Colorimetric Identification and Quantitation of Methaqualone in Toxicological Specimens Joerg N. Pirl, Veronica M. Rotterman, and Frank F. Fiorese Bureau of Toxicology, Department of Public Health, Division of Laboratories, State of Illinois, 1800 West Fillmore Street, Chicago, Ill. 60612
METHAQUALONE HYDROCHLORIDE is a sedative and hypnotic and has been sold since 1956 under various trade names. The usual adult dosage is 200 to 400 mg one-half hour before retiring or 75 mg four times daily. Because of the high fat solubility of this drug, plasma levels are usually low and detection requires very sensitive methods. In 1966, Lawson and Brown reported maximum plasma levels greater than 3 mg in patients who had ingested 5 grams of the drug. Mildly poisoned patients had levels ranging from 0.2 to 2 mg on admission to the hospital ( I ) . Brown and Smart showed that therapeutic dosages gave values of 0.9 to 2.2 pg/ml 12 hours after ingestion. Daily ingestion of 500 mg of methaqualone leads to blood levels of about 0.5 mg (2). Until recently, the usual method of quantitation of methaqualone in toxicological specimens was extraction followed by ultraviolet spectrophotometry (3). Methaqualone shows ultraviolet maxima at 225, 265, 305, and 316 nm. Concentrations as low as 1 pg/ml can accurately be measured by this method in neat samples, but toxicological extracts are often contaminated and require successive dilutions resulting in false negative results. Several indirect methods for the identification and quantitation of methaqualone have been devised. These methods require hydrolysis of the drug to form diazotizable amines, but these methods are lacking in specificity (4-6). In 1969, Brown and Smart reduced methaqualone to the tetrahydroquinazolinone which shows intense untraviolet fluorescence (2). This laboratory has devised a technique for the detection of levels as low as 0.3 pg/ml of methaqualone in toxicological specimens. This method is such that interference from normal body constituents or other drugs is unlikely.
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EXPERIMENTAL
Equipment. All spectrophotometric measurements were performed on a Beckman DK2-A ratio recording spectrophotometer. Reagents. The reagents used were: 0.1N sodium hydroxide; 0.1N hydrochloric acid; Reagent Grade chloroform; absolute methanol; freshly prepared sodium methoxide in methanol (100 mg %); freshly prepared p-N,N-dimethylaminobenzaldehyde (p-DMAB) in methanol (200 mg %); and formic acid (88 %)-methanol mixture (3 : 5), freshly prepared (0.04M HCI in methanol can be substituted). (1) A. A. H. Lawson and S. S. Brown, Scot. Med. J., 12, 63-8, (1967). (2) S. S. Brown and G. A. Smart, J. Pharm. Pkarmacol., 21, 466-8 (1969). ( 3 ) M. Akagi, Y . Oketani, and M. Takada, Chem. Pharm. Bull., Tokyo, 11, 62-7 (1963). (4) E. Maggiorelli and G. Gangerni, Bull. Chim.-Farm., 103, 74852 (1964). ( 5 ) M. Nakano, Yakuzaigaku, 24, 49-52 (1964); Chem. Abstr., 61, 4155 (1964). (6) K. S. Okurnura, T. Oine, Y . Yamada, G. Hayashi, and M. Nakarna, J . Med. Chem., 11, 348-52 (1968).
Procedure. The extraction of methaqualone from blood and tissues is performed in the following manner. The specimen is made alkaline with 0.1N NaOH and extracted with ten times its volume of chloroform. After separation, the chloroform layer is filtered and washed with one-tenth its volume of 0.1N HCI, then filtered, dried, and evaporated. This residue is taken up with methanol, transferred to a 125 mm by 15 mm test tube, and evaporated to near dryness on the steam bath. When the sample is cool, 1 ml of sodium methoxide in methanol is added, followed by 1 ml of pDMAB in methanol. A blank is similarly prepared. The tubes are placed on the steam bath in a water-filled beaker until the liquid portion of each sample has evaporated. To assure completeness of reaction, 0.5 ml of methanol is added to each sample and again allowed to evaporate. After cooling, 5 ml of formic acid-methanol mixture is added. An orange color indicates the presence of methaqualone. It is advisable to run a standard solution concurrently with the sample. One milliliter of a 10 pg/ml methanolic methaqualone solution is placed into a 125 mm by 15 mm test tube, evaporated to dryness and treated as outlined. RESULTS
The condensation product of methaqualone with p-DMAB acts as an indicator, being yellow in alkaline medium and colorless in strong acid. In weakly acidic medium, this compound is orange and absorbs strongly at 503 nm obeying BeerLambert law. The methaqualone concentration is calculated according to pg % in sample
=
Absorbance, sample Absorbance, standard
5 -x gram sample
Concentration standard The unique behavior of the condensation product makes the analysis for methaqualone in the presence of other colored matter possible. The addition of a drop of strong acid to the spectrophotometric sample will convert all methaqualone-pDMAB reaction product into its colorless version. The difference in A at 503 nm after strong acidification is due to methaqualone. Comparison of Condensation Product Using Drugs Other than Methaqualone. The following drugs or drug mixtures gave either a colorless or faintly yellow solution after addition of formic acid-methanol mixture to their respective reaction products with p-DMAB in alkaline MeOH : Acetaminophen, amphetamine, amytal, antabuse, APC, atromid-S, atropine, mixture of diallyl barbituric acid, butobarbital, amytal, seconal and phenobarbital, bemegrid, benzoic acid, bromural, brucine, caffeine, carbinoxamine, carbromal, carisoprodol, cantharidine, chlorpheniramine, cholesterol, clemisole, cocaine, compazine, dartal, darvon, demerol, DDT, digitalis, dilantin, diuril, doriden, elavil, ephedrine, ethotoin, ethsuximide, fluphenazine, hydroxyamphetamine, librium, listica, LSD, rnebaral, meclizine, mellaril, mephenesine, meprobamate, mescaline, methadone, metaraminol, methapyriline, methamphetamine, methocarbamol, methsuximide, morphine,
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nembutal, nicotine, noludar, nardil, norpramine, pacatal, papaverine, paveril, permitil, phenacetin, phenobarbital, phenyle'phrine, phensuximide, primidone, promazine, pronestyl, proketazine, pyrilamine, mixture of pheniramine, phenylpropanolamine and pyrilamine, quide, quinine, pyronil, resperine, ritalin, salicylic acid, salicyluric acid, salicylamide, sinequan, strychnine, styramate, surital, talwin, tapazole, taractan, tentone, temaril, theobromine, theophylline, thorazine, Valium, warfarin.
Of these, only p-aminobenzoic acid showed an orange color after addition of the acid mixture, but this color remained after the addition of strong acid. ACKNOWLEDGMENT
We are deeply indebted to the cooperation of the staff of the Toxicological Laboratory of the State of Illinois. RECEIVED for review November 22, 1971. Accepted April 19,1972.
Determination of Hexahydro91,3,5-Trinitro-s-Triazine (RDX) in Octahydro-1,3,5,7-Tetranitro-~-Tetrazocine(HMX) by Infrared Spectrophotometry J. W. Grindlay Ministry of Defence, Explosives Research and Development Establishment, Waltham Abbey, Essex, U.K.
A NUMBER OF DIFFERENT methods, employing various techniques, have been developed for the analysis of RDX in HMX, and Scullion ( I ) has reviewed some of this work. It has also been summarized by Schwartz and Mark ( 2 ) and, later, a gaschromatographic method (3) was described for amounts up to 5 %. For the routine examination of HMX for RDX contaminant (in the range 0.2-25 %), a speedy and straightforward method was desirable. The method described here, based on infrared measurement, was suitable in these respects and provided the desired accuracy. Some previous investigators of infrared methods have used Nujol mulls but this technique is susceptible to errors due to, for example, variations in mulling, and is inferior to solution measurement in cells of standard thickness. A method has been described ( 2 ) where dimethyl sulfoxide is used as sample solvent for amounts of RDX up to 8 %. Since this technique necessitated the use of a more elaborate spectrophotometer than was available, and assay of samples containing up to 25 RDX was required, an alternative method was sought. It was also considered that separation of the RDX before analysis would be advantageous and a satisfactory technique was evolved using 1,2-dichloroethane to extract the RDX and measuring the intensity of its absorption peak at 1590 cm-l. EXPERIMENTAL
A gravimetric method has been widely used in which the loss of RDX on treatment of the sample with 1,2-dichloroethane is estimated, the insoluble HMX being weighed after filtration. This technique entails mechanical shaking for 2 hours with HMX-saturated solvent before filtering, washing, and drying, and may give satisfactory results with standard mixtures but could be inaccurate when applied to some batches of manufactured HMX; it is also time-consuming. RDX, in dichloroethane solution, when measured by the differential method using a solvent-containing reference cell, shows an absorption peak at 1590 cm-1, which is suitable for (1) H. J. Scullion, Chemical Inspectorate Memorandum No. 169, (1965).
(2) M. Schwartz and E. A. Mark, ANAL.CHEM., 38, 610 (1966). (3) M. L. Rowe, J. Gas Chromatogr., 5 , 531 (1967). 1676
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Figure 1. Typical absorption curves for standard solution of RDX a. 0.05 % RDX (w/v) 6. 0.10% RDX (w/v) c. 0.16 % RDX (w/v) d. 0.20% RDX (w/v)
purposes of quantitative measurement (see Figure 1). For the preparation of standard RDX solutions, HMX-saturated dichloroethane was used as solvent and, initially, appropriate quantities of RDX were dissolved, as in the gravimetric method, by shaking with solvent for 2 hours. It was subsequently found, however, that the RDX could be dissolved in a few minutes by gently refluxing on a water-bath, thus eliminating the long period of shaking. After cooling the solution to room temperature and diluting it to standard volume, the peak height at 1590 cm-l was measured, for each solution, against a reference cell containing HMXsaturated solvent, and a calibration curve was drawn up by plotting absorption us. concentration. This extraction technique was applied to standard RDX/HMX mixtures and to laboratory synthesized samples of HMX. Apparatus. The instrument used was a Perkin-Elmer Model 237 Infrared Grating Spectrophotometer, the normal (minimum) slit-width and fast scan speed being used. Sample and reference solutions were contained in a matched pair of rock-salt cells of 0.5-mm path length, the spectra being
ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972
