Analyst, May, 1983, Vol. 108, p p . 603-607 603

Development and Evaluation of a Radioimmunoassay for the Detection of Amphetamine and Related Compounds in Biological Fluids

Peter Alan Mason, Tara Singh Bal, Brian Law and Anthony Christopher Moffat Central Research Establishment, Home Ofice Forensic Science Service, Aldermaston, Reading, Berkshire, RG7 4PN

A radioimmunoassay has been developed for the detection of amphetamine and its analogues in blood and urine without any pre-treatment of the samples. It is based on a commercially available antiserum and a [1*5I]iodinatecl derivative of amphetamine.

The assay can detect low levels of amphetamine (less than 10 ng ml-l) in very small samples (50 pl) of blood and urine. It is cheap (3 pence per test) , rapid, simple to perform and is specific for compounds closely related to amphetamine.

A high, positive correlation was obtained ( r = 0.93) when results of the analyses of urine samples from volunteers who had ingested amphetamine were compared with those produced by gas chromatography - mass spectrometry.

The assay has proved very useful for the detection of amphetamine and closely related compounds in biological fluids.

Keywords : A nzphetainine detection ; blood ; urine ; radioinzrnunoassay

Inimunoassays are now accepted as a very efficient means of screening samples of biological materials for the presence of drugs. This is particularly so in forensic science where only a small percentage of the samples tested are likely to be positive for any given drug. The reasons for the popularity of these types of assays are that they are relatively cheap, rapid and simple to perform. Immunoassays generally lack complete specificity, and although this is a draw- back in some applications, i t can be a distinct advantage in screening for the presence of groups of drugs in biological samples.

Amphetamine has a low relative molecular mass (135), which imposes considerable constraints on the development of immunoassays for its detection, Only two such assays are available commercially : an enzyme immunoassay (Emit Amphetamine DAU) marketed by Syva (UK) Ltd. and a radioimmunoassay (RIA) (Amphetamine Abuscreen) marketrd by Roche Diagnostics Ltd. Although both are excellent for the analysis of urine samples,l the Emit assay is not suitable for use with blood samples and the RIA is not sufficiently sensitive to detect the low levels of amphetamine likely to be present during therapy or in some instances of drug abuse. Apart from the above kits, the authors are not aware of a commercial source of anti-amphetamine antiserum.

We have therefore developed and evaluated an RIA suitable for analysis of both blood and urine samples submitted for forensic analysis.

Experimental

Materials and Equipment

Dorset. Unless otherwise stated, all chemicals were of AnalaR grade from BDH Chemicals, Poole,

Radio immuiz oassa? Phosphate buffer (0 .1 AT, pH 7.4) containing 0.2% m/V of bovine gamma-globulin (Cohn

Fraction 11, Sigma Chemical Co., Poole, Dorset) and 0.01% m/V of sodium azide was used throughout the assay. Antiserum, obtained from an Emit Amphetamine DAU kit [Syva (UK) Ltd., Maidenhead, Berkshire] was diluted with assay buffer (1 + 99) immediately before use.

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604 MASON et a,?. : RADIOIMMUNOSSAY OF AMPHETAMINE Analyst, VoZ. 108 The radiolabelled amphetamine derivative N-(3-[1251]iodo-4-hydroxyphenyl)isobutyl-

amphetamine (1251A), specific activity 3.32 TBq mmol-l (8.15 hlBq pg-l), was prepared by iodination of N-(4-hydroxyphenyl)isobutylamphetamine by the chloramine-T reaction2 as previously described3 and stored in ethanol a t 4 "C. I t was diluted with assay buffer (typically 1 + 399) as required, to give approximately 546 Bq (0.067 ng) per 100 pl.

Polypropylene microcentrifuge tubes were obtained from Hughes and Hughes Ltd., Romford, Essex .

Gamma counting was carried out using an NE1600 gamma-counter (Nuclear Enter- prises Ltd., Beenham, Berkshire), which had an efficiency of 61% for [1291]iodine. The gamma- counter was interfaced to a Commodore, Model 3032, PET computer.

Standard solutions of amphetamine were prepared in synthetic urine4 at concentrations of 0, 25, 50, 125, 250, 500, 1000, 5000 and 10000 ng ml-l.

Polyethylene glycol (PEG, Mr 6000) was obtained from Sigma Chemical Co. and was used to prepare a 27.5% m/V aqueous solution for use as a separation reagent.

Gas chromatography - mass spectrometry - mailti$Ze ion detection Gas chromatography (GC) was carried out using a Perkin-Elmer Sigma 3B gas chromato-

graph. A glass column (2.0 m x 2.0 mm i.d.) was packed with 3% OV-17 on Chromosorb W HP (100-120 mesh). Helium was used as carrier gas a t a flow-rate of 25 ml min-l ; the oven temperature was 135 "C (isothermal) and the injection temperature was 260 "C. The gas chromatograph was interfaced to a VG Analytical 70/70 H mass spectrometer via a single-stage jet separator. The mass-spectrometric (MS) conditions were as follows: electron energy 70 eV with filament current 200 mA, source temperature 200 "C and interface temperature 200 "C. Multiple ion detection (MID) measurements were made by magnetic switching under electron- impact conditions to monitor the base peak and second most intense peak for the trifluoroacetyl derivatives of amphetamine (m/z 140 and 118) and methylamphetamine (m/z 154 and 118). A residence time of 200 ms was used for each ion.

Methods Radioimm.unoassay

Solutions of sample or standard (50 p1) were pipetted into duplicate sets of polypropylene microcentrifuges tubes. The tubes were then capped, shaken and incubated at room temperature for 30 min. Equilibrium was attained after 15 min and was stable for up to 24 h after that time. PEG solution (250 p1) was then added and the tubes were re-capped and vortexed until the contents appeared homogeneous before being centrifuged (3.5 min at 9 000 g). The supernatant was removed by aspiration and the tubes containing precipitates were counted in a gamma-counter. Urine and blood samples were assayed directly.

In order to determine the background levels of cross-reactivity, 100 samples of urine and 43 samples of blood were obtained from normal subjects who were not receiving amphetamine type medication, and assayed as described above. The condition of the blood samples ranged from fresh unhaemolysed to haemolysed/putrefied.

A study was also carried out to determine whether the assay was affected by the presence of preservative agents. To known blank urine samples (2.5 ml) were added phenylmercury(I1) nitrate and sodium fluoride (50 + 100mg), sodium sulphate and sodium fluoride (300 + 300 mg) and sodium azide (50 mg). Blood samples (1 ml) were dispensed into vials containing sodium fluoride and potassium oxalate (37.5 + 18.7 mg). The concentrations of preservatives in the urine samples were approximately five times greater than those recommended for forensic science use ; the concentrations in blood samples were approximately 2.5 times greater than those recommended.

Cross-reactivities of a number of compounds structurally related to amphetamine (Table I) were determined by comparison of their calibration graphs with that of amphetamine (Fig. 1) .

To these were added 1251A and antiserum (100 pl of each).

GC - M S - M I D Samples and standard solutions were extracted and processed according to a previously

Some modifications were found to be necessary : standard solutions were described m e t h ~ d . ~

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May, 1983 AND RELATED COMPOUNDS IN BIOLOGICAL FLUIDS 605

prepared in synthetic urine and methylamphetamine was used as the internal standard at a concentration an order of magnitude greater than that suggested (i.e., 7 320 ng ml-l).

Under the GC conditions described above, the trifluoroacetyl (TFA) derivatives of ampheta- mine and methylamphetamine had retention times of 2 min 52 s (A) and 5 min 6 s (B), respec- tively. A calibration graph was prepared by plotting the ratio of the signals of m/z 118 at retention times A and B against the concentrations of amphetamine in the standard solutions (0-5 pg ml-l). Each of the extracts was analysed in duplicate and the mean value was calcu- lated. This gave a correlation coefficient of 0.999 and intercept of 0.013 (n = 7). A similar calibration graph was prepared by plotting the ratio of the signals at m/x 140 and 154 (for amphetamine and methylamphetamine derivatives) against standard concentrations of amphetamine. The amphetamine concentrations in the samples were determined by measuring either of these ratios and comparing them with the corresponding calibration graph.

This gave a correlation of 0.9993 and intercept of 0.026 (n = 7).

OraL ingestion of amphetamine Four volunteers each took an oral dose of dexamphetamine sulphate (two 5-mg tablets).

Venous blood was taken into Monovette syringes (lithium heparin) before and at 2 , 4 and 6 h after ingestion of the drug. Urine samples, obtained before and at 2, 4, 6, 12, 24 and 36 h after ingestion of the drug, were stored at -18 "C.

The dosage level was approved by the Medical Ethics Committee, Chemical Defence Establishment, Porton Down.

The blood samples were stored at 4 "C before analysis.

This experiment was carried out on volunteers from laboratory staff.

Results and Discussion An example of a calibration graph obtained with the amphetamine RIA is shown in Fig. 1.

The assay has a very wide dynamic range that extends to 10000 ng ml-l whilst still maintain- ing sensitivity to less than 25 ng ml-' of amphetamine. This is a very useful feature of an assay for amphetamine because blood and urine samples from a subject who has consumed amphetamine may show enormous differences in concentration, urine levels often being up to 1000 times higher than blood levels. Thus, the wide range of this assay allows analysis of both blood and urine samples with the same calibration graph.

\ ' X

X \

X

'X

\ x\

x\

z 0 "0 102 103 1 04

Concentration of amphetaminehg ml-'

Fig. 1. Calibration graph for amphet- amine obtained using the radioimmuno- assay.

The cross-reactivities of several compounds closely related to amphetamine are listed in Table I. I t is apparent that the assay is specific for amphetamine, methylamphetamine and a few closely related compounds. In particular it is not very sensitive to /3-phenylethylamine. This is particularly important in an assay designed for forensic use, as /3-phenylethylamine is one of several amines produced during putrefaction. However, even the low level of cross- reaction seen in this assay may result in interference by this compound in severely putrefied

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606 MASON et al. : RADIOIMMUNOSSAY OF AMPHETAMINE Analyst, VoZ. 108 TABLE I

CROSS-REACTIVITIES OF SEVERAL COMPOUNDS CLOSELY RELATED TO AMPHETAMINE

Relative reactivity Relative reactivity Compound in RIA* Compound in RIA*

Amphetamine . . . . . . Methylamphetamine . . . . Phentermine . . . . . . Benzphetamine . . . . Tranylcypromine . . . . Ephedrine . . .. . . /3-Phenylethylamine . . . . Fenfluramine . . . . . . Phenylpropanolamine . . . . Mephentermine . . . .

1 1.4 3.7

11 16 27 29 34 57 79

Chlorphentermine . . . . Phenmetrazine . . .. . . Diethylpropion . . . . . . 4-Hydroxyamphetaniine . . Methylphenidate . . . . Nialamide . . . . .. Phendimetrazine . . . . Phenelzine . . . . . . Phenylephrine . . . . . .

79 97

> 100 > 100 > 100 > 100 > 100 > 100 > 100

* Determined a t 60% depression of binding (equivalent to 660 ng ml-l of amphetamine).

blood samples. It is unlikely that an immunoassay for amphetamine could be developed that did not detect P-phenylethylamine to some extent because they differ only in one methyl group.

The distribution of background levels of cross-reactivity in 100 samples of urine from normal subjects was 6.4 & 6.3 (S.D.) ng ml-l. A positive/negative cut-off was set a t 30 ng ml-l to ensure a 99% probability of obtaining a true positive result. Similar analysis of 43 blank blood samples showed that the background level of cross reactivity was 1.4 & 1.2 (S.D.) ng ml-l. A positive/negative cut-off was set a t 6 ng ml-l to ensure a 99% probability of obtaining a true positive result.

Of the preservatives tested, only the combination of phenylmercury(I1) nitrate and sodium fluoride in urine gave an elevation of background levels (equivalent to 100 ng ml-l of amphet- amine). It is therefore recommended that neither of these preservatives is used in urine samples. Background levels in the blood samples were not affected by the presence of the preservatives tested.

Coefficients of variation for urine samples containing 300 and 2 500 ng ml-l of amphetamine were 12.2 and 10.Oyo intra-assay (n = 20), respectively, and 15.1 and 12.2% inter-assay (n = 12), respectively.

The results of RIA analysis of blood samples from volunteers who had ingested a single dose of dexamphetamine sulphate are shown in Table 11. Amphetamine blood levels reached a plateau between 2 and 4 h after ingestion of the drug, at a mean concentration of 12.5 ng ml-l. Results of analysis of urine samples from the same experiment are shown in Table 111. In marked contrast to the blood concentrations, amphetamine concentrations in the urine samples do not follow a uniform pattern. This has been shown to be due to changes in urinary pH causing fluctuations in the rate of excretion of am~hetamine.~, '

TABLE I1 BLOOD AMPHETAMINE CONCENTRATIONS, MEASURED BY RIA, OF FOUR

VOLUNTEERS FOLLOWING AN ORAL DOSE OF 10 mg OF DEXAMPHETAMINE SULPHATE

Time after oral dose/h

Mean amphetamine blood concentration f standard deviationlng ml-l

0 2.3 f 1.6 2 10.4 f 2.1 4 12.5 f 1.0 6 12.3 f 0.6

Twelve of the urine samples were also analysed by GC - M S - MID for comparison with the results obtained by RIA. The agreement between the two methods was good (correlation coefficient = 0.93, intercept 83.4, slope 2.36, n = 12). However, from this study it is apparent that RIA over estimates the value obtained by the more specific technique by a factor of approximately 2.4. This is not surprising as i t is likely that the RIA detects metabolites of amphetamine that do not interfere with GC - MS - MID analysis.

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May, 1983 AND RELATED COMPOUNDS IN BIOLOGICAL FLUIDS 607 TABLE I11

URINARY AMPHETAMINE CONCENTRATIONS, MEASURED BY RIA, OF FOUR VOLUNTEERS FOLLOWING AN ORAL DOSE OF 10 mg

OF DEXAMPHETAMINE SULPHATE

Time after oral dose/h

2 4 6

12 24 36

Amphetamine concentration/pg ml-I

kubject 1 Subject 2 Subject 3 Subject 4 0.9 0.2 0.3 0.1 2.2 1.5 0.6 1.1 1.1 3.1 1.0 1.0 0.8 0.9 1.1 0.2 3.1 0.5 2.3 2.1 0.2 0.4 0.9 0.4

In conclusion, the amphetamine RIA described is suitable for screening biological fluids for It is rapid, simple to use and is

In terms of reagent expense, each test costs approximately 3 pence. the presence of amphetamine and closely related compounds. very cheap.

We thank Dr. R. Gleadle (Chemical Defence Establishment, Porton Down) for his help in the collection of blood samples.

References 1. 2. 3. 4.

5.

6. 7.

Bost, R. O., Sutheimer, C. A,, and Sunshine, I., Clin. Chem., 1976, 22, 789. Hunter, W . M., and Greenwood, F. C., Nature (London), 1962, 194, 495. Mason, P. A., Law, B., and Moffat, A. C., J . Immunoassay, accepted for publication. Rodgers, R., Crowl, C. P., Eimstad, W. M., Hu, M. W., Kam, J. K., Ronald, R. C., Rowley, G. L., and

Ullman, E. F., Clin. Chem., 1978, 24, 95. Foltz, R. L., Fertimen, A. F., Jr., and Foltz, R. B., “GC/MS Assays for Abused Drugs in Body

Fluids,” National Institute on Drug Abuse, Research Monograph 32, Washington DC., 1980, p . 150. Beckett, A. H., and Rowland, M., J . Pharm. Pharmacol., 1965, 17, 59. Beckett, A. H. , Salmon, J . A., and Mitchard, M., J . Pharm. Pharmacol., 1969, 21, 251.

Received November 17th, 1982 Accepted December 13th, 1982

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