Metabolic Profile of Amphetamine and Methamphetamine Following Administration of the Drug Famprofazone ',+

Transcription

1 Journal of Analytical Toxicology, Vol, 27, October 2003 Metabolic Profile of Amphetamine and Methamphetamine Following Administration of the Drug Famprofazone ',+ Brandy Greenhill 1, Sandra Valtier 2 and John T. Cody3, * IGraduate Program in Clinical Laboratory Sciences, Department of Clinical Laboratory Sciences, University of Texas Health Science Center, San Antonio, Texas ; 2Clinical Research Squadron, 59th Medical Wing, Lackland AFB, Texas ; and 3Academy of Health Sciences, MCCS-HMP PA Branch, Fort Sam Houston, Texas Abstrad ] There are a several drugs that lead to the production of methamphetamine and/or amphetamine in the body which are subsequently excreted in the urine. These drugs raise obvious concerns when interpreting positive amphetamine drug testing results. Famprofazone is an analgesic found in a multi-ingredient medication (Gewodin used for pain relief. Two Gewodin tablets (50 mg of famprofazone) were administered orally to healthy volunteers with no history of amphetamine, methampbetamine, or famprofazone use. Following administration, urine samples were collected ad lib for up to six days, and ph, specific gravity, and creatinine values were determined. In order to determine the quantitative excretion profile of amphetamine and methamphetamine, samples were extraded using liquid-liquid extraction, derivatized with heptafluorobutyric anhydride, and analyzed by gas chromatography-mass spectrometry (GC-MS). The ions monitored were 91, 118, 240 for amphetamine and 254, 210, 118 for methamphetamine. Amphetamine-de and methamphetamine-du were used as internal standards. Peak concentrations for amphetamine ranged from 148 to 2271 ng/ml. and for methamphetamine 615 to 7361 ng/ml. Concentrations of both compounds peaked between 3 and 7 h post-dose. Amphetamine and methamphetamine could be detected (limit of detection = 5 ng/ml) at 121 and 143 h post.dose, respectively. Using a cutoff of 500 ng/ml, all subjects had individual urine samples that tested positive. One subject had 14 samples above the cutoff with the last positive being detected over 48 h post-dose. The profile of methamphetamine and amphetamine enantiomers was also determined using liquid-liquid extraction, derivatization with N-trifluoroacetyl-/-prolyl chloride and analysis by GC--MS. 9 This work was supported by the USAF Surgeon General's Office (FWH H). The voluntary fully informed consent of the subjects used in this research was oblained as required by AF The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Defense or other Deportments of the U.S. Government. ' Portions of the data in this manuscript were presented at the annual meetings of the American Academy of Forensic Sciences and The Society of Forensic Toxicologists. This study was completed in partial fulfillment of the Master of Science degree and was awarded the Educational Research Award by The Society of Forensic Toxicologists, l Author to whom correspondence should be addressed: John T. Cody, Ph.D., AMEDD C&S, MCCS-HMP PA Branch, 3151 Scott Road, Ft. Sam Houston, TX Data showed the famprofazone metabolites amphetamine and methamphetamine to be both d- and/-enantiomers. The proportion of I.methamphetamine exceeded that of its d-enantiomer from the first sample collected. Initially, the proportion was approximately 70%/-methamphetamine and this proportion increased over time. Amphetamine results showed/- and d-amphetamine were virtually the same in the early samples with the proportion of /-amphetamine increasing as time progressed. Forensic interpretation of drug testing results is a challenging critical part of forensic drug testing area because of the potential repercussions the results found may have on an individual's life. The finding of each enantiomers by itself differentiates famprofazone use from the most commonly abused form of methamphetamine and all medicinal methamphetamine available in the U.S., which is either d-methamphetamine (prescription medication) or /-methamphetamine (Vicks inhaler). Coupling this information with the concentrations of amphetamine and methamphetamine helps to determine the potential for use of this drug. Introduction Analysis and interpretation of a positive urine drug test result, especially in the case of methamphetamine, can be a complicated task. It has been documented that at least 14 different medications are metabolized to methamphetamine and/or amphetamine and can therefore be responsible for a positive result for amphetamines (1). It is beneficial for forensic toxicologists and medical review officers (MROs) to understand the metabolic pattern of these drugs as well as the enantiomeric composition of the methamphetamine and amphetamine subsequently excreted in the urine. This information will facilitate the proper interpretation of drug tests performed on individuals using these medications. Famprofazone is an analgesic and anti-pyretic available in several foreign countries. It is one component in a multi-ingredient tablet (Gewodin, Geistlich, Baden-Baden, Germany) containing 25 mg of famprofazone, 250 mg paracetamol Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 479

2 /ournal of Analytical Toxicology, Vol. 27, October 2003 (acetaminophen), 75 mg isopropylphenazone, and 30 mg of caffeine (2). This drug has been reported to be metabolized to methamphetamine and amphetamine (Figure 1) and can result in an amphetamine- or methamphetamine-positive drug test (3-6). Additional research on famprofazone metabolism focused on detecting the parent drug and unique metabolites (7-9). Shin and colleagues documented famprofazone and p-hydroxydesmethylfamprofazone could be detected in urine (7) and later demonstrated the enantioselective nature of this metabolism (9). Detecting famprofazone, desmethylfamprofazone, or p-hydroxydesmethylfamprofazone in a urine sample would clearly demonstrate the use of famprofazone. Unfortunately, these compounds are not available commercially and, therefore, cannot be quantitated without custom synthesis of the pure compounds to allow preparation of analytical standards. In addition, according to the Health and Human Services Guidelines, testing for "other compounds" is not allowed unless there is reasonable suspicion of drug use and the drug in question is a Schedule I or II drug (10). For this reason, forensic toxicologists and medical review officers involved in regulated drug testing programs must rely on current testing methodologies available such as methamphetamine and amphetamine quantitation and enantiomeric composition to assist in identifying the possibility of famprofazone use in the case of a methamphetamine positive. Previous studies involving quantitation of methamphetamine and amphetamine metabolized from famprofazone in urine monitored the concentrations for a relatively short period of time (24 and 72 h) using pooled samples (5,6). Although these studies provide valuable information, only one previous study (3) collected and analyzed every urine void following administration of the drug until amphetamine and methamphetamine were no longer detectable. This study was significant because it represented the first report of the enantiomeric composition of amphetamine and methamphetamine derived from famprofazone. Although a single subject can clearly establish the enantiomeric composition of the compounds excreted, it does not demonstrate the interindividual variation in concentrations or enantiomer proportions. In addition, this study indicated the rate of metabolism for l-famprofazone was greater than for the d-enantiomer. In addition, it showed the proportion of methamphetamine in samples collected shortly after drug administra- Do~mothyl~mp~fazono Fomprofazone '~_/ ', ~. ~..~. ",~ I L ', tion was approximately 70% l-methamphetamine, not what would be predicted from a racemic drug. As an extension to that study, an additional four subjects were enrolled and administered drug and samples were collected under the same protocol conditions to further establish the results obtained. The current study was designed to determine the concentrations of methamphetamine and amphetamine in each sample, peak concentrations, percent conversion of each metabolite from the parent drug, and evaluate the ratio of methamphetamine to amphetamine in urine from the time of drug administration until amphetamine and methamphetamine are no longer detected. Enantiomer composition of amphetamine and methamphetamine was also evaluated over time. Materials and Methods Materials Amphetamine, methamphetamine, amphetamine-d6, and methamphetamine-dn were obtained from Cerilliant. d-amphetamine, d-methamphetamine,/-amphetamine, and l-methamphetamine were obtained from Sigma Chemical. The derivatizing reagents, heptafluorobutyric anhydride (HFBA) and N-trifluoroacetyl-l-prolyl chloride (I-TPC), were obtained from Sigma and Regis Chemical, respectively. The famprofazone administered to experimental subjects, in the form of Gewodin, was obtained through a pharmacy in Germany. Drug administration and sample collection Fifty milligrams of famprofazone (as two Gewodin tablets) was administered to healthy volunteers. Following administration, urine was collected ad lib for the next six days. The subjects recorded the total volume of urine voided, date and time of void. Urine samples were collected daily and refrigerated until time of analysis. No attempt was made to control urine ph, but the ph was measured for each sample along with specific gravity and creatinine. Sample preparation and analysis Sample ph was measured using Fisher Accumet AR50 ph meter. Specific gravity was measured using the AO Scientific Refractometer. Creatinine levels were determined at the Wilford Hall Medical Center clinical laboratory using standard clinical laboratory procedures. Gas chromatographic-mass spectrometric (GC-MS) analyses were performed using a Hewlett- Packard 6890 GC coupled with an HP 5973 MS using a 7683 autoinjector. Arn;)heUlmlne Figure 1. Famprofazone metabolism, H ydroxyrnemyl~opmpylphe nozono Melharnphldemlnl Quantitative analysis Two-milliliter aliquots of each urine sample containing 500 ng/ml each of amphetamine-d6 and methamphetamine-du as internal standards were extracted by adding 0.3 ml 1M NaOH and 5 ml of 1-chlorobutane. Tubes were shaken for 10 min at 120 cycles/min. If an emulsion was present after shaking, the samples were centrifuged for 5 rain at 1500 rpm to separate the layers. The top organic layer was transferred to a dry, clean glass screw-top tube and the drugs were back extracted by adding 2.0 ml of 0.15M sulfuric acid. The tubes were again shaken and 480

3 Table I. Sample ph, Creatinine, Specific Gravity, Amphetamine and Methamphetamine Concentrations, and I-Enantiomer Composition of Amphetamine and Methamphetamine* Specific Hours Amp MA % % Sample ph Gravity Creatinine Post-Dose (ng/mt) (ng/mt) /-Amp /-/VIA SubiectA Pre-Dose: : f : : , : : : : : : : : : : : : : , : : , : , : : : , : : ,52 1, : : : : : : , : ,6 87: , ,8 88: : : , , : : : : : : : , : : : : : : Subject B Pre-Dose: , : : "d-enanliorner propo~ion can be calculaled by subtracting the I-enantiomer percentage from 100. t indicates enantiomer analysis was not completed because of the low concentration or lack of drug in the sample. centrifuged as described. The top organic layer was aspirated to waste followed by adding 1 ml of 1M NaOH and 5 ml of 1-chiorobutane to the bottom aqueous layer. Samples were again shaken and centrifuged as described. The top organic layer was then transferred to a clean dry glass tube to which 200 IJL of 1.0% HCI in methanol was added. Samples were then placed in a water bath (50-60~ and evaporated to dryness under a stream of nitrogen. Derivatization was accomplished by reconstitution in 100 IJL of ethyl acetate and 25 IJL of HFBA and incubated at 60-70~ for 15 rain. The extract was then evaporated under a stream of nitrogen, reconstituted in ethyl acetate and injected into the GC-MS. Instrumental conditions were splitless injection; injector and interface temperature were set at 270~ An HP-1 (12 m x 0.2-mm i.d., 0.33-mm film thickness, Hewlett-Packard) or equivalent column was used with a temperature program 80~ for 1 rnin, programmed to 180~ at 20~ with a 2 min final time. Ions monitored were m/z 240, 118, 91 for amphetamine; rn/z 244, 123 for amphetamined6; m/z 254, 210, 118 for methamphetamine; and m/z 213 and 260 for methamphetaminedn. A single-point calibration using a standard with 500 or 25 ng/ml (depending on expected sample concentrations) of amphetamine and methamphetamine was used. Samples with high concentrations (> 100 ng/ml) were analyzed with a 500- ng/ml calibrator. Each batch contained controls at 0, 100, and 2500 ng/ml. Samples with low concentrations (< 100 ng/ml) were analyzed with a 25-ng/mL calibrator. Each batch contained controls at 0, 10, and 100 ng/ml. The assay is linear to 10,000 ng/ml for both amphetamine and methamphetamine with a limit of detection (LOD) of 5 ng/ml for both analytes (11). Enantiomer analysis ~o-milliliter urine samples were analyzed using amphetamine-ds and methamphetamine-d~ as internal standards. Extraction was accomplished by adding 0.3 rnl of 1M NaOH and 5 ml 1-chlorobutane. Tubes were shaken for 10 min at 120 cycles/rain. If an emulsion was present after shaking, the sampies were centrifuged for 5 rain at 1500 rpm to separate the layers. The top organic layer was transferred to a clean, dry glass tube followed by adding 50 rnl of I-TPC, and incubated at room temperature for 15 min. Three milliliters of 0.01M NaOH were then added and the samples shaken for 15 rain. The 481

4 Table I, (continued) Sample ph, Creatinine, Specific Gravity, Amphetamine and Methamphetamine Concentrations, and/-enantiomer Composition of Amphetamine and Methamphetamine* Specific Hours Amp MA % % Sample ph Gravity Creatinine Post-Dose (ng/mt) (ng/mt) /-Amp /-MA Subject C Pre-Dose: I : : : ,2 20: ,2 24: : : I :00 I : : :00 40 I : : : : : : ,4 74: : : : : ,9 92: : I : : , : : , : ,2 115: ,2 121: , : , : : , : : , : : , , : : , : ,012 89,8 29: ,3 35: , ,0 45: , : : , : , : : : : * d-enantiomer proportion can be calculated by subtracting the l-enantiomer percentage from 100. t indicates enantiomer analysis was not completed because of the low concentralion or lack of drug in the sample. organic layer was transferred, evaporated under nitrogen at 50-60~ followed by reconstitution in ethyl acetate and injected in to the GC-MS. Instrumental conditions were as follows: splitless injection; injector and interface temperature at 270~ oven temperature 120~ for 2 rain to 200~ at 4~ Ions monitored were rn/z 237, 241, 251, 255 for d- and/-amphetamine; d,l-amphetamine-ds; d- and l-methamphetamine; d,l-methamphetamine-ds, respectively (12). Each batch of samples was calibrated for retention time using a sample containing 50% of both enantiomers of amphetamine and methamphetamine and analyzed with control samples containing 0% l-enantiomer and 100% d-enantiomers of amphetamine and methamphetamine; and 0% d-enantiomer and 100% l-enantiomer of amphetamine and methamphetamine along with a negative control. Results and Discussion As expected, all subjects metabolized famprofazone to methamphetamine and amphetamine. The quantitative results for methamphetamine and amphetamine are shown in Table I along with the l-enantiomeric composition of methamphetamine and amphetamine. The first detectable methamphetamine (LOD = 5 ng/ml) was seen from 1 h 41 min (1:41) to 4 h 15 min (4:15) post dose. The number of methamphetamine positives (~ 500 ng/ml) ranged from I to 14 for the subjects and the time of first positive ranged from 2:05 to 7:05 h post dose. The last positive methamphetamine was seen up to 53:20 h post dose. Methamphetamine was last detected from 104:15 to 143:15 h post dose. The first detectable amphetamine (LOD = 5 ng/ml) ranged from 1:41 to 4:15 h post dose. Only one subject had samples that tested positive (~_ 500 ng/ml) for amphetamine. The first amphetamine positive was seen 5:20 h post dose with the last positive seen 28:15 h post dose. Amphetamine was last detected from 74:15 to 120:30 h post dose. All but one of the subjects had samples that tested positive using the current HHS cutoffs (13) for reporting positive methamphetamine (_~ 500 ng/ml methamphetamine plus 200 ng/ml amphetamine). Peak concentrations for both methamphetamine and amphetamine were seen in samples collected 3 to 7 h post dose where concentrations reached 615 to 7361 ng/ml 482

5 Table I. (continued) Sample ph, Creatinine, Specific Gravity, Amphetamine and Methamphetamine Concentrations, and l-enantiomer Composition of Amphetamine and Methamphetamine* Specific Hours Amp MA % % Sample ph Gravity Creatinine Post.Dose (ng/mt) (ng/mt) /-Amp /-MA : : : : : : , ,7 t09:t : : : : : : Subject D Pre-Dose: : : : : : : , : : : : , : , : : : : : : : : : : , : : : "d-enantiomer proportion can be calculated by subtracting the/-enantiomer percentage from 100. ~" indicates enantiomer analysis was not completed because of the low concentration or lack of drug in the sample. and 148 to 2271 ng/ml for methamphetamine and amphetamine respectively. The previous single-dose study (3) showed peak concentrations of 420 ng/ml for amphetamine and 1976 ng/ml for methamphetamine at 14:00 h post dose. Yoo et al. (5) reported peak urine concentrations occurred h post dose (pooled samples) with values ranging from 1.4 to 5.0 and 0.5 to 0.7 mg/rnl for methamphetamine and amphetamine, respectively. Previous studies pooled the urine samples collected at specific time intervals and reported the concentration of the pooled sample, which represents an average of the individual samples collected during the specified collection intervals (5,6). In a typical forensic urine drug-testing program, a single random sample is collected and analyzed. As a result, to compare this value to that of a pooled sample may be misleading since a pooled sample does not show the highest or the lowest concentration during the collection time frame. The use of pooled samples may also ex- 89.o7 plain the difference in the time to peak seen in the present study from that reported in pre vious studies. To address these issues, this study collected each individual urine sample l over a seven-day period. Samples were ana- lyzed and reported individually, more closely loo.o0 emulating the circumstances encountered in random drug testing. It is difficult to directly compare the current study to previous studies because one study only monitored the drug excretion for 24 h in three subjects (5), and another for only 72 h in two subjects (6) as well as the use of pooled samples. In the present study, the percent conversion from famprofazone to methamphetamine ranged from 4.4% to 15.8% and amphetamine represented 1.5% to 3.6% of the dose. Shin et al. (4) reported 6.6% to 20.0% conversion to methamphetamine and 0.69 to 0.78% to am phetamine (n = 3). Oh et al. (6) found 7% to 83.o3 8% of a 50 mg dose of famprofazone was ex creted as methamphetamine (n = 2) Studies following the administration of 1 oo.oo methamphetamine reported the concentration loo.oo loo.0o N^ of amphetamine in urine samples ranged from 5 to 20% of the concentration of methamphetamine (14,15). In the current study, the amount of amphetamine relative to methamphetamine started at approximately 20% for all subjects. Several subjects showed increases to as high as 55%; however, it must be noted that these high values represent low concentration samples associated with terminal excretion of the drugs. Evaluation of samples that met criteria for a positive sample, the proportions ranged up to approximately 30%. If, under normal circumstances, the concentration of amphetamine is up to 20% that of methamphetamine, the higher percentage found in this study suggests another pathway contributing to the amount of amphetamine found in the samples. It is reasonable to assume from data provided by previous studies (4,7) that famprofazone is metabolized to desmethylfamprofazone (Figure 1). N-Dealkylation of desmethlyfamprofazone could also produce amphetamine and would account for the higher percentage of amphetamine relative to methamphetamine found in these studies. Analysis showed the famprofazone metabolites amphetamine and methamphetamine to be both d- and l-enantiomers as previously reported (3,9,16). In earlier studies performed from the ingestion of racemic methamphetamine, the initial composition 483

6 of d- and l-methamphetamine averaged nearly 50:50, reflecting the composition of the ingested drugs (14). In this study, l-methamphetamine predominated from the first sample collected and its percentage increased over time in all subjects relative to d-methamphetamine. The proportion of l-methamphetamine was approximately 70% for all but one of the subjects whose initial sample (at 3 h post dose) was approximately 60% but then increased to over 70% in the next sample collected a few hours later (see Table I). Amphetamine results showed/-amphetamine and d-amphetamine remained fairly close in early samples at % and increasing to 65-75% in the later samples. The change in enantiomer proportion was quite consistent between subjects and did not vary with changes in ph or specific gravity/creatinine as did the concentrations. This is not surprising because the proportion of the enantiomers changes because of differing rates of metabolism of the d- versus l-enantiomer and is largely independent of dilution (reflected by specific gravity and creatinine) or reabsorption in the kidney (influenced by ph). The presence of l-methamphetamine following the use of famprofazone differentiates its use from most medicinal and even illicit methamphetamine use because prescription methamphetamine in the U.S. is the d-enantiomer only as is most of the illicitly produced drug. The Vicks inhaler contains only l-methamphetamine, therefore the presence of d-methamphetamine demonstrates the use of something other than the inhaler. Use of racemic methamphetamine results in the excretion of essentially equal amounts of both enantiomers shortly after administration. Following that, the proportion of l-methamphetamine exceeds that of the d-enantiomer, and this proportion increases with time. Eventually the proportion of d- and 1-enantiomers of methamphetamine will reach levels consistent with that of famprofazone use, but whereas the proportion is near 50:50, famprofazone can be excluded as the sole source of the drug found. As the l-enantiomer proportion approaches and exceeds 70%, these proportions alone will no longer allow easy interpretation. Drug concentration is also of value, because the levels seen with famprofazone use are also much lower than seen in abuse cases. Together these data can help assess the possible involvement of this drug in a sample positive for amphetamines. References I. J.T. Cody. Precursor medications as a source of methamphetamine and/or amphetamine positive drug testing results. J. Occup. Environ. Med. 44(5): (2002). 2. Gewodin Geistlich (Package Insert). Vol /9308/BB. Gewo Chemie GmBH, Baden-Baden, Germany, J.T. Cody. Enantiomeric composition of amphetamine and methamphetamine derived from the precursor compound famprofazone. Forensic 5ci. Int. 80: (1996). 4. H.S. Shin, B.B. Park, S.N. Choi, J.]. Oh, C.E Hong, and H. Ryu. Identification of new urinary metabolites of famprofazone in humans. ]. Anal. ToxicoL 22:55-60 (1998). 5. Y. Yoo, H. Chung, and H. Choi. Urinary methamphetamine concentration following famprofazone administration. J. Anal Toxicol. 18: (1994). 6. E.S. Oh, S.K. Hong, and G.I. Kang. Plasma and urinary concentrations of methamphetamine after oral administration of famprofazone to man. Xenobiotica 22(3): (1992). 7. H.S. Shin, J.S. Park, P.B. Park, and S.J. Yun. Detection and identification of famprofazone and its metabolite in human urine. ]. Chromatogr. B 661(2): (1994). 8. M. Neugebauer. Some new urinary metabolites of famprofazone and morazone in man. J. Pharm. Biomed. Anal 2(1): (1984). 9. H.S. Shin. Stereoselective metabolism of famprofazone in humans: N-dealkylation and beta- and p-hydroxylation. Chirality 9(1): (1997). 10. Mandatory guidelines for federal workplace drug testing programs. Fed. Regist. 59(110): (1994). 11. S. Valtier and J.T. Cody. Evaluation of internal standards for the analysis of amphetamine and methamphetamine. ]. Anal ToxicoL 19: (1995). 12. D. Hensley and J.T. Cod,/. Simultaneous determination of amphetamine, methamphetamine, methyienedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), and methylenedioxyethylamphetamine (MDEA) enantiomers by GC-MS. J. Anal. Toxicol. 23: (1999). 13. Cutoff Concentrations as specified in the Mandatory Guidelines for Federal Workplace Drug Testing Programs. November 1, 2001, Substance Abuse and Mental Health Services Administration, Rockville, MD. 14. J. Caldwell, L.G. Dring, and R.T. Williams. Metabolism of [14C] methamphetamine in man, the guinea pig and the rat. Biochem. J. 129:11-22 (1972). 15. A.H. Beckett and M. Rowland. Urinary excretion kinetics of methylamphetamine in man. J. Pharm. Pharmacol. 17(Suppl.): 109S- 114S (1965). 16. M. Neugebauer, A. Khedr, N. EI-Rabbat, M. EI-Kommos, and G. Saleh. Stereoselective metabolic study of famprofazone. Biomed. Chromatogr. 11 : (1997). 484