ANALYTICAL APPROACH

ιί Using Infrared Spectrometry and Chromatography

Daniel J. Brown, Louis F. Schneider

U.S. Food and Drug Administration 1560 E. Jefferson Avenue Detroit, Ml 48207 James A. Howell Western Michigan University Department of Chemistry Kalamazoo, Ml 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 investi­

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 problems 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 (i.e., 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 was carried out on the urine specimens. The bro-mophenol blue complex of the pancur­onium ion was 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). Sub­sequently 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 III: 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

This article not subject to U.S. Copyright Published 1988 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988 · 1005 A

SOLVING MYSTERIES

Figure 1. Chromatogram of extracted il­licit injectable anabolic steroid 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 μg of sample and 7-10 mg of KBr were made for FT-IR examination using a 4X beam condenser.

On one occasion, we attempted to an­alyze 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, methyl-testosterone, and nortestosterone pro­pionate (Figure 2a) were readily identi­fied and confirmed. However, a fourth peak partially resolved from the methyl-testosterone peak was not conclusively identified. A good match between the IR spectrum (employing Nicolet's "ab­solute derivative" algorithm) and the Georgia State Crime Laboratory Li­brary was not possible because the methyltestosterone absorption inter­fered with the spectrum of the uniden­tified component. Interactive subtrac­tion 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-methylene-dioxymethamphetamine (MDMA, Fig­ure 2b).

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

Sesamin Oxandroione

Figure 2. Structures of (a) several anabolic steroids, (b) the psychotropic drug 3,4-methylenedioxymethamphetamine (MDMA), (c) sesamin, and (d) oxandroione.

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 2c), which contains two 1,3-benzodioxole 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 case, we encoun­tered another illicit steroid sample dis­solved in a sesame oil carrier that con­

tained a white precipitate. The precipi­tate was removed by filtration and identified by FT-IR as oxandroione (Figure 2d). The precipitate was then assayed gravimetrically to determine the amount of oxandroione present in the sample. We first had to establish that 100% recoveries could be achieved by mixing standard oxandroione in ses­ame oil and petroleum ether. The mix­ture was then filtered and washed with petroleum ether, and the oxandroione precipitate was weighed. Another por­tion of the sample was extracted using a 9:1 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

1006 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988

Nortestosterone Methyltestosterone

3,4-Methylenedioxy-methamphetamine

(MDMA) Nortestosterone propionate

Figure 3. Chromatograms of extracted illicit anabolic steroid in sesame oil contain­ing a precipitate. Detection at (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 HPLC, collecting frac­tions, evaporating the solvent, and ana­lyzing the KBr disks by 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 be oxandrolone, the sample precipi­tate. Apparently, oxandrolone co-elutes to some extent with 19-nortes­tosterone; but because it weakly ab­sorbs in the UV range, it is virtually undetected using a UV detector.

Case IV: 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 ma­

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 cross-contamination 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 at fault. Ob­viously a quick and reliable method was of utmost importance.

The two gases involved were halo-thane (2-bromo-2-chloro-l,l ,l-tri-fluoroethane) and forane (1-chloro-2,2,2-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 sus­pected 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 1000 cm - 1 , forane exhib­its 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 its linearity. Aliquots of stan­dards (50.0 /iL) containing various ra­tios of halothane and forane were indi­vidually introduced into a 10.0-cm IR gas cell and allowed to vaporize at am­bient temperature and pressure. The cell was then placed in the FT-IR spec­trometer and its absorption spectrum obtained. Figure 6 illustrates the ab­sorption spectra in the vicinity of 1000 cm - 1 for various volume percent mix­tures of forane. Subsequently, 50.0 μϋ.

Figure 4. FT-IR spectra of oxandrolone. Oxandrolone standard spectrum (lower). Result of the interactive subtraction of 19-nortestosterone stan­dard from the spectrum of impure peak (upper).

1008 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988

s c a S Ε en c a

F

#

Wavenumbers (cm-1)

Figure 6. FT-IR absorbance spectra of various concentrations (v/v%) forane in halothane: (a) 0 % , (b) 1 %, (c) 5%, (d) 10%, (e)subsample 4.

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 forane 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% v/v 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 vari-

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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.

The authors are indebted to Milda J. Walters, who carried out the pancuronium studies and per­formed the HPLC studies on the steroid mixtures; James S. Jasinski for the NMR analysis; and Rus­sell J. Ayers for the GC/MS analysis.

Louis F. Schneider is the director of the Detroit District Laboratory of the FDA. Schneider received a B.S. degree in chemistry and mathematics from Western Kentucky University. His re­search interests include the applica­tion of chromatography and spectros­copy to analytical problems in the areas of food and drugs.

Daniel J. 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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