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CLIN. CHEM. 20/2, 255-265 (1974) Routine Use of a Flexible Gas Chromatograph-Mass Spectrometer-Computer System to Identify Drugs and Their Metabolites in Body Fluids of Overdose Victims C. E. Costello, H. S. Hertz, T. Sakai, and K. Biemann We have assembled a Computer-searchable collecspectra of drugs, metabolites, normal body-fluid constituents, and common sample contaminants, for use in conjunction with a gas chromatograph-low resolution mass spectrometer system. The system is designed for analysis of drugs in body fluids of comatose victims of accidental or deliberate ingestion of a drug overdose. The speed and generality of the method make it particularly useful for such emergency situations. It has now been in routine use for longer than two years, and its usefulness is illustrated by results obtained for several actual samples. This goal has been achieved in the intervening time and the results have been presented periodically in preliminary form.2 The present paper represents a detailed report and discussion of the results of about two years of use of the technique for some 600 patients. In many cases a drug ingested in an overdose can easily be identified by rather simple, conventional techniques such as color reactions, thin-layer chromatography, gas chromatography, or ultraviolet spectroscopy, alone or in combination. Very often there is some direct or circumstantial evidence avail- Additional Keyphrases: emergency ed, which then merely needs to be confirmed in the laboratory. In many instances, however, there is no information available; or a number of drugs have been ingested, which may mutually interfere in the reliable identification; or the specimen may contain tion of 300 mass able toxicology #{149}library #{149} rapid identification of drugs and of mass spectra drug metabolites An earlier paper (1) described chromatograph-mass to rapidly the use of a gas spectrometer-computer system the toxic agent-a large dose of the body fluids of a patient who had identify “Darvon”1-in attempted suicide. Although the instrumentation and techniques (2, 3) used had been developed for quite different problems in chemical research, they served well in that emergency. In that case the resulting mass spectra had to be interpreted from basic principles, and it was imme- diately obvious, as stated then, that the technique could be highly automated and accelerated if a collection of the mass spectra of commonly used drugs and related substances were available for immediate automatic comparison gas-chromatographic recorded in real time with fractions, the mass which by a computer. spectra were of the already concerning the only metabolized a compound nature drug; that of the or, finally, compound one may is not a common as is often the case with illicit require a more sophisticated ingest- drugs. deal with medicinal Such drug, situations analytical technique; the cases dealt with in our laboratory belong to these categories exclusively. It is, of course, desirable to use an approach that not only identifies any one of a predetermined set of compounds, if present, but also reveals the identity of all the other components that are extracted from the original specimen. This not only assures the investigator that no potentially harmful component has been overlooked but also leads to detection identification of unexpected compounds, be they drugs (particularly illicit ones), new or known and new me- The availability of such a system would not only greatly shorten the time required to identify these substances but it would also reduce the need for involvement of personnel intimately acquainted and experienced with the interpretation of mass spectra of organic compounds. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass. 02139. 1 Darvon is the trade name for 4-dimethylamino-3-methyl-1,2diphenyl-2-butanol proprionate hydrochloride (Eli Lilly, Inc., Indianapolis, md.). Received Nov. 30, 1973; accepted Dec. 2, 1973. 2 () Biemann, K., Biller, J. E., Costello, C. E., and Sakai, T., The potential of GC-MS-computer systems in biomedical and clinical research, Pittsburgh Conf. on Anal. Chem. and Applied Spectroscopy, Cleveland, Ohio, March 1972. (b) Costello, C. E., Sakai, T., and Biemann, K., Identification of drugs in body fluids, particularly in emergency cases of acute poisoning. 20th Annu. Conf. on Mass Spectrometry and Allied Topics, Dallas, Texas, June 1972. (c) Costello, C. E., and Biemann, K., On call for poisonings: The mass spectrometry facility as adjunct to the emergency room. Pittsburgh Conf. on Analytical Chemistry. and Applied Spectroscopy, Cleveland, Ohio, March 1973. (d) Biemann, K., Costello, C. E., and Andresen, B. D., Mass spectrometnc identification of drugs and their metabolites in body fluids. 166th National Meeting, American Chemical Society, Chicago, Ill., August 1973. CLINICAL CHEMISTRY, Vol. 20, No. 2, 1974 255 tabolites that help in the identification or confirmation of the original drug, or even artifacts and contaminants introduced into the sample before it reached the laboratory. The identifications of all these for materials the represent continuous analysis, an important improvement because once they prerequisite of the are recognized their sponsive to human analysis intervention, that is re- supervision, and improvement-is provided by designing the data processing along two parallel lines: (a) the continuous mass-spectrometric matogram is compared, with the authentic record of the entire gas chroone spectrum after another, data tion of all components for the automatic of which identifica- authentic spectra are available and can be successfully matched; (b) at the same time, the data are also processed in the normal manner, well i.e., presented as individual as individual mass mass chromatograms spectra (plots as of in- m/e values over the entire gas chromatogram) (4) and reproduced on 16-mm microfilm for individual inspection by the investigator.3 This process is the most convenient, practical way of surveying and making detailed interpretation of such a set dividual of data. Automatic searching simple and reliable are available, techniques are very if mass spectra of high quality the extract of a blood or urine but specimen represents a complex mixture. Because these are emergency situations, there is no time to separate the extract into various sub-fractions and to optimize choice pound the gas chromatographic separation of varied column materials types or by derivatization be “contaminated” This a problem poses for different comof the materials to terpretation, and with other materials. in effecting various means the automatic to remedy are represented handled to date Sample-Handling Procedures the Gastric body contents, fluids clotted usually blood, supplied and (or) urine are by the hospital, although spinal fluid and bile have also been used. Quantities available vary, but the recommended amounts are 10 ml of gastric contents or urine or 5 ml of blood. Much smaller quantities can be used successfully when necessary, but less conveniently. The extraction procedure is illustrated in Scheme 1. Methylene chloride was chosen because of its good solvent property for most drugs; its availability in high purity at a reasonable cost (“Nanograde,” Mallinckrodt Chemical Works, St. Louis, Mo. 63160); its simple, unconfusing mass spectrum; and its low boil- ing point, tract which without facilitates losses. concentration Solid sodium of the ex- bicarbonate sure that the more basic drugs (amphetamines, sodium orator. sulfate, and concentrated with a rotary evapThe water bath used during evaporation is kept at or slightly below ambient temperature and the time for this step is kept as short as possible, to prevent loss of volatile drugs such as ethchlorvynol or amphetamines. The of methylene chloride. subjected computer residue is dissolved in 100 /41 A 2- to 5-/4l portion is then to gas chromatograph-mass analysis (Scheme Gastric contents (10 ml) 2). filter Blood (5 ml) or \ centrifuge spectrometerCo-injection Urine centrifuge this (10 ml) ntrifuge extract with 5 vol CH2C12 will data to permit the investigator to either verify the results of the computer interpretation or inspect and interpret the mass spectra of fractions that may add solid NaHCO3 to aqueous phra8e not re-extract with 5 vol CH2C12 automatic proce- dures. Materials The hospitals and Methods body fluids affiliated investigated with the were Boston obtained Poison combine dry with from 3Biller, J. E., Hertz, H. S., and Biemann, K., The application of a GC-MS-computen system and various interpretive programs to problems in natural products chemistry. 19th Annu. Conf. on Mass Spectrometry and Allied Topics, Atlanta, Ga., May 1971. CLINICAL CHEMISTRY. Vol. 20, No.2, 1974 CH2C12 fractions, Na2SO4 Informa- tion Center at the Children’s Hospital Medical Center. Only a few hospitals submit most of the samples, 256 of a in- all the by the mor- phine) are removed from the aqueous layer. The organic extracts are combined, dried over anhydrous discussed. It also makes it even more important to provide the fast and efficient means of processing characterized (5 g, ACS Certified, Fisher Scientific Co., Pittsburgh, Pa. 15219) is added before the second extraction, to en- be be fully among by this by be separated (5). Thus, ideal gas-chromatographic separation is rarely achieved, and the system has to be able to cope with mass spectra that may to vari- ous degrees 30 hospitals 600 cases laboratory. automatic spectra are added to the computer-searchable collection. From then on they will be automatically identified whenever they are present in a specimen. One may say the system “learns.” This flexibility-an automated but more than the approximately Concentrate CH2C12 solution 100 /41 extract Scheme 1 2-5 of solution 1/41 p1 extract of standard Gas chromatrograph Mass Mass hydrocarbons To facilitate ranged 455 - 4-Sec scan cycle Total ion plot Barplots of spectra Mass chromatromatograms Library search and spectrometer is manner then can be evaporated which is inserted via a the ion source of the mass slowly continuously as during it possible (6). the presence of a drug behavior (e.g., aspi- 2-pl portion sample-holder, directly into spectrometer makes vaporized scanned while in the the gas-chromatographic the same run. Instrumentation The puter gas chromatograph-mass system used in this spectrometer-com- work consists of a Model 990 gas chromatograph (Perkin-Elmer Corp., walk, Conn. 06852), a Hitachi RMU 6L mass trometer (Perkin-Elmer Corp.), and an Nor- specIBM 1800 Corp., Ar- data acquisition and control system (IBM monk, N. Y. 10504). The computer is configured with 32 768 words of core memory, six IBM 2315 magnetic disk packs (capacity: 512 000 16-bit words each), and two IBM 2401 magnetic tape drives. The gas chromatograph is coupled to the mass spectrometer by a low-volume splitter linked to both a flame ionization detector and a porous fritted-glass tube pressure-reduction unit (7) by a heated stainless-steel capillary and valve. The exit of the pressure-reduction unit is connected leads into the ionization trometer. This configuration, ber of research more powerful to a glass chamber which was line that of the mass specdesigned available. lection was distributed in 1972 and is now indices of hydrocarbons of pertinent in computer-searchable they became 2 to determine retention indices If there is reason to suspect with poor gas-chromatographic a second a collec- tained from two other laboratories (Baylor College of Medicine and the National Heart and Lung Institute, through the courtesy of Dr. Marjorie Horning and Dr. Henry Fales, respectively) were added when m/e 28 - Scheme onto a glass vacuum lock compiled MS Identification rin, LSD), we have of mass spectra drugs and their metabolites. In addition, the mass spectra of abundant normal constituents of human body fluids and the mass spectra of commonly occurring contaminants were included. This “library” was ar- for a num- applications, is more elaborate and than that needed for the work de- format. Spectra (The first version ob- of the col- to the biomedical community also being used in commercial are unambiguously identified in actual extracts, or are reported in the literature, their spectra are also added to the collection. The 304 spectra in the collection at this time include 240 drugs, 24 metabolites, 18 normal constituents (e.g., cholesterol, dimethyl sulfone, piperidone, fatty acids), 19 artifacts (e.g., phthalates, adipates, and other plasticizers, and antioxidants such as 2,6-di-t-butyl-4-methylphenol or “lonol”) and three other harmful materials (e.g., pinene, the major constituent of turpentine). When a series of similar non-drug-related materials is likely to be encountered, only one typical member of the set is added to the collection, which systems.) mixture analyses, tion (library) Retention standard Library GC on 150 cm X 3 mm column 3% OV-17 on 100/200 mesh GC-Q 12 or 16 #{176}C/min from 80 to 330 #{176}C spectrometer Computer Spectra As additional drugs identifies such materials from consideration as or metabolites sufficiently potential to exclude them causative agents without adding unnecessarily large numbers of nondrug spectra to the collection. For example, the entry “BHA-type antioxidant,” which appears in the search output in Figure 7, is of this type and covers the class of butylated hydroxyanisole food additives, all of which produce mass spectra having m/e 135 and 197 as the most abundant ions. Only one hydro- carbon spectrum, collection and that of n-C24H50, is included it is labeled as n-alkane in the (Figures 2 and 3). Computer Comparison Algorithm (TPLI B) The computer-searchable collection (“library”) of drug mass spectra is stored on magnetic tape. The routine TPLIB (tape library) is designed for library generation, (i.e., library addition searching, of new The user states to the the mode of operation execution of the program. Each compound, with data, occupies one record cluded in the its abbreviated and library spectra record mass updating existing tape). desired upon its essential spectrometric on the magnetic tape. In- is the name of the compound, spectrum, the most abundant scribed in this paper; most of the procedures and programs we describe here can be adapted for use with more limited systems such as those now also peak, to which all other peaks are normalized (“base peak”), the number of peaks in the abbreviated commercially ments. of time ed system available. or flexibility provides Some compromises are necessary, much useful data but even (8). in terms a limit- spectrum, and the five highest The description are published rectangular-array details of these concepts of the spectrum-comparison and ele- a detailed algorithm (9), and will only be summarized. CLINICAL CHEMISTRY, Vol. 20, No. 2, 1974 257 The abbreviated mass spectrum contains the two most intense peaks in each 14 mass-unit interval (i.e., the intervals 6-19, 20-33, 34-47, etc.). Use of the abbreviated spectrum allows one to retain the significant details of the full mass spectrum, while minimizing the size of the searchable data base. The elements of the rectangular array are obtained by summing the intensities of the homologous ions at m/el+14n,2+14n,...14+14n,wheren0,1, 2.... These fourteen sums are then arranged in decreasing order and the homologous series represented by the five highest sums are stored on tape. Comparison of the unknown and known mass spectra is accomplished by the computer in two parts: (a) the pre-search and (b) the detailed comparison of abbreviated mass spectra. The pre-search is performed to eliminate obviously dissimilar spectra, for which a detailed comparison would be a waste of time. Basically the pre-search requires that: the largest peak in the known spectrum be at least 25% relative intensity in the unknown spectrum; the mass range covered by the known and unknown spectra differ by not more than a factor of approximately three (calculated by using the number of peaks in the abbreviated spectrum); and the total abundance of homologous series of ions be similar in the known and unknown spectra (using the rectangular array elements). For each known spectrum that satisfies the three successivepre-search requirements, a detailed comparison with the unknown spectrum is performed. The result of this detailed comparison is a “similari- ty index,” which ranges between 0.000 (complete mismatch) and 1.000 (perfect match). Essentially, the similarity index is the weighted ratio of the intensities in the known abbreviated spectrum to those in the unknown abbreviated spectrum taken mass for mass. Any or all of the pre-searches can be bypassed when it seems preferable to do so. For example, if a spectrum has a high background because of incomplete gas-chromatographic separation, the rectangular array pre-search could remove from consideration reference spectra that are actually quite similar to the unknown. Alternatively, removing the base peak or reverse base peak requirements permits similarity index calculations, to help identify metabolites that still have much of the structure of the parent drug but whose spectra exhibit a very significant shift in the base peak. For example, demethylation or dealkylation of the amino side chain of tricyclic drugs converts these drugs to metabolites that retain the ring structure and hence give rise to mass spectra similar in some respects to that of the parent drug, but for which the base peak derived from the amine moiety is shifted to a lower m/e value. Because removal of pre-searches results in the computation of many more similarity indices, and a longer search time, the pre-searches are retained unless preliminary results indicate that such manipulation of the search procedure would yield additional information. 258 CLINICAL CHEMISTRY, Vol. 20, No.2, 1974 Data Acquisition The operating and Interpretation Procedure conditions gas of the chromato- graph-mass spectrometer system for the analysis of the body fluid extracts are shown above in Scheme 2. Mass-spectrometric data for the complete gas chromatogram are accumulated by the computer and peak centers are calculated in real time. Perfluoroalkane is introduced from a reservoir before each gas chromatogram as calibration for time-to-mass conversion. After the run is completed, the summed intensities of each consecutive spectrum are plotted and the spectra are processed (2, 3). This corresponds essentially to a conventional total ionization plot (see Figure 1) and represents a computer-generated gas chromatogram, with the numbers along the abscissa corresponding to the spectrum index numbers of the individual spectra taken during the course of the gas chromatogram. Mass chromatograms (4) are calculated for each m/e value and the oscilloscope display of each mass spectrum and mass chromatogram is microfilmed during processing.3 The mass chromatogram, of one specified number, can be thought recorded with a detector mass value. tensity The processed data a plot of unnormalized in- mass vs. spectrum index of as a gas chromatogram that is specific for a single are automatically written on magnetic tape, and are thus available for comparison with the drug library a very short time after data acquisition is completed. When TPLIB is executed for searching purposes, the spectrum collection is transferred to a temporary file on disk for use during searching. (A disk file can be manipulated much more rapidly than a tape file.) At this point, two alternatives are available to the user: individual spectra can be compared to the collection, or the whole GC-MS run can be compared to the collection. In the latter case, the most probable compound for each unknown spectrum is printed, together with its similarity index, along the contours of the total ionization plot for the GC-MS run (see Figure 2). In addition, at the end of the search, the 10 most abundant compounds thus identified are printed out in order of decreasing abundance (see Figure 3). Thus, this results in a rapid, almost fully automatic analysis of the sample for a wide variety of toxic substances. In the few cases where the coma may be caused by a drug in so low a concentration in the body fluid as to be obscured by normal constituents, it is still often possible to detect the presence of such a foreign material by visual examination of the filmed mass chromatograms. These clearly resolve even minor peaks, as is illustrated below (see discussion of Figure 5). Results The disposition of Case 1 is typical of the majority of samples handled by this approach. When a deeply comatose 24-year-old man was brought to Massachusetts General Hospital after first being admitted to a BLUIJO 595 O-l TOTP,L IONIZMIC4 9PE(CTR : MOST PLOT P1)4 O.1887E 0.5650E O.4161E O.2895E O.1217E O.4132E O.3090E j9Q DUX Fig. tract 1. Total ionization from Case 1 plot for 08 08 07 07 07 06 06 COMPOUNDS MEPERIDINE N-ALKANE CHOLESTEROL ME TH AQUA LO NE DIAZEPAM OLEIC ACID CHOLESTA-3.5-DIENE Fig. 3. Summary of library search results for extract of blood sample, Case 1, for which Figure 1 is the total ionization plot i iU PROBABLE 2389 M GC/MS run of blood ex- Fig. 2. Library search output in profile format for the experiment for which the total ionization plot is shown in Figure 1. To avoid reduction to the point of illegibility, only the section from scan 75 to scan 130 is shown suburban hospital, a blood specimen was sent to our laboratory for analysis. Little history was available; there was some possibility of meperidine ingestion, but other information had indicated the absence of meperidine but the possible presence of amitriptylene. The patient’s condition was not consistent with this result and it was suspected that he had ingested a combination of drugs. In our laboratory, 5 ml blood serum was extracted as shown in Scheme 1 and the GC-MS data acquired and processed as in Scheme 2. The total ionization plot for this sample is shown in Figure 1. A section of the library output along the contours of the total ion plot appears in Figure 2. This output format consists of an entry for the result of the library search for each individual spectrum in the run. The scan number is printed together with the similarity index found for the most similar spectrum in the reference collection. The name of the matched compound also appears, displaced by a distance indicative of the total ionization in that spectrum. Here the most intense spectrum was that recorded in spectrum number 80, which was identified as meperidine. The summary of the library search for the entire run is shown in Figure 3. This was printed after the profile output was completed, and it lists the most abundant compounds found in the spectrum-byspectrum search, together with an exponential expression of their total ionization in the entire run. In this instance, the names of significance were meperidine, methaqualone, and diazepam, since they indicated ingested drugs-cholesterol and oleic acid are normal blood constituents and cholestadiene is an artifact produced by dehydration of cholesterol on the injector port of the gas chromatograph. The nalkane (C26H54) reference spectrum was found most similar to each of the three hydrocarbon-retentionindex standards (C14H30, C22H54, C32H66) added to the sample, since only one such compound is included in the library. It was thus apparent that the patient had ingested a very large amount of meperidine together with lesser amounts of both methaqualone and diazepam. There was no indication that any other drugs, including amitriptylene, were present. In this situation, as in most cases, no further manipulation of the data was necessary and the results were reported immediately to the hospital, less than an hour after the sample had been received. Cases 2 and 3 are among the less-common types of samples, but are included because they illustrate the flexibility of the system and its potential for rapidly dealing with samples that pose difficulties by containing uncommon or unexpected drugs, contaminants unrelated to drugs, and extremely complex mixtures. The urine specimen submitted for Case 2, a young child afflicted with severe hallucinations, arrived in a plastic tube. The gas chromatogram obtained with an extract of this sample appeared to be devoid of drug-related peaks: the peaks observed correlated in both retention time and mass spectra with the common plasticizers dioctyl adipate and several isomers of dioctyl phthalate.. Upon closer examination it was noted, however, that spectrum 348, Figure 4a, contained ions in addition to those caused by dioctyl phthalate. Subtraction of spectrum 357, which also contained the ions characteristic of dioctyl phthalate but lacked the additional ions observed in spectrum 348, gave a spectrum of a component that was centered at spectrum 348, as indicated by the maximum observed in the mass chromatogram of m/e 287, Figure 5. Computer comparison of this “cleaned” spectrum (Figure 4b) with the drug library gave a close fit for the anti-allergy drug cyproheptadine (I), and CLINICAL CHEMISTRY, Vol. 20, No.2,1974 259 LPThS 94, DSC 2 972 976--348 0 #{149} La I r#{149}’ .1LL i’r pe e .ri#{149}’1 visual comparison of this spectrum with the reference spectrum (Figure 4c) confirmed the identity of the unknown with this drug. The presence of cyproheptadine had not been suspected and it is doubtful whether the conventional methods of analysis would have detected it at all, because most methods are limited to recognition of a compound or group of compounds whose presence is anticipated and they are consequently unable to detect or to identify compounds outside their limited range of application. An excessive dose of cyproheptadine would result in a state such as that observed for the child and its identification in this urine extract satisfactorily explained the child’s condition. ,- .Ld.U. - . aoo N CYPPU-[PTAOII[ Fig. 4. (a) Scan CH3 348 from GC/MS run of urine extract from Case 2. Spectrum is predominantly that of dioctyl phthalate. (b) Scan 348, above, after subtraction of scan 357, effectively removing dioctyl phthalate ions. (c) Reference spectrum of cyproheptadine 267 TOTAL IONZATION- Case 3 involved a young man, a participant in a methadone maintenance program, who was brought to the hospital in a state of deepening coma. It was not known whether he had consumed an overdose of methadone or had fallen back into the use of other drugs. Blood and urine samples were provided to our laboratory. The total ionization-retention index plot obtained for the urine extract is shown in Figure 6, labeled to show the results of the library search. [The hydrocarbon standards C14H30, C22H46 and C32H66 were coinjected in order to provide reference data for the retention index calculations (6).] These results indicated the ingestion of the drugs methadone and methaqualone. The identification of the metabolites as well as the unchanged drugs enabled the analyst to characterize additional major chromatographic fractions and thereby confirm the identifications of the drugs. The metabolite spectra had already been placed in the reference collection, and so these compounds were completely characterized by the automatic procedure, but some indication of their structures could have been obtained from the library search even if the exact compound spectra had not yet been added. Figure 7 shows the results of the comparison of the spectrum assigned to HI with the library. This output format presents the 10 compounds in the reference collection found to be most like the spectrum 550 Fig. 5. Overplot O of m/e ion of cyproheptadine, 550 550 287 on plot (light trace) for the extract 260 (heavy trace), the of the total of urine, Case 2 a section CLINICAL CHEMISTRY, Vol. 20, No.2, 1974 molecular ionization MW 287 under consideration, together with an index of their similarity. For this spectrum the highest similarity was found for methaqualone metabolite 1 (III) and indeed the retention index also agrees well with that previously determined for this compound. The search program also retrieved two closely related compounds: another methaqualone metabolite (IV) and the drug itself (II). The inclusion of these two compounds on the list of most similar compounds would have provided valuable clues LPET#{128} . 43 DEN _ TNN 5 23 73 DT#{128}. METHADONE M E TA AOL lIE 5 DEN 73 73 2O21-lP METHADONE 7 (21) UEIHAOUALOME (2 METNAOUALONE iil (21)110) CIMEIHOL SULEONE NICOTINE LAIE ( cnoLEsrERoL THEOBROMINE c.Hji (‘a, ‘nap Fig. 6. Total ionization-retention index plot (6) for urine extract described in text as Case 3 Labeled peaks indicate library search results obtained for this GC/MS run. A mixture of hydrocarbons was coinjected and these are labeled C14(n-C14H35), C22(n-C22H46), and C32(n-C32H66) URINE 480, 161 NON RUN NO. H cTrAm[ 2096 RESULTS II). METHAOUALONE METAPOLITE METHAQUALONE METABOLITE THIORIDAZINE METAPOLITE 2,6-01 -T-BUTVL-4-METI,YLPHENOI.. BHA-TYPE ANTIOXIDANT THIORIE,AZINE METABOLITE NARCE 1 NE METHAOUALONE MEPHOBARBI PHRNVL8UTAZ I 2 2 I TAL ONE SIR. 0.173 0.104 0.085 0.079 0.069 0.068 0.065 0.064 0.059 0.059 247 248 250 252 280 249 229 225 203 236 Fig. 7. Library search output, identifying methaqualone metabolite (III), for scan taken at maximum of GC peak, listing 10 compounds in collection most Similar to the spectrum of the compound found in this extract, to the identity of the unknown, had the spectrum of III not yet been in the collection. Case 3 reference 235 H3C MW 250 2511 ‘C H3 MW 266 251 LI iL Scan 137. from GC/MS run of 3, taken at maximum of peak that similar to methaqualone; (b) reference methaqualone (II); (C) reference spectrum (V) Fig. 8. (a) Case most urine extract, library found spectrum of of cocaine Comparison of spectrum number 137 in the urine extract chromatogram, Figure 8a, or the corresponding spectrum in the blood extract chromatogram, with the reference spectrum of methaqualone (II) (Figure 8b), reveals the presence of extraneous peaks and thus points to the presence of yet another substance that is unresolved from methaqualone under these conditions. Inspection of mass chromatograms of the unmatched masses to determine those that maximize at scan 137, as illustrated in Figure 9 by the mass chromatograms of m/e 182 and m/e 303, permits one to assemble a list of the more abundant ions originating from the unknown. Comparison of this list with an eight-peak index of our drug spectra collection4 suggests that the unknown compound is cocaine (V) and a visual comparison of its spectrum (Figure 8c) with scan 137 shows that, indeed, all the peaks of the reference spectrum of cocaine are present in this scan, in addition to those peaks appropriate for methaqualone. Retention index data from other samples also indicated the similar behavior of the drugs cocaine and methaqualone under the gas- NkH3 I(YOH MW 266 4 This index was compiled (and has been distributed upon request) by the authors’ laboratory. It is patterned after the Eight Peak Index of Mass Spectra, Table 3, published by the Mass Spectrometry Data Centre, AWRE, Aldermaston, Reading RG7 4PR, U.K. CLINICAL CHEMISTRY, Vol. 20, No. 2. 1974 261 LIIFE 420, 104 RiM . TOTAL I55IZAT!55 PLOT IF 9’#{128}CT#A200 s’cciaa M/E t02 I1X F o I I P WE I. j)Jv2I F z H LIr 55% Fig. (b) I I l I II 4i IP/’’lT”)’E j.77 Fig. I chromatographic conditions used here. All these considerations led to the conclusion that cocaine was present, even though it was masked in the gas chromatogram by the co-eluting methaqualone, the major component. The unambiguous identification of cocaine was important, not only for the immediate treatment of the patient’s critical condition but also provided a piece of useful information for those responsible for the patient’s participation in the methadone program. C #{231}COCH3 -.. JJi4h LI 20- II II.JIIj. -188 MW303 1821 In the blood-serum extract, in addition to methadone and methaqualone and its metabolites, diazepam and its N-demethyl metabolite were identified. The presence of diazepam in the blood specimen and its absence in the urine sample illustrate the importance of examining more than one body fluid whenever possible. Discussion The GC-MS-Computer system is particularly well suited for samples where the poisoning agent is unknown and could include any or a number of various classes of compounds. The extraction procedure and gas-chromatographic separation are rapid and will accommodate a wide variety of toxic agents. Because no class separations or derivatizations are involved, they are not so limited in scope as are more specific techniques. The method also tolerates the presence of other materials (fatty acids, phthalates) that comCLINICAL CHEMISTRY, Vol. 20, No.2, 1974 10. Mass spectrum 168 of urine extract, Iiili ii ‘‘‘‘ 200 1.00 3. Case 3 262 73 0.70 9. (a) Total ionization plot for urine extract, Case 3. Mass chromatogram for m/e 182, urine extract, Case (c) Mass chromatogram for m/e 303, urine extract, 0 5 23 I&AR scan I 480, 16-I of methaqualone Case 300 metabolite (IV) 3 monly occur in large concentrations and can pose (recognized or unrecognized) interferences in other techniques. The result is the identification of a specific drug, rather than simple indication of its being a member of a class of drugs. This capability can be of significance in discriminating between long- and short-acting barbiturates, for example, for which different clinical treatments will be required. Since the reference spectra are already in the tape library, it also obviates the need to have a standard available for simultaneous analysis or coinjection. In the case of the less-common drugs, this can be a critical factor, because valuable time could be lost locating an authentic sample of a drug whose presence was suggested by thin-layer chromatographic, GC, or ultraviolet data alone. On the other hand, if the spectra obtained should include substances not contained in the collection, the basic interpretability of mass spectra makes it possible for the chemist to deduce the nature of the substance even in the absence of an authentic spectrum. This capability is particularly useful in the analysis of new or unusual “street” drugs, for which no reference material may be available. It is also essential in recognizing that some of the components of the mixture are metabolites of drugs that may or may not also be present in the extract. For example, it is a fairly simple matter to recognize that spectrum 168, Figure 10, belongs to a hydroxylated metabolite of methaqualone (IV). The shift of the major peaks in the spectrum to m/e 251 and 266 and the difference in retention index indicate aromatic hydroxylation (6) . In contrast, the spectra of some drug metabolites are quite different from those of the parent compound. The mass spectrum of methadone (VI) is dominated by the ion at m/e 72, resulting from cleavage fi to the nitrogen: CH3CH=N(CH3)2. There are no ions at m/e values greater than 72 that have an abundance of more than 10%. Spectrum 116, Figure 11, is obviously quite different from that of Nau, H., and Biemann, K., Utilization of automatically signed retention indices for computer interpretation of mass tra, Anal. Lett., in press. asspec- Lrnr’E #{149}. Am, so-i 5 A LJ.l.Li Mass spectrum #{149} L I 100 Fig. 11. e3 9i15 73 J 2121 of methadone metabolite (VII) scan 116 of urine extract, Case 3 methadone. metabolized Methadone, however, is known to be to a cyclic compound (VII) (10), and spectrum 116 does correlate quite well with the mass spectral behavior expected of such a structure. It exhibits an abundant molecular ion, reflecting the stability of the phenyl-substituted heterocyclic ring and the only facile loss is that of a methyl group. \\ CH Cli N’ 112 CH 7 CH3 CH “CH3 - N3 - Cli -N; <CH2 262 CI I 72 MW While 309 during assignments 277 such as these can be it is more efficient and requires less expertise on the part of the individual analyst if these metabolite spectra are included in the reference collection. Accordingly, after each of these metabolites was first identified by deduction on the basis of spectra obtained during analysis of emergency samples, their authenticated spectra were added to the library, so that their identification in the case illustrated here was automatic. It is essential that metabolites be recognized as such for two reasons: (a) The parent drug may have been completely metabolized and can thus be recognized only by identifying the resulting metabolites. Chloral hydrate, for instance, is converted to 1,1,1trichloroethanol and none of the drug itself appears in the blood or urine of the patient. (b) An analysis of the type described here cannot be considered complete until all of the significant gas-chromatographic peaks are satisfactorily accounted for. The total number of drugs ingested is usually unknown, so that unmatched peaks in the chromatogram must be considered as potential evidence of further drugs unless a relationship to a drug already identified in the extract can be established. Contaminants may also be present and give rise to gas-chromatographic peaks as large as or even larger deduced structure MW an analysis, than the drugs cation of these themselves. contaminants Fast and reliable identifieliminates the possibil- ity of confusing them with the causative agent(s) and makes it possible to suggest modifications of sample acquisition and transfer procedures that minimize or eliminate this interference in the future. For example, it was noted quite early in this work that some blood samples produced a major peak with a retention index 2665 (OV-17). Interpretation of its low and high resolution mass spectra led to its identification as tri-(2-butoxyethyl) phosphate and its source was traced to the rubber stoppers used on blood-collection tubes (“B-D Vacutainers”). (To eliminate this interference, we recommend that all blood samples submitted to our laboratory be drawn with glass syringes.) Information such as the identity and source of contaminants is useful not only for GC-MS procedures but can be of assistance to those using more limited methods in which unknown contaminants interfere even more. Simple procedural modifications can then be devised to eliminate contaminants, once their nature and source are established by GC-MS. The additional dimension of specific data provided by mass spectra means that drugs whose gas-chromatographic behavior is identical on OV-17 (e.g., glutethimide and meprobamate; diazepam and diphenylhydantoin) may be easily distinguished by their mass spectra. The GC-MS approach to this problem is preferable to direct admission of the sample into the ion source of the mass spectrometer, as has been discussed elsewhere (11). Samples of biological origin, extracted by a deliberately nonselective procedure to assure that a very wide range of drug types will be retrieved, contain a large number of compounds such as fatty acids and cholesterol, which may be present in concentrations much greater than are the drugs of interest in these analyses. Multiple drug ingestion may also result in a very complex extract. If this mixture is admitted directly into the mass spectrometer, ions derived from an individual drug will be accompanied by (and possibly obscured by) ions derived from all the other materials present. It is then a task, which can be difficult and may at times be impossible, to determine successfully which observed masses correspond to the individual components and thereby identify the causative agent(s). While slow controlled heating of the sample and the use of highresolution mass spectrometry do help to alleviate these difficulties somewhat, we have found that use of the gas chromatograph to achieve separation is the most routinely useful approach for extracts of body fluids. The spectra obtained from GC peaks are those of relatively pure compounds and are therefore amenable to immediate automated comparison, since the data-acquisition system provides digital information that is quickly converted to a searchable format. The separation of components ensures that even relatively minor components produce definitive spectra. CLINICAL CHEMISTRY, Vol.20, No.2, 1974 263 In order to make maximum use of the data provided by the gas-chromatographic separation, we routinely calculate retention indices for all runs (6). This additional dimension of information is useful in several ways. It provides a means of distinguishing clearly between compounds whose common structural feature makes their mass spectra very similar, e.g., the base peak at m/e 58 (and only major ion) of many compounds containing the -CH2N(CH3)2 grouping, a very common feature of many local anesthetics and tranquilizers, among others. A comparison of the retention index of the compound retrieved by the library search with that of the spectrum being searched helps the analyst to decide whether the search has found the identical compound or, instead, the most similar compound in the collection. In the event of incomplete gas-chromatographic resolution, the presence of extraneous peaks in the experimental spectrum may cause the similarity index between known and unknown spectra to be quite low, even when the exact compound is being matched. Coincidence of the retention indices of the known and unknown gives a further degree of confidence in the analysis when relatively impure spectra must be used for the comparison, as is illustrated in Figure 8, where the spectrum of a small gas-chromatographic peak has been successfully matched despite the presence of a relatively high background. Examination of the body fluids of persons who have taken abnormally high dosages of drugs also provides an opportunity for studying metabolism under circumstances that could not be induced deliberately. Such studies can give valuable information concerning the metabolism of therapeutic doses of such drugs by providing larger amounts of metabolites or may indicate alternative metabolic pathways that become important at much higher doses. The retention-index data generated in the GC-MS experiment can be used for further studies with gas chromatography alone and also have an independent information content, because differences in retention indices among a related group of compounds give clues to their structural differences-a valuable tool in interpreting the mass spectra of new metabolites The procedure described here has been designed as a qualitative technique, but does permit rough estimation of quantities involved by comparison of the area of the gas-chromatographic peak of the unknown with that of co-injected standards. An allowance must be made for nonquantitative extraction and variability of flame detector response to different types of compounds. Usually it suffices to report to the attending medical personnel the approximate concentration at the time the identification of the agent is reported. If a greater accuracy is indeed required, the conventional clinical methods of quantitative analysis can be more reliably used, once the identity of the substance to be quantitated and the nature of the contaminants have been established. GC-MS methods with stable isotopes have unique advantages for quantitative analysis of drugs and . 264 CLINICAL CHEMISTRY, Vol. 20, No.2, 1974 drug metabolites in body fluids (12 ); but the simpler methods are usually adequate for these emergency situations, once the qualitative results have been obtained as described. The selection of drugs for inclusion in the searchable library was originally based on the likelihood that they would be ingested in suicide attempts. Experience has shown, however, and Poison Bureau data (13) also indicate that many of the poisoning victims who arrive at the emergency room of local hospitals are suffering the consequences of accidental poisoning either because of deliberate ingestion of drugs purchased from illegal sources, or, in the case of young children, random ingestion of drugs or other toxic materials available in the home or elsewhere. We have therefore expanded the library to include a number of illicit drugs (LSD, psilocybin, morphine, phencyclidine, etc.) and potential contaminants (strychnine), as well as drugs and other substances not usually considered as possibilities in adult poisonings, but which the unsupervised child may ingest in toxic quantities (doxylamine, camphor, etc.). In order to make it possible to rapidly identify all the major peaks in the gas chromatogram, the spectra of commonly-occurring blood and urine constituents and contaminants have also been added to the library. This ensures that the chemist’s attention will be directed toward those components of toxicological significance. Although efforts have been made to instruct the attending physicians as to the proper manner of sample handling to minimize contamination, the very number of physicians and hospitals who may potentially send samples for examination increases the likelihood that impurities such as plasticizers will still be observed occasionally. It is a simple matter to add such spectra to the library and thereby avoid the chance that this can cause a false lead as to the poisoning agent. We have received more than 600 samples during the past two years; we have identified at least one toxic substance in about 75% of the cases. The remaining 25% includes those patients whose coma was caused by factors other than ingested substances and those overdose victims whose body fluid did not contain a detectable amount of the toxic agent at the time of sampling. Multiple ingestions have been most common with barbiturate poisonings. We have, so far, found a total of 72 different drugs, as well as 26 other toxic materials, results that constitute a strong argument in favor of this broad-based approach. The age distribution of the patients from whom we have received samples over the same period is indicated in Table 1. In general, we have observed that accidental ingestions account for most of the poisonings of small children, that young teenagers are frequently the victims of illicit drugs that have been misrepresented to them, while the comas of older teenagers usually involve excessive or unwise use of known drugs and those of adults are most often the result of deliberate suicide attempts. Table 1. Age Distribution of Patients Age, yrs No. of patients tional Institutes of Health, RR 00317 from the Biotechnology Resources Branch, Division of Research Resources, and Training Grant GM 01523. Dr. James E. Biller’s and Mr. Edward M. Ruiz’s contributions in the continuous improvements of the data handling procedures 0 29 0-2 3-6 95 47 are gratefully acknowledged. 38 References 7-10 11-15 111 16-20 child, ?yrs. adult 91 13 177 Total 601 It has to be kept in mind that both gas chromatography and mass spectrometry require the substance to have a measurable vapor pressure. Fortunately, the vast majority of drugs, or at least the corresponding free acids or bases (if the drug is administered as a salt) or some of the major metabolites, fall into this category. Inorganic poisons like arsenic and cyanide, however, are, of course, not detectable. We conclude that the GC-MS-Computer system described is well suited to rapid identification of toxic materials in body fluids of poisoning victims. The system is general enough to quickly identify a wide variety of agents responsible for the coma or other emergency condition of the patient. The system has advantages in both speed and specificity over other methods and it therefore is expected that the method will be widely applicable and extremely useful for clinical chemists, particularly in view of the ever-increasing number of drugs appearing on the legal and illegal market. We are grateful to Professor John Hedley-Whyte and Mr. Hans Laasburg of Beth Israel Hospital, Boston, for their advice and for providing us with samples of many of the drugs needed to accumulate the collection of authentic mass spectra, and to Dr. Frederick Lovejoy, Jr. of the Boston Poison Information Center for arranging communication with the appropriate personnel in area hospitals. This work is supported by research grants of the Na- 1. Althaus, J. R., Biemann, K., Biller, J., et al., Identification of the drug Darvon and its metabolites in the urine of a comatose patient using a gas chromatograph-mass spectrometer-computer system. Experientia 26, 714 (1970). 2. Hites, R. 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