the mode of action of the myotomia-inducing agent 20,25-diazacholesterol (XI, DAC). This drug was developed in the early 1960's during a search for hypercholesterolemic agents (13). In the latter regard, DAC is extremely effective; indeed, had it not been for its myotomia-producing side effects, it might be in widespread use today. Early work on the molecular level mode of action of DAC was not com pletely definitive and indicated that several sites might be involved along the cholesterol biosynthetic pathway (14-17). Inexplicably, this conclusion was disregarded in favor of the sim pler rationalization that DAC inhibit ed what in the 1960's was thought to be the last step in the pathway: the conversion of desmosterol (24-dehydrocholesterol) to cholesterol (see ref. 18 for current thinking). This possibil ity emerged from the observation that in man and animals, DAC treatment not only reduces total plasma sterols but also increases markedly the ratio of desmosterol to cholesterol (19-25). Several laboratories have implicated the high desmosterol levels as a caus ative agent in the DAC-induced myotomia (see ref. 26). RGC/MS technology, particularly in the SIM mode, has proved invalu able in a reexamination of the mode of action of DAC since it allows end products and intermediates to be ex amined for mass and radioactivity si multaneously. Figure 4 (top panel) was obtained by treating a 15-cm petri dish of seven-day-old cultured chick embryo pectoral muscle cells with DAC (1 Mg/mL culture medium) for 12 h in the presence of l- 1 4 C-acetate (25 ^Ci). Following the experiment, a Folch extract was made of the cells, and it was presented to the RGC/MS underivatized. Figure 4 (bottom panel) was obtained from a control culture of the same age that was ex posed to l- 1 4 C-acetate for the same period of time. Both treated and con
Figure 4. Traces of mass (middle), radioactivity (upper), and m/e 27Ί ion profile (lower) of total extracts of cultures of chick pectoral muscle cells following 12-h incubation with 1-14C-acetate. Top panel: cells were treated with 1 Mg/mL diazacholesterol; bottom panel: untreated control cells. Zones A, B, and C correspond to elution positions of cholesterol, desmosterol, and dihydrolanosterol, respectively
trol cultures were established in a growth medium that lacked serum 4% h prior to and during the experiment. In the panels, the upper trace is the radioactivity profile, the middle is the total ion current output of the mass spectrometer, and the lower is the se lected ion current profile at m/e 271. This ion is of low abundance in choles terol, lanosterol, and dihydrolanoster ol but is abundant in desmosterol. From Figure 4, several facts are evi dent. Firstly, DAC indeed inhibits the transfer of isotope from l- 1 4 C-acetate to cholesterol. Secondly, rather than increasing over the 12-h exposure pe riod, the desmosterol/cholesterol ratio decreases approximately twofold. Thirdly, the transfer of isotope from l- 1 4 C-acetate to desmosterol is also re duced by DAC. Fourthly, there is an other cell constituent with an abun d a n t ion at m/e 271 which suffers no reduction in mass (relative to choles terol) and no loss of radioactivity on
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treatment of the cells with DAC. T h e nature of this substance is presently uncertain although it has approxi mately the same retention time as d i hydrolanosterol. Two conclusions can be made from the data. T h e first is that the block placed by DAC in cholesterol biosyn thesis is not between desmosterol and cholesterol in muscle cells. As far as any DAC-induced accumulation of desmosterol is concerned, this would need to be a secondary (or even more distal) effect. The second conclusion is that continued use of RGC/MS technology employing 1 4 C-acetate, l4 C-mevalonate, and other radiola beled precursors should reveal the full impact of DAC on the cholesterol bio synthetic pathway. R G C / M S Technology as a Moni tor for Cell Development. The me tabolism of cells changes as they exe cute their normal development cycle or are caused to depart from that cycle
Already, however, two outcomes are evident. Firstly, it is an extremely powerful approach to understanding what cells are accomplishing metabolically at any given time and to defining their developmental state in molecular terms. Coupling the use of RGC/MS with morphological studies conducted by light and electron microscopes should provide considerable insight into the problem of cell development/ differentiation. Secondly, the technique we have applied to the spore development problem needs much refinement. We see advantages in making multiple samplings during the 2-h isotope incorporation experiment to allow the kinetic aspect of the metabolic flux to be more evident. Compartmental analytical techniques could productively be applied to the resulting data (see ref. 28). Fortunately, such is the speed and comprehensiveness of RGC/MS methods that they are quite able to cope with the large number of samples that such an experimental design would produce. Figure 5. Traces of mass (right-hand panel) and radioactivity (left-hand panel) obtained from acid extracts of germinating spores of P. brevicompactum at five time points Figures on left-hand panel indicate count rate and percentage error of that rate at 95% confidence (in parentheses) for the largest peak in trace. Radioactivity peaks associated with methyl esters of malonate (1). succinate (2). malate (3), citrate (4), and palmitate (4)
by a variety of circumstances, some beneficial, some deleterious. These changes are initiated at the maeromolecular level but are usually expressed at the small molecule level. This being the case, judicious use of RGC/MS methodology should allow particular developmental states to he defined and should also permit the progress from one state to another to be monitored in metabolic terms. Recently, we have been trying to realize this potential of RGC/MS by examining the germination and subsequent development of spores of the fungus Pénicillium brevicompactum (27) (the organism that produces the mycotoxin mycophenolic acid, discussed above). Figure 5 shows the traces produced when samples (75 mL) of a suspension of P. brevicompactum spores were incubated for 2 h with l- 1 4 C-acetate (50 //Ci) 0, 3, 6, 9, and 12 h following establishment of the culture. None of the spores in the colony had produced germ tubes during the first 15 h of the experiment. The right-hand panel shows the output of the total ion current monitor; the left-hand panel shows the trace produced by the radio counter. The samples for analysis were obtained by: a) extracting the combined biomass and culture medium with ethyl acetate and methylating the residue with diazomethane; and b) acidi-
fying the residue from a) to pH 1.0 and extracting and derivatizing as before. Mass spectra were taken throughout each run. From the neutral extracts (traces not shown), it could be learned that fatty acid biosynthesis was active from the earliest times. Characteristics of the later stages of pre-emergent germination were intracellular pools of the fatty acids Ci2:o, Ci2:i. Ci4;o, and Cj, 1:1 . The acidic extract traces contain much more information on the various phases of pre-emergent development, particularly the left-hand panel wherein the radioactivity profile is contained. The fate of an equal charge of l- 1 4 C-acetate varied significantly over the 12-h time span studied. Initially, the bulk of the activity in an acidic extract of the developing spores was found in succinate; eventually, it was found in palmitate. At intermediate time points, malate and citrate carried significant quantities of the administered isotope. To underscore the point made earlier regarding the effectiveness of the radioactivity counter, Figure 5 contains on the lefthand side measures of the disintegration rate associated with the largest peak in each trace and the probable error (95% confidence) in making that measurement. Work with this particular aspect of RGC/MS technology has only begun.
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Conclusions It is hoped that the above examples document our confidence in the present-day utility and the future potentials of RGC/MS-based methods of analysis in biological science. It is our experience that both factors are rate limited more by the vision and ingenuity of the biological scientist rather than by any inherent defect of the instrumentation. Acknowledgment The work described in this article has been a joint undertaking of a group of individuals over eight years, to wit, C. D. Rartman, D. L. Doerfler, L. A. Ernst, E. Farish, E. W. Grotzinger, R. Kadlec, E. L. McGandy, I. R. McManus, J. M. Malloy, J. D. Naworal, C. P. Nulton, E. R. Rosenblum, and J.R.R. Slayback. I thank R. Bentley for constructive criticism of the manuscript. References (1) Π. C. Hobbs. Am. Soc. Mass Spectrom., Annual Meeting, Abstract R57, 1970. (2) C. P. Nulton, J. D. Naworal, I. M. Campbell, and K. W. Grotzinger, Anal. Riochem., 75,219(1976). (3) W. H. Braun, E. O. Madrid, and R. J. Karbowski, Anal. Chern., 48, 2284 (1976). (4) P. R. Yackovich and R. L. Swann, American Chemical Society, 173rd Meet ing, Abstract Pest. 74, New Orleans, La., March 1977. (5) B. E. Gordon. W. R. Erwin, M. Press, and R. M. Lemmon, Anal. Chem., 50, 179(1978). (6) A. G. Netting and C. Barr, Anal. Biochem., 84, 136(1978). (7) I. M. Campbell, D. L. Doerfler, S. Λ. Donahey, R. Kadlec, E. L. McGandy, J. I). Naworal, C. P. Nulton, M. Venza-
Raczka, and F. Wimberly, Anal. Chcm., 49, 1726 (1977). (8) M. Matucha and E. Smolkova, J. Chromatogr. (Chromatogr. Rev.), 127, 16S" (1976). (9) I. Kiricsi, K. Varga, and P. Fejes, ibid., 12.·!, 279(1976). (10) Π. C. Hobbs, Antimicrob. Agents Chemother., 2,272(1972). (11) E. Grotzinger and I. M. Campbell, Phytochemistry, 13,923 (1974) (12) H. Bentley, Pure Appl. Chem., 41, 47 (1975) (Hi) R. K. Counsell, P. I). Klimstra, and R. E. Ranney, J. Med. Pharm. Chem., 5, 1224 (1962). (14) R. R. Ranney and D. L. Cook, Arch. Int. Pharmacodyn. Ther., 154,51 (1965). (15) R. Niemiroand R. Fumagalli, Biochim. Biophys. Acta, 98, 624 (1965). (16) P. D. Klein, R E. Ranney, R. E. Counsell. and P. A. Szczepanik, Fed. Proc, 22,591 (1963). (17) M. J. Thompson, J. Dupont, and W. E. Robbins, Steroids, 2, 99 (1963). (18) VV. R. Nes and M. L. McKean, "Bio chemistry of Steroids and other Isopentenoids," Chap. 9, University Park Press, Baltimore, Md., 1977. (19) N. Winer, J. M. Martt, J. E. Somers, L. Wolcott, H. E. Dale, and T. W. Burns, J. l.ab. Clin. Med., fifi, 758 (1965). (20) B. A. Sacks and L. Wolfman, Arch. Int. Med., 116,366(1965). (21) R. Fumagalli and R. Niemiro, Life Sci., 3,555(1964). (22) R. E. Ranney and E. G. Daskalakis, Proc. Soc. Ëxp. Biol. Med., 16, 999 (1964). (23) R. A. Ahrens, .1. Dupont, and M. .J. Thompson, ibid., 118, 436 (1965). (24) T . W. Burns, H. E. Dale, and P. L. Langley. Am. J. Physiol., 209, 1227 (1965). (25) R. A. Singh, J. F. Weiss, and E. C. Maber, Poult. Sci., 5 1 , 449 (1972). (26) J. B. Peter and D. S. Campion, in "Pathogenesis of Human Muscular Dystrophies," L. E. Rowland, Ed., p 739, "Excepta Medica," Amsterdam, T h e Netherlands, 1977. (27) D. L. Doerfler, C. P. Nulton, C. D. Bartman, V. -J. Gottlieb, and I. M. Campbell, Can. J. Microbiol., 24, 1490 (1978). (28) I. M. Campbell, Phytochemistry, 15, 1367 (1976). (29) T. G. Cooper, " T h e Tools of Biochemistry," ρ 108, Wiley-Interscience, New York, N.Y., 1977. '
Iain M. Campbell is an associate pro fessor of biological sciences. His gen eral research interest is the biochem istry of small molecules. The biologi cal roles played by secondary metabo lites in the producing organism, and the mechanisms of drug action are particular current concerns. Financial assistance provided by the National In stitute of Health (RR-00273 and A l l 1819), the National Science Foundation (PCM 73-01024 and PCM77-03966), and the Muscular Dystrophy Association of America.
For most organic acid applications, Dionex recom mends the dual system Ion Chromatograph 16. Actually two complete ion chromatographs in one instrument, the IC 16 analyzes complex mix tures containing both organic acids and inorganic ions— at trace levels. This includes most ions with pKa less than approximately 7. Examples are inorganic ions such as F~ C l r PQ»,3N0 3 ~ and SO,, 2- as well as organic acids such as C-1-C10 carboxylic acids, organic phosphonic and sulfonic acids, and carbonic acids. All are easily handled by the IC 16 and its advanced column technology. Applications include organic determination in milk products, wine, coffee, wastewater, fuels and in body fluids such as urine and blood serum. Circle the appropriate number for applications and product data. Organic acids 51 IC16 52 Air Quality 53 Water Quality 54 Elemental Analysis 55 Brine Analysis 56 57 Clinical Power Production 58 59 QC—Process 60 QC—Foods 61 Soil Analysis
DIONEX
Dionex Corporation In the US: 1228 Titan Way Sunnyvale, CA 94086,408-737-0700 479 Green Street Woodbridge, NJ 07095, 201-634-1127 In Europe: c/o 4, The Buchan, Camberley, Surrey, GU15 3XB, England
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