Quantitative Analysis by Isotopic Dilution Using Mass Spectroscopy The Determination of Caffeine by GC-MS
J. Chem. Educ. 1988.65:907. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 01/23/19. For personal use only.
Devon W. Hill, Brian T. McSharry, and Larry S. Trzupek Furman University, Greenville, SC 29613
The development of reliable, easy-to-use, and reasonably priced systems for coupled gas chromatography-mass spectroscopy (GC-MS) has led to the proliferation of these sophisticated instruments, even in academic settings with a predominantly undergraduate emphasis. For example, the NSF-CSIP program made at least 12 grants to colleges in 1985 for GC-MS systems to be used for instructional purposes, a number large enough that a special section on the incorporation of GC-MS in the undergraduate curriculum was included in a program on “NSF/CSIP Catalyzed Innovations in the Undergraduate Laboratory” at the 1987 National ACS meeting in New Orleans. In many cases, the most straightforward use of these instruments in undergraduate experiments involves qualitative analysis of mixtures or structure determination of reasonably pure compounds using the mass spectrometer component in its “scan” mode. However, many of the most interesting practical applications of GC-MS involve its use in quantitative analysis of components in complex mixtures, using the “specific ion monitoring”, or “SIM” mode. In this configuration, the mass spectrometer samples the effluent GC stream only for ions of specific, designated masses, typically cycling through a small number of such ions many times per second. In this way, both the sensitivity and the analytical specificity of the technique are enhanced. The most sophisticated of these procedures use quantitation through isotopic dilution, in which the intensity of a specific analyte ion is compared to that of an analogous ion of a suitably labelled form of the compound of interest, added to the analytical matrix in a known amount. Such methods have been extensively used in both environmental and forensic analysis. For example, EPA Method 8280 describes the GC-MS analysis for polychlorinated dibenzo-p-dioxins, a procedure which requires the addition of C13-labeled TCDD’s as internal standards for quantitation. In addition, the most reliable methods for urinalysis of drugs and drug metabolites are those which involve GC-MS and isotopic dilution; a typical case involves the GC-MS analysis of urine for ll-nor-deIta-9-tetrahydrocannabinol-9-carboxylic acid, the major metabolite of (—)-trans-delta-9-tetrahydrocannabinol (THC), an active component of marijuana (1-3). Despite the technical elegance of such methods, practical problems (such as the expense of the standards involved) complicate their introduction into the undergraduate lab. In an effort to make available to undergraduates an isotopic dilution method for GC-MS analysis that could be used even for urinalysis, we have developed a method for caffeine. Caffeine appears to be one of the most popular targets for undergraduate experiments involving qualitative and quantitative analysis (4-13); additional procedures involving caffeine analysis have been described in other technical literature (14-18). Our desired GC-MS method for caffeine would require a labeled analogue for the compound that would be conveniently accessible. Although several forms of mono-deuterated caffeine are commercially available, they are less desirable in this application than polydeuterated analogues, due to the complications caused by the M + 1 peaks found for the
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Figure 1. Mass spectra for caffeine and synthetic caffeine-cfe, highlighting peaks suitable for SIM analysis by GC-MS. The spectra were obtained using a Hewlett-Packard 5890A gas chromatograph equipped with a 5970 mass selective detector.
natural substance. A trideuterated analogue would be ideal for this application. Preparations of caffeine by methylation of various of the dimethylxanthines date back to the early German literature (19); these reactions are easy enough to carry out that they can be readily incorporated into an undergraduate experiment (11). In a similar fashion, one can prepare 7,7,7-caffeine-ef3 from two inexpensive and readily available materials, theophylline (1,3-dimethyl xanthine; $5.15/50 g from Sigma) and iodomethane-dg (99+ atom %; $30/5 g from Aldrich), using the simple procedure described in the Experimental section. The product of this reaction elutes as a single peak by HPLC, and exhibited both the retention time and UV spectrum of authentic caffeine when analyzed with the diode-array detector on our instrument. Analysis of this material on a Hewlett-Packard 5890 gas chromatograph coupled to a 5970 mass selective detector (MSD) operating in the scan mode gives a total ion chromatogram with a single peak at the retention time of authentic caffeine. Extraction of the mass spectrum from the total ion chromatogram yields a fragmentation pattern very closely related to that of caffeine, as illustrated in Figure 1. Examination of these mass spectra readily suggests peaks for analytical use in the SIM mode: those at m/Z of 194,109, and 82 Volume 65
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October 1988
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