Identification of dangerous drugs by mass spectrometry

Identification of dangerous drugs by mass spectrometry

221 CLINICA CHIMICA ACTA IDENTIFICATION OF DANGEROUS N. C. LAW, AANDAHL, VIRGINIA DRUGS H. M. FALES AND BY MASS SPECTROMETRY G. W. A. MILN...

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221

CLINICA CHIMICA ACTA

IDENTIFICATION

OF DANGEROUS

N. C. LAW,

AANDAHL,

VIRGINIA

DRUGS

H. M. FALES

AND

BY

MASS SPECTROMETRY

G. W. A. MILNE*

Suburban Hospztal, Bethesda, Md. 20014, Davision of Computer Research and Technology, Nataonal Instztutes of Health, and National Heart and Lung Institzcte, National Institutes of Health, Bethesda, Md. 200x4 (U.S.A.) (Received

November

6, 1970)

SUMMARY

The low resolution mass spectra of fifty-eight commonly used drugs have been recorded. These drugs, some of which are available without prescription, may be lethal when taken in excessive quantities. Gas chromatographic and mass spectrometric techniques which can be used to obtain the mass spectrum of the toxic principle in biological materials such as gastric lavage, serum or urine are described. A system has been developed by which an unknown drug can be identified from the salient features of its mass spectrum. This employs a program written for a DEC PDP-81 computer with 4K words of memory and disk storage.

In the last twenty years, there has been a marked increase in the number of drugs available with analgesic, sedative and tranquilizing action. Predictably, there has been a corresponding increase in the number of cases in which quantities far in excess of prescribed amounts have been consumed either accidentally or purposely, sometimes with fatal results. Identification of the drug in question is a prerequisite to optimal treatment in such cases. For example, when a patient has a serum level of 3-4 mg/roo ml of a sedative such as glutethimide or seconal, it is essential to lower this level rapidly, before irreversible damage can occur 1. The most effective means for removing the offending drugs utilizes the patient’s own kidneys or in the event of renal overload via artificial kidney or peritoneal dialysis. However, use of the artificial kidney is a major surgical procedure; the necessary equipment is scarce and its use is expensive. Peritoneal dialysis is simpler and cheaper but is of less value for drugs of a basic character such as meperidine. The nonbasic nature of the drug in question should therefore be established before peritoneal dialysis can be optimally employed for its removal. The patient himself can often identify the drug or drugs that he ingested and this source of information is often very reliable. However, if the patient is comatose * Reprint requests can be addressed to Dr. G. W. A. Milne, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. Clin. China. Acta,

32 (1971) 221-228

LAW

222

et al.

upon arrival at the hospital, as is sometimes the case, the information is not available. In such cases, the clinical chemist is required to identify the drug or drugs from an examination of biological specimens, the most readily available of which are urine, blood and stomach contents. If ancillary symptomatic evidence is available, indicating the presence of a specific drug the chemist may be required merely to seek confirmatory evidence. In either case, he is obliged to embark upon a series of tests to differentiate between hundreds of possibilities in order to arrive at a definitive conclusion. Generally, an attempt is made only to identify and quantitate the major classes of drugs by chemical meanP. Specific analysis is a simple matter only in the case of a few drugs such as ethchlorvynol (placidyl) whose chemical properties are sufficiently distinctive to permit accuracy and reliability. Within other classes of drugs such as the barbiturates or the amphetamines, it can be predicted that truly specific analytical methods based upon chemical reactions will be difficult to develop. While several hospitals have recently employed thin-layer chromatography for such analyse9, it has been our experience that these methods are time-consuming and require careful attention to details such as freshly prepared reagents and so on. A further difficulty is that the certainty with which compounds can be identified by thin-layer chromatography is often insufficient to provide the necessary confidence in the results. Gas chromatography provides more positive identification since Rt values are more accurate than Rf values and a program was inaugurated to identify by this method drugs commonly encountered in suicide attempts, particularly glutethimide and seconal. It soon became evident that gas chromatography alone (GC) has severe limitations. Serum from various patients was found by GC to contain several low molecular weight compounds which could not be identified by GC alone. When the effluent from the GC was examined, peak by peak, in a mass spectrometer, a study of the resulting mass spectra permitted the identification of many of the compounds as for example, metabdlites of glutethimide and seconal, normal serum constituents such as cholesterol and frequently, common plasticizers such as dibutyl and dioctyl phthalate, which are readily leached from plastic tubes by organic solvents. From several experiences of this sort, it became clear that GC alone was often insufficient to provide positive identification. The decision was then made to investigate the unknown specimens by coupled gas chromatography-mass spectrometry (GC-MS). In this method, the components of a mixture may be separated by GC and the mass spectrum of each of them can be obtained as the material passes from the gas chromatograph into the mass spectrometer. As will be seen, the mass spectrum of a compound is a very reliable “fingerprint” of that compound and permits immediate and accurate identification of the material, All the compounds considered here were originally identified by a manual study of their mass spectra. As the number of cases handled increased and the number of drugs to be considered increased, it became clear that the identification phase of the problem could be dealt with most effectively with a computer. METHODS AND MATERIALS

All drugs discussed below were derived from one of three sources: Clkn. Chim. Acta, 32 (1971)

221-228

I.

specimens

DANGEROUS

223

DRUGS BY MASS SPECTROMETRY

available in N.I.H. laboratories; 2. from the appropriate manufacturer; 3. by physical extraction from the compounded tablets, capsules, etc. which were obtained from the Pharmacy Department at N.I.H. and Suburban Hospital. Biological specimens from patients suffering from drug overdose were obtained at the Suburban Hospital, Bethesda, Md. All samples were handled at the time of acquisition according to a standard procedure applicable to serum, urine and gastric lavage. Following chloroform extractions at acidic and basic pHs, the organic layers were dried and evaporated to dryness or near dryness with some care as various drugs, for example, ethchlorvynol and the amphetamines, are relatively volatile. This procedure was adopted as an expedient technique in order to obtain the extracts as rapidly as possible. The materials so isolated were immediately submitted to GC-MS analysis by the staff of N.I.H. and Suburban Hospital. Mass spectra were measured on an LKB9000 gas chromatograph-mass spectrometer with a source temperature of 250’ and an electron beam voltage of 70 eV. The standard samples of drugs were in most cases admitted to the mass spectrometer via the direct insertion probe while samples derived from biological specimens were admitted via either the direct insertion probe or the gas chromatograph inlet. In all cases, a 1% OV-17 column was used and the carrier gas was helium, at a flow rate of 20-40 ml/min. The temperature of the column typically was programmed up from 80” at 3O/min. The computer used in this work was a DEC PDP-81 with a memory of 4096 rz-bit words and 65536 words of disk storage. Programs were all written in assembly language and use the SERF disk monitor designed by Applied Data Research, Inc., Princeton, N. J.“. These programs are available upon request6. RESULTS AND DISCUSSION

Data extracted from the low resolution mass spectra of fifty-eight commonly used drugs are given in Table I. A representative selection of dangerous drugs appears on this list. Additions can be easily made to this “master file” however, and if a new drug is encountered the appropriate data are entered into the file. Equally importantly, artifacts encountered in the GC analysis of extracts from biological specimens may be entered upon the list. Thus when they are encountered again, they can be identified with certainty and not mistaken for drugs or drug metabolites. Each entry in the master file consists of the m/e values of the five biggest peaks in the mass spectrum, listed in order of decreasing abundance. As can be seen from the table, this description of each spectrum is unique. No ions of m/e less than 40 are considered so as to prevent confusion from the varying abundances of ions arising from background air and water. The entire spectrum is normalized to the most intense ion above m/e 40, and if two ions should be of equal abundance, they are listed in order of increasing m/e value. The drugs whose spectra are included in the file fall into seven main categories with respect to their biological activity. They are therefore classified as sedatives, tranquilizers, narcotics, stimulants, analgesics, antihistaminics or alkaloids. In addition to these classes, there is a small miscellaneous category of drugs that fit none of these descriptions; there also are a few entries for the artifacts mentioned above. If the data are available for any given drug, the appropriate entry is made in the file even if Cl&.

Chim.

Acta,

32 (1971) 221-228

LAW et al.

224 TABLE MASTER

I FILE

OF DRUG

MASS

SPECTRA

Class*

COWl@4VZd

GIVING

FIVE

1

2

283 I72 I56 44

282

Levallorphan Alphaprodine Amobarbital Amphetamine Aprobarbital Butalbital Chlordiazepoxide Chlorpromazine

M N S St S S T T

Cocaine Cyclobarbital (+)-Desoxyephedrine Dextropropoxyphene Diacetylmorphine Ethchlorvynol Glutethimide Hexethal

A S St An N S S S

Hexobarbital Histamine Meperidine Meprobamate Methadone Methapyriline Methylphenidate Morphine

S M N T N Ah St A

s; 9I 58 84 285

Nalorphine Phenazocme Phenobarbital Primaclone Probenecid Pyrimethamine Scopolamine Secobarbital

M An S M M M A S

Chlorothiazide Triamterene Barbital Chlorpheniramine Acetylsalicylic acid Quinacrine Chloroquine Antipyrine

~67 168 282

58

I87 I4I 9I 4I 4I 283 318

LARGEST

PEAKS

4

5

256 84 I57 65 168 I24 284 86

I76 57 4I 42 I24 181 299 272

I57 42 55 43 I67 241 85

Librium

IO5

94 67 43 9I 43 53 160 I57

77 79 56 Io5 3Io IO9 II5 55

Methamphetamine Darvon Heroin PlacIdyl Doriden

80

120

82 I4I 9I 57 369 II7 II7 I4I

59 268

81 81 70 84 223 97 85 162

I57 44 247 43 294 72 56 215

54 57 56 57 7’ 9I 42

I55 55 42 55 42 191 55 286

271 230 204 I46 256 247 94 168

44 231 63 190 248 I38 I67

270 58 I46 II7 I85 249 42 4I

214 IO5 232 118 224 250 108 43

272 44 II7 I89 257 219 I36 I24

M M S Ah An M M An

295 253 I56 203

297 43 98 205 43 259 73 77

97 254 I55 204 92 58 87 56

57 IO4 55 72 I2I

86 86 188

268 252 I4I 58 I38 126 48 96

Sulfapyridine Promethazine Tripelennamine Zoxazolamine Talbutal Procaine Acetaminophen Heptabarbital

M Ah Ah M S An An S

I84 72 58 168 I67 86 IO9 221

I85 73 91 I79 168 99 I5I I4I

92 I98 I97 II3 4’ 120

108 213

:I

65 180 72 78 97 58 80 79

Ethinamate Diphenylhydantoin Carbromal Thiamylal

S S S S

81

91 209 69 41

106 IO4 4I I*4

95 223 208 168

79 252 210 I67

Clin. Chim.

,4&a,

32 (1971)

182

207 58 58 327 II5 I89 I56 221

82

120

180 44

43 221-228

121

Other name

3

81 59

89

I32 4’

Demerol

Ritalin

Primadone

Seconal Diuril

Aspirin

112

319 IO5

:J I24 87 81 222

Phenergan Flexin Tempra

Dilantin

225

DANGEROUS DRUGS BY MASS SPECTROMETRY TABLE

I (continued)

Compound

Class

I

2

3

4

5

Other name

Diazepam Dibutyl phthalate Pentachloroethane Cholesterol

Tr Ar Ar Ar

256 I49 I67 368

283 4I II7 386

284 205 II9 275

257 223 I65 353

254

Valium

Iso-octyl phthalate Stearic acid Pentazocine Phencyclidine Tetrahydrocannabinol Pentobarbital Codeine Methyl salicylate Normeperidine

Ar Ar An M M S An M Ar

I49 44 217

I67 73 II0 9I 299 I4I 162 92 233

57 57 70 243 231 43 229 152 42

7I 60

70 55 270 186 243 I57 3oo 65 I58

200

314 I56 299 120 57

202

242 271 41 I24 121 56

?3: I47

* M = Miscellaneous. N = Narcotic. A = Alkaloid. An = Analgesic. ulant. Ah = Antihistaminic. T = Tranquilizer. Ar = Artifact.

S = Sedative.

St = Stim-

the drug is unlikely to be encountered as an overdose. Thus histamine is to be found in the file even though it is now never used except under direct medical control. Irrelevant entries such as this do not detract from the value of the file or appreciably affect the computer searching time and their automatic inclusion circumvents the alternative judgment decision. The complete mass spectra from which the data in Table I have been abstracted have been submitted to the Mass Spectrometry Data Centre of the U.K. Atomic Weapons Research Establishment, Aldermaston, England, and also to the Atlas of Mass Spectral Data’ from where they are available upon request. In order to search this list effectively, a computer program has been written to compare experimental data with the entries on the list. The computer retrieves the disk-stored master file and checks each entry on it against the input data. Any entry on the list that consists of the same five numbers as the input data is considered to be a solution and is presented as such. If no such “five-peak fit” is found, the operator is so notified and asked if a four-peak fit will suffice. An affirmative response to this causes the computer to refer again to the master file, seeking this time any entry which has any four out of five numbers in common with the input data. This process can be repeated if necessary until a one-peak fit is attempted. Solutions are not repeated; a four-peak fit is not considered subsequently to be also a three-peak fit. When a solution at any level of correspondence is found, this entry is typed out in the format shown in Fig. I. The data given in such a solution consist basically of reference data or items of information which File + MS7403

CompoundName Promethazine Phenergan, S

s

I

2

72

73

3 I98

4 180

5 213

Mol.Wt. 284

P/A P

Fig. I. Output from a file search.

can be used by the operator in seeking corroborative evidence in support of the solution. The molecular weight is given, and in the last column, headed P/A, an entry “P” indicates that the molecular ion, with this mass, is present and should have an abundance of at least 1% of the base peak of the spectrum. An entry “A” reveals that this molecular ion should be absent. Cl&. Chim. Ada,

32 (1971)

221-228

226

LAW et al.

The five most intense ions in the spectrum are given in order of decreasing intensity. This order is not considered during the search because each file entry is unique, irrespective of the order in which the ion m/e values are listed. The order can be checked at this stage however by the operator. The second line of the output contains the page in the Merck Index* upon which the drug is listed. Thus “M870” refers to page 870 of this publication. This is sometimes followed by comments such as common synonyms for the drug (e.g. “phenergan”), and other information that may be considered to be useful and any heteroatoms (i.e., Cl, Br, I, S, etc.) present. Thus promethazine contains sulfur. Noting this, the operator can check that the expected isotope peaks are present in the spectrum. In Table II are given some representative cases in which GC-MS has been used to identify drugs taken in overdose quantities and subsequently extracted either

TABLE CASES

II

OF DRUG

OVERDOSE.

IDENTIFICATION

BY

MASS

SPECTROMETRY

Date

Patient

Specamen

Identi$catzon

Remarks

2/24/70

K. M.

Serum

Dextropropoxyphene Pentobarbital Dibutyl phthalate

High level

3127170

F.

c.

Serum

Glutethimide

4/21/70

M. B.

Serum

Phenobarbital Seconal Dibutyl phthalate

51”/70

C. M.

Gastric

Meprobamate Dibutyl phthalate

5/14/70

G. H.

Gastric

Meprobamate Dibutyl phthalate

5/~6/70

D. B.

Serum

Pentobarbital Amobarbital

5/22/70

P. M.

Serum

Secobarbital Amobarbital Dibutyl phthalate

5/22/70

C. J.

Gastric

Meprobamate Chlordiazepoxide

6/8/70

R. I.

Gastric

Phenobarbital

6124170

G. P.

Gastric

Secobarbital Dibutyl phthalate

717170

R. W.

Gastric

Chlordlazepoxide

719170

A. B.

Gastric

Ethchlorcynol Dibutyl phthalate

7118170

R. C.

Gastric

Phenobarbital Secobarbital Diazepam Dibutyl phthalate

7119170

T. W.

Gastric

Secobarbital

9/20/70

P. F.

Gastric

Phenobarbital Methapyrllene Acetylsalicylic acid Methyl salicylate

Clin. Chim.

Acta,

32 (1971)

221~-228

85% 15%

Octyl phthalates also present

DANGEROUS

DRUGS BY MASS SPECTROMETRY

227

from serum or from gastric lavage. These cases, in which routine chemical inspection of the material failed to give uneq~voc~ results represent examples taken from IO*~ of the overdose cases admitted by this hospital. Almost all the drugs identified were either sedatives (particularly barbiturates) or tranquilizers. These types are frequently found together in a sample either because they are so compounded or because persons attempting suicide are prone to have both types in their possession. Identification in gastric Iavage of a drug or drugs taken in overdose quantities is a problem in analytical organic chemistry but differs from most such problems in one important respect, viz., the quantity of material available, A prescribed dose of the common barbiturate seconal, for example, might be IOO mg*. An overdose of the drug may well be ten times this quantity and even under conditions favoring rapid absorption (i.e., presence of alcohol, full stomach, long delay) a So-ml sample of gastric lavage will still yield a few mgs of the unchanged drug. For this reason stomach contents undoubtedly are the best source of the unchanged drug and we have concluded that it is extremely important to acquire gastric lavage samples for analysis. These samples, together with circumstantial evidence (e.g., pills, vials) can be collected in the emergency room and education of the appropriate personnel in this regard has led to a much higher incidence of successful analysis. In many cases, the serum of the patient also contains considerable quantities of the unchanged drug, but metabolites may also be present. As time progresses, only the metabolites may be detectable and their positive identification becomes more critical. With this end in view, drug metabolites are also included in the master file, as the necessary mass spectral data becomes available. For example, the last compound in the file, normeperidine, is an important metabolic product of meperidine (demerol) formed very rapidly by demethylation in the liverlo. Unfortunately, the metabolic products of many drugs, even the older established ones such as the barbiturates, have not all been clearly identified and overdose specimens examined in this way present an unusual opportunity to study human drug metabolism. Urine is a rather unpromising source of data for drug identifica~on when compared to the other two biological fluids discussed above, because many drugs fail to emerge intact in urine and one must methodically search for more polar metabolites which are often somewhat harder to deal with than the drug itself. For example, aspirin (acetyl salicylic acid), is a relatively simple aromatic acid which can be extracted into organic solvents and easily identified by GC-MS. It is excreted in urine, however, as its glucuronide l1 which is water-soluble and too polar to permit satisfactory GC-MS analysis. At the same time, it should be noted that the nonbarbiturate sedative glutethimide (doriden) is excreted exclusively as an underivatized metabolite, a-phenylglutarimide12~13 and a method for identification of this drug, based on the mass spectrometric detection of this metabolite in urine has been developed’*. A GC-MS technique for the detection of dextropropoxyphene (Darvon) in urine has also been publishedl5. The organic materials extracted from biological specimens initially may be submitted to mass spectrometric analysis using the direct insertion probe to admit the sample to the mass spectrometer. In the case of chronic overdoses, there is often so much of the drug present that a rapid and unequivocal identification may be arrived at in this way. The GC-MS method used here is a somewhat slower procedure but it has important advantages over the probe method. Each mass spectrum is obtained C&n. Gkim. Acla, 32 (rg7r)

221-228

LAW et al.

228

from a pure compound whose identification is therefore extremely simple by reference to the master file. Furthermore, many retention times have been recorded in the literature2 and can be used to assist in the identification. Each of the components of a mixture, present at very different levels, may be identified in this way whereas the probe technique usually permits the identification of only the major component. Finally, the total ion monitor, like the detector of a simple gas chromatograph, provides a crude but very useful measure of the amount of drug present. In the case of serum samples, this can be approximated relative to serum cholesterol which is usually present at levels of 130-200 mg/roo ml and is eluted from an OV-17 column at about 250’ after most of the commonly encountered drugs. Mass spectrometric analysis is a relatively costly process at present. The majority of hospitals do not possess such sophisticated instrumentation or employ personnel trained to utilize this technique. However, this preliminary work suggests that it could be of great assistance to clinicians at least to have access to such a facility on a twenty-four hour basis. It does not seem unreasonable that one such facility might be located near each large population center, staffed by one technician equipped with mass spectrometer and data handling systems. REFERENCES A. L. LINTON, Scot. Med. J., II (1966) 295. I. SUNSHINE E(d.), Handbook of Analytical Toxicology,

Chemical Rubber Co., Cleveland, Ohio,

1969. B. DAVIDOW, N. L. PETRI AND B. QUAME, Tech. Bull. Registry Med. Tech., 38 (1968) 298. We are grateful to Drs. E. MAY, A. GUARINO, J. AXELROD, AND L. KA~YAK for gifts of samples of many of these drugs. SERF, Applied Data Research, Inc., Princeton, N. J. Inquiries should be directed to Miss V. A. AANDAHL, Division of Computer Research and Technology, National Institutes of Health, Bethesda, Md. 20014. Atlas of Mass Spectral Data, John Wiley and Sons, Inc., New York, N.Y., 1971. The Merck Index, 8th ed., Merck and Co., Rahway, N. J., 1968. Physzcian’s Desk Reference, Medical Economics, Inc., Oradelle, New Jersey 1969, p. 842. J. J. BURNS, B. L. BERGER, P. A. LIEF, A. VOLLACK, E. M. PAPPER AND B. B. BRODIE, J. Phavmacol. Exp. Therap, 114 (1955) 289. E. M. KAPP AND A. F. COBURN, J. Btol. Chew., 145 (1942) 549. A. J. MCBAY AND C. G. KATSAS, New Engl. J. Med., 257 (1957) 97. H. SHEPPARD, B. S. D’ASARO AND A. J. PLUMMER J. Amer. Pharm. Assoc., Sci. Ed., 45 (1956)

681. G. BOHN AND

R. RUCKER, Arch. fiir Toxzkol., 23 (1968) 221. J. R. ALTHAUS, K. BIEMANN, J. BILLER, P. F. DONAGHUE, D. A. EVANS, H.-J. FGRSTER, H. S. HERTZ, C. E. HIGNITE, R. C. MURPHY, G. PRETI AND V. REINHOLD, Experientia, 26 (1970) 697. Clin. Chim. Acta, 32 (1971) 221-228