ANALYrICAL APPROACH
Daniel J. Brown, Louis F. Schnek U S . Food and Drug Administration 1560 E. Jefferson Avenue Detroit, MI 48207 James A. Howell Western Michigan University Department of Chemistry Kalamazw, MI 49008 Product tampering. Unexplained deaths. These are typical examples of real-life situations that must be investigated by the field office laboratories of the Food and Drug Administration (FDA). The analytical methods used to solve these mysteries must be highly reliable. Speed is essential because the laboratory findings often dictate the immediate and final course of action taken by the FDA to protect the public. Furthermore, the reliability of the results obtained must be unequivocable because litigation at a later time is possible. Thus confirmation of results by an alternate method is required in all cases in which food and drug laws may have been violated. Although laboratories like ours have used dispersive infrared (IR) spectrometry since 1957, it was not always the technique of choice and was used almost exclusively for qualitative analysis. The use of IR spectrometry was restricted because of the limited sensitivity of the available instruments. In addition, it was difficult to perform spectral searches on manual systems, and a tedious work-up of the sample was often necessary to obtain good quality spectra. In some of our investiThis article mat subject to U.S. Copyright Published 1988 American Chemical Society
gations, however, IR spectrometry was necessary to provide conclusive evidence of a compound's identity. In 1984 we acquired a Fourier transform infrared (FT-IR) spectrometer, and we have successfully applied this technique to a variety of problems and sample matrices. In this article, we present several examples of the prohlems that are investigated in our laboratory. The first scenario describes a situation in which dispersive IR spectrometry was used. The last three examples discuss situations in which FT-IR spectrometry was implemented in conjunction with chromatography. Case I: mysterious deaths We received a request from hospital authorities to investigate the unexplained deaths of several patients that had occurred at the Veterans Administration Hospital in Ann Arbor, MI. The hospital suspected that intravenous (iv) solutions were contaminated. Screening of the iv solutions using thin-layer chromatography (TLC) revealed no irregularities. However, upon examination of the deceased patients' urine, we discovered the presence of pancuronium bromide, a powerful muscle relaxant with a curare-type action (Le., it hinders involuntary functions of the respiratory system) that should not have been present. As a result of this finding, a lengthy extraction and purification procedure waa carried out on the urine specimens. The bromophenol blue complex of the pancuronium ion waa formed and extracted. A series of separations using TLC and re-
extractions followed. Ultimately the presence of the drug was verified using dispersive IR spectrometry. Further confirmation was made using two additional TLC procedures as well as gas chromatography (GC). Suhsequently the FBI analyzed exhumed body samples and confirmed the presence of pancuronium bromide by mass spectrometry (MS). The FBI concluded that several patients had been murdered, and several members of the hospital staff were arrested.
Cases II and 111: steroids On numerous occasions, the FDA has received complaints about steroid mixtures that were illegally sold to athletes for muscle-building purposes. The illicit steroids were used without the supervision of a physician: this posed a serious health problem, and thus we had to verify what was in the steroid mixture before the FDA could take any legal action. As in most cases when we are dealing with mixtures, chromatographic separation is the obvious method of choice. Because these mixtures generally require derivatization prior to GC analysis, high-performance liquid chromatography (HPLC) is preferred; it provides both qualitative and quantitative information without destroying the sample. Because the various steroids of these mixtures were structurally similar, we had to confirm the results by using a technique capable of distinguishing the individual components. This was achieved satisfactorily using FT-IR once the components were separated from one another. Thus fractions
ANALYTICAL CHEMISTRY. VOL. 60, NO. 17, SEPTEMBER 1. 1988
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Figure 1. Chromatogram of extra1 licit injectable anabolic sterold In sesame oil. usually were separated and collected first by HPLC. The solvent from the samples was then evaporated, and micro KBr disks (1.5 mm) containing 35-50 pg of sample and 7-10 mg of KBr were made for FT-IR examination using a 4X beam condenser. Onone occasion, weattempted toanalyze an illicit injectable anabolic steroid labeled as fluoxetine hydrochloride. The liquid chromatogram of this sample exhibited four peaks; three were identified on the basis of their retention times and confirmed by FT-IR (Figure 1). Nortestosterone, methyltestosterone, and nortestosterone propionate (Figure 2a) were readily identified and confirmed. However, a fourth peak partially resolved from the methyltestosterone peak was not conclusively identified. A good match between the IR spectrum (employing Nicolet’s “ahsolute derivative” algorithm) and the Georgia State Crime Lahoratory Library was not possible because the methyltestosterone absorption interfered with the spectrum of the unidentified component. Interactive suhtraction of the methyltestosterone from the absorption of the unidentified material provided a corrected spectrum presumably characteristic of the unidentified component. A spectral search tentatively identified this material as a psychotropic drug, 3,4-methylenedioxymethamphetamine (MDMA, Figure 2h). An ultraviolet absorption spectrum was obtained that was consistent with that of MDMA. However, finding this psychotropic drug in the presence of anabolic steroids was viewed with some degree of skepticism; one would not ordinarily expect to find muscle-building 1006 A
xtestostemne propionate
3,4Methylendioxymethamphetamine
Figure 2. Structures of (a) several anabolic steroids, (b) the psychotropic drug methylenedioxymethamphetamine (MDMA), (c)sesamin, and (d) oxandrolone.
drugs with mind-altering drugs. As is routinely the case, a blank determination of the carrier of the illicit drug, sesame oil, was obtained using HPLC. A peak was present that corresponded to the unidentified material (MDMA). Because it seemed improbable that the sesame oil would contain MDMA, a naturally occurring compound resembling MDMA was suspected. An examination of the literature revealed that sesame oil contains sesamin (Figure Zc), which contains two 1,3-henzodioxole groups that are also found in MDMA. The similarity of the two structures readily explains the similar spectral properties of the two compounds. We concluded that the unidentified component of the illicit anabolic steroid was a component of the carrier, sesame oil. In a more recent c w , we encountered another illicit steroid sample dissolved in a sesame oil carrier that con-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17. SEPTEMBER 1, 1988
tained a white precipitate. The precipitate was removed by filtration and identified by FT-IR as oxandrolone (Figure 2d). The precipitate was then assayed gravimetrically to determine the amount of oxandrolone present in the samole. We first had to establish that 100% recoveries could be achieved hy mixing standard oxandrolone in sesame oil and petroleum ether. The mixture was then filtered and washed with petroleum ether, and the oxandrolone precipitate was weighed. Another portion of the sample was extracted using a 9:l methanol-water mixture and screened by HPLC for additional steroids. Using a UV-vis diode array detector, we obtained the chromatograms shown in Figure 3. Two major peaks corresponding to 19-nortestosterone (5.5 min) and methyltestosterone (7.7 min) were found. Both peaks exhibited an absorption maximum in the vicinity of
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Figure 3. Chromatograms of extracted illicit anabolic steroid in sesame oil containing a preclpltate. Detectionat (a) 280 nm. (b) 210 nm, and (c) 240 nm.
240 nm, which is consistent with the UV spectra of steroids. Although no peak was evident for oxandrolone, this was not surprising in light of the fact that the oxandrolone chromophoric system is not a strong absorber in this region of the spectrum. We confirmed the identity of the peaks by separating the sample via $PLC, collecting fractions, evaporating thesolvent, and analyzing the KBr disks hy FT-IR. The peak at 7.7 min was a good spectral match with the methyltestosterone standard. However, the 19-nortestosterone peak, although identifiable, appeared to be contaminated with another compound. Although there was no doubt as to the identity of methyltestosterone and 19-nortestosterone, complete characterization of the sample depended on identifying the impurity. Interactive subtraction (Figure 4) of a 19-nortestosterone spectrum from the impure sample revealed the impurity to he oxandrolone, the sample precipitate. Apparently, oxandrolone coelutes to some extent with 19-nortestosterone; but because it weakly ahsorbs in the UV range, it is virtually undetected using a UV detector. Case I V anesthesia victim? In another instance, we found it necessary to employ quantitative analysis using FT-IR in a case involving the death of a child during surgery. The child went into shock while under general anesthesia. The anesthesia maloo8A
chine was capable of handling two different anesthetics by means of a selection valve. Because the patient's symptoms were consistent with those of someone who had received a crosscontamination of the anesthetics, a malfunction of the machine was suspected. There was concern that if one of these machines had failed, another could also fail. We had to ascertain
whether the machine was a t fault. Obviously a quick and reliable method was of utmost importance. The two gases involved were halothane (2-hromo-2-chloro-l,l,l-trifluoroethane) and forane (l-chloro2,2,2-trifluoroethyl difluoromethyl ether). The patient should have received halothane only; we needed to determine if a significant amount of forane was also inadvertently introduced by the machine. We sampled the gas coming from the machine by condensing it in a cold trap of slurried dry ice and ethanol. Two samples of SIXpected forane-contaminated gas were collected. One was taken under normal conditions (subsample 3), and another was taken while the selector lever on the anesthesia machine was jiggled (subsample 4). To verify the reliability of our results, we analyzed the condensate by FT-IR, NMR, and GC/MS. The superimposed IR spectra of the two gases are shown in Figure 5. Examination of the spectra revealed that in the vicinity of 1WO cm-', forane exhihita an absorption band whereas halothane does not. Thus we chose this wavenumber for the determination of forane in halothane. Next, we had to establish a calibration plot and determine ita linearity. Aliquota of standards (50.0 p L ) containing various ratios of halothane and forane were individually introduced into a 10.0-cm IR gas cell and allowed to vaporize a t ambient temperature and pressure. The cell was then placed in the FT-IR spectrometer and ita absorption spectrum obtained. Figure 6 illustrates the absorption spectra in the vicinity of 1000 cm-I for various volume percent mixtures of forane. Subsequently, 50.0 fiL
Figure 4. FT-IR spectra of oxandrol Oxandmlone standard s p e c " (lower). Result of the interactive subtraction of lsnptesmsterone Standard lrom ttm w&um of Impure peak (upper).
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17. SEF'TEMBER 1, 1988
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aliquots of the sample condensates were introduced into the gas cell and treated in the same manner as the.standards. Subsample 3 gave a spectrum that coincided with the 0% standard; subsample 4 gave an absorbance equivalent to a forane concentration of 3.6%. The FT-IR analysis was in good agreement with the results obtained by
NMR and GC/MS. Both techniques failed to detect any forana in subsample 3 and found values of 3.4%and 4.0% forane, respectively, in subsample 4. Even in the case of subsample 4, one must consider that the flow of the halothane is normally adjusted to administer the amount equivalent to approximately 2% vlv to the patient. Under the
worst-case scenario, the patient would have been receiving only 0.07% forane. Based on this data, medical experts determined that the anesthesia machine did not cause the patient’s death.
Summary We have found that IR spectrometry and chromatography used with a var-
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ety of other analytical techniques are valuable tools for solving a wide assortment of analytical problems applicable to regulatory decision making. Highly automated instrumentation and computerized data systems are a great asset, but one must never underestimate the importance of planning and developing a logical approach for analytical problem solving. Theaurhorssre indebted toMilda J. Wslters.who carried out the paneumnium studies and performed the HPLC studieson thesteroid mixtures; .lames S. dssinrki for the NMR analysis: and Rus. sell d. Ayers for the C.C/MSanalysis,
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DanielJ. Brown (left) is a chemist and IR spectroscopist at the FDA’s Detroit District Laboratory and is responsible for analyzing food, drug, and cosmetic samples. He received a B.S. degree from Wayne State University (1965). His research interests include IR spectroscopy, chromatography. and laboratory automation and computerization. James A. Howell (right) is a professor of chemistry at Western Michigan University. Since 1976 he has been a science advisor for the FDA’s Detroit District Laboratory. Howell received a Ph.D. degree from Wayne State University. His research interests lie in the areas of absorption spectroscopy, chromatography, and analytical instrumentation.
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