Metabolism

Important consideration for the interpretation of amphetamine and methamphetamine results includes metabolism and excretion of the drugs and their metabolizes. There is no indication in any published research in this area that any of these drugs or their metabolites is racemized in the body. As a result, the enantiomeric form of the drug that is taken into the body is the same as what is excreted. For this reason, knowledge of the configuration of the drug is invaluable in the interpretation of results. Thus, a drug such as deprenyl, which is the I-enantiomer, gives rise to methamphetamine and amphetamine, which are also in the l-configuration.

However, enantiomeric configuration does influence the metabolism of amphetamine and methamphetamine, as well as the precursor drugs. When racemic methamphetamine or amphetamine is taken into the body, the d-enantiomer is metabolized more rapidly than the l-enantiomer. As a result, the composition of methamphetamine and its metabolite amphetamine in urine are not equally distributed. As time elapses following the administration of the drug, the enantiomeric distribution continues to change the implications of these differences are discussed in the individual sections for the drugs to which they apply and the reader is referred to several other references for further information on this subject.

Amphetamine and Methamphetamine

The metabolism of amphetamine (Refer to Figure 9-1) and methamphetamine (Refer to Figure 9-2) has been at length described in a number of publications. The understanding of amphetamine and methamphetamine results is multifaceted due in large part to the influence of urinary pH on the excretion rate of these drugs; the pK of amphetamine and methamphetamine are reported to be 9.9. The movement across the membrane is strictly limited for the charged form of the drug but not for the neutral form. The impact is significant re-absorption of the drugs under alkaline conditions but rapid excretion of the drugs under acid conditions.

Several controlled studies of amphetamine and methamphetamine have been completed that show their metabolism and excretion. A study examining the excretion of d-amphetamine administered a lamp dose to subjects. Peak concentration reached nearly 6,400ng/mL. Peak concentrations were reached from 2 to 21 h post-dose. Samples were positive (≥500ng/mL) for approximately 44 h. Evaluation of mixed enantiomer amphetamine involved a 20-mg dose of After all administered to 5 subjects. Peak concentration was slightly less than 6,000ng/mL and the last positive sample was seen approximately 48 h post-dose 61001. On administration of multiple doses of after all, subjects showed a peak concentration of slightly over 19,000ng/mL. Samples remained positive for as long as 60 h after the last dose 61014.

Studies involving methamphetamine include those using a variety of different conditions. One study involved the administration of 30 mg of methamphetamine by smoking. Peak concentration of urine was seen at 9-h post-dose. Subjective effects were reported in less than 20 min. No significant differences were seen between smoking and IV administration of the drug. A study of oral administration (30 mg/70 kg) showed time to peak concentration of 4 to 6 h. A primary purpose of this study was to evaluate whether or not there was a significant difference between morning and evening administration. There appeared to be no difference between the administration times. An interesting observation of this study was the vast number of samples greater than 500ng/mL that contained less than 200ng/mL of amphetamine.

Another study examined the potential difference in metabolism of methamphetamine precipitated by previous use. The study involved administration of methamphetamine over a period of 16 days and examining the parameters seen with the metabolism on day 1 and day 16. The data showed no significant difference. In a study designed to assess the concentration of methamphetamine that can be seen in urine drug testing samples following use of a Vicks Inhaler, 3 subjects inhaled every 20 min for 6 h. Peak concentrations were 6,000 and 455 ng/mL for methamphetamine and amphetamine, respectively. It is noteworthy that these data were from pooled samples and do not represent concentrations from a single sample.

Legitimate use of amphetamine and methamphetamine is accompanied by a valid medical prescription, making interpretation of legitimate use relatively straightforward. However, because of the nature of these drugs, having a legitimate prescription does not eliminate the potential for abuse. In fact, inappropriate medical use of the drugs can lead to dependence and ultimately to abuse. Even with a valid medical prescription, it is important to evaluate the enantiomeric composition of the drugs Figures 9-1 and 9-2.

Prescription-based methamphetamine is available in the United States only as the d-enantiomer. Therefore, the occurrence of both enantiomers could not come from use of prescription methamphetamine. Amphetamine, however, is prescribed as either the d-enantiomer or as a mixture of d-, 1-enantiomers. In this case, understanding of the enantiomeric composition is based on the composition of the prescribed drug For example, if the prescribed drug was methamphetamine, the presence of both enantiomers is most likely due to the illegal use of racemic methamphetamine. If d-amphetamine were prescribed, then only the d-enantiomer should be seen. Likewise, if the racemic form was used, there should be obvious evidence of the appropriate metabolic profile. The occurrence of methamphetamine in a sample from an individual for which amphetamine was prescribed is a clear indication of methamphetamine use because amphetamine is not metabolically converted to methamphetamine.

Although several nasal inhalers sold in the United States contain methamphetamine, elsewhere in the world they do not. Therefore, when evaluating enantiomeric results, it is important to check the inhaler to ensure it actually contained the drug l-methamphetamine. For these reasons, interpretation of illegal use requires evaluation of the enantiomers even if a prescription for the drug is available.

Precursor Compounds Genera/ Considerations

For those precursor compounds that are also excreted intact, the detection of the parent compounds when related to the amount of the amphetamine or methamphetamine present can be a powers tool to help properly interpret the analytical results. Other measures to consider are whether the drug in question fits its prescribed means and whether it correlates with its relevant pharmacological activities. The combination of these data can allow for appropriate evaluation of the origin of amphetamine and/or methamphetamine.

Detection of parent drug, in most cases, affirms the use of its respective precursor compound. When parent drug cannot be detected or very little is excreted, such as the case with deprenyl, fenproporex, benzphetamine, amphetaminil and famprofazone, there may be one or more metabolites unique to the compound that can be used to demonstrate use of the precursor.

Investigation of the pharmacological activity of several of these drugs is an active and ongoing process and their uses may change. As a result, it is important to maintain awareness of all reasonable clinical uses for these drugs. For example, if the only indication for use of deprenyl was for treatment of Parkinson disease, then simply knowing that the individual did not have Parkinson disease would resolve the issue. Studies have shown deprenyl to be effective not only in the treatment of Parkinson disease, but also in Alzheimer disease, depression, prevention of stress ulcers, motion sickness, increased longevity in rats as well as  humans, and sexual activity in rats, and humans. The hyper-sexuality induced in humans extended from virtually nonexistent to the point of being a problematical side effect in some patients. The impact of deprenyl treatment on dementia has also been studied and shown by several investigators to have a significant positive effect.

As deprenyl and other precursor drugs come into wider use, the interpretation of amphetamine and methamphetamine positive results will require an even more thorough consideration of possible medicinal uses of these precursor drugs.

Fenproporex is an example of a compound that is essentially entirely metabolized. Studies of the excretion of the fenproporex in humans have shown very little of the parent drug excreted and even then only for a few hours, while the metabolite amphetamine could be detected for days. Benzphetamine is another example of a precursor drug that cannot be detected in urine following a single dose id). Inability to detect the parent drug has also been reported for amphetaminil, famprofazone, and deprenyl, although some have been detected with more sensitive procedures and several of these do produce unique metabolizes. Even for those that can he detected, often amphetamine or methamphetamine can be detected after they are no longer detectable in the sample. In cases where the presence of the parent precursor compounds or metabolites cannot be demonstrated. There are other analytical data that can be used to help evaluate the possibility of their use. For example, prescription deprenyl is the l-enantiomer. Therefore, the methamphetamine and amphetamine metabolically produced from deprenyl are the l-enantiomers. A finding of d-methamphetamine or the mixture of d and l-methamphetamine would not be consistent with the administration of deprenyl.

From a forensic point of view, it is important to consider the fact that these precursor drugs are characteristically controlled, and if legally available can only be dispensed with a prescription. Lack of a suitable medical prescription may have a strong influence on the understanding of the presence of either amphetamine or methamphetamine. In addition, some of the drugs are for very specific medical conditions that are clearly provable either by assessment of the patient or by appropriate review of the medical records.

Many of these precursor compounds are relatively safe, but some are prone to be abused. Concern for the abuse potential of these drugs has caused some to be placed on the list of scheduled drugs or removed from the market. There have also been reports of several of these drugs being associated with significant complications and, in several extreme cases, fatalities. For example, benzphetamine was determined to be the cause of death in a suicide case invoking a young male  who showed high concentrations of the drug in blood, Anne, and a variety of tissues. Clobenzorex has been shown to be responsible for cardiac arrest in at least one case, a woman who had a long history of taking abusing the drug. Prenylamine has also been associated with dangerous ventricular arrhythmias. These hazards are potentially useful information in the clinical emergency or postmortem environment.

Examination of the chemical structure of these compounds reveals the portion of the molecule that gives rise to either amphetamines amphetaminil, clobenzorex, ethylamphetamine, fenethylline, fenproporex, lisdexamfetamine, mefenorex, mesocarb, prenylamine or methamphetamine, benzphetamine, deprenyl, dimethylamphetamine, famprofazone, fencamine, furfenorex. As expected, the methamphetamine derived from these drugs is subsequently metabolized to amphetamine.

Numerous studies have examined the extent of conversion of these precursor compounds to methamphetamine and/or amphetamine. Not surprisingly, there is considerable variability in the extent of metabolism. Although there are a number of possible reasons for this variability, the most likely are the fact that urine pH has such a significant effect on the rate of excretion of amphetamine and methamphetamine. In the majority studies, there was no attempt to control the urine ply of the experimental subjects. One exception to that lack of control is seen in the study by Elsworth et al., which did take into account the effects of pH. Their findings, as expected, indicated that urine pet had a significant effect on the excretion. The excretion of amphetamine and methamphetamine is also dramatically affected by adjustment of urine pH.

Analysis of the precursor compounds is described later in this chapter under the specific analyte. In most cases, these procedures were developed as part of the investigation of the drug in question. There are a number of procedures that have been developed for purposes of identification of multiple compounds. In several cases these general procedures evaluated the ability of the assay to identify some of the precursor drugs among others. An example is the systematic toxicological analysis procedure described by Maurer and colleagues, which has been applied to the analysis of several studies of precursor compounds. These and other reports describe the analysis of a variety of drugs to include one or more of the precursor compounds. Regulated workplace drug testing (WPDT) does not allow testing for other compounds without specific approval by the Agency. As a result, determination of involvement of precursor compounds is severely limited due to the prohibition of conducting most of the analysis described below. For those laboratories not limited boy these rules substantial insight can be obtained by evaluation of the samples as described to evaluate the involvement of these compounds in the origin of the amphetamine or methamphetamine in the samples.

Amphetamine-Producing Drugs

Amphetaminil (Refer to Figure 9-3). Studies have shown that amphetamine is not detected in the urine after administration of the drug to humans, or rats. After administration of amphetaminil, amphetamine concentrations in blood increased over time and closely correlated with the pharmacological activity. After only 30 min. two-thirds of the amphetamine was already metabolized to amphetamine. Over the first ^90 min after administration of amphetaminil, the majority of the drug was metabolized to other products. This rapid disappearance of the parent drug and appearance of other metabolizes, including amphetamine, suggest that amphetamine, rather than amphetaminil, is the active agent. Tissues (E.g., brain) were shown to contain 2 to 3 times the amount of amphetamine found in blood, counter to what would be expected where the more hydrophobic form, such as seen with methamphetamine, would lie expected to find its way into the central nervous system ACNE than a more water-soluble form of the drug, such as amphetamine.

Extraction of amphetaminil from blood, brain, and adipose tissue was accomplished using benzene after adjustment of the pH to 5. Because of the low pH, separate extraction conditions must be used to isolate both amphetamine and amphetaminil from the same sample. A consideration in the analysis of amphetaminil is the instability of the drug in polar solvents. Substantial degradation of the drug was noted in polar solvents, requiring the use of non-polar, non-aqueous solvents. Analysis of amphetaminil, among a variety of other amphetamine-related compounds, has been described using the REMEDi system. This system includes sample preparation as part of the process, malting it appealing for rapid results for small numbers of samples rather than off-line sample preparation. The system does suffer from relatively high detection limits, which may or may not be problematic for some purposes. In the case of amphetamine, the limit of detection was reported to be 100mg/mL. The REMEDi system was also used in another report, which also showed a relatively high detection limit for amphetaminil.

Clobenzorex (Refer to Figure 9.4):

Even though there is some discrepancy in the literature regarding the enantiomeric composition of clobenzorex, it is the d-enantiomer only. Although this alone cannot absolutely differentiate whether the amphetamine originated from this precursor it can eliminate the possibility of this drug as the origin if the amphetamine found contains any l-enantiomer.

The cross-reactivity of samples from subjects that used clobenzorex has been described for 2 fluorescence polarization immunoassays. A gas chromatography-mass spectrometry GC-MS procedure for the analysis of a number of drugs, including clobenzorex, from urine using solid-phase extraction method has been reported. The quantification limits for the drugs in this procedure were 200 ng/mL for derivatized and 500ng/mL for underivatizable compounds. Incorporation of clobenzorex and other amphetamine analogs into hair has also been described.

An early investigation of the metabolism of clobenzorex was studied in rats using radio-labeled drug. This study identified the drug and several metabolizes, including 4-hydroxyamphetamine, 4-hydroxyclobenzorex, amphetamine, and hippuric acid. Significant amounts of the hydroxy forms were corrugated. An extensive study of the metabolism of clobenzorex by Maurer and colleagues described a number of metabolizes and their detection times, including 2 hydroxy metabolizes, 1 of which was detected for a long period of time relative to the others and in some cases, comparable to amphetamine. Other studies also looked at the metabolism and urinary excretion of clobenzorex and several metabolizes in urine. This research group identified the long-lasting hydroxy metabolite as 4-hydroxyclobenzorex by comparison to analytical standards synthesized for that reason. This metabolite was found in all urine samples positive for the amphetamine following both single and multiple doses of clobenzorex and many, but not all, that contained detectable amphetamine. The authors described the modus operandi for the identification and quantification of 4-hydroxyclobenzorex using its 3-regioisomer as internal standard. The procedure was a minor modification of the laboratory’s amphetamine procedure. An account of clobenzorex and its metabolites from the point of view of interpretation of analytical results was reviewed together with a number of related precursor compounds by several authors.

Another procedure used solid-phase micro-extraction (SPME) followed by MS analysis of the drug from urine samples.

Ethyl-amphetamine (Refer to Figure 9.5) is excreted intact and metabolized to amphetamine and 4-hydroxyethylamphetamine (Refer to Figure 9.6). Following administration of a 30-mg dose, 9 to 14.5% of the dose was excreted as amphetamine. The percentages changed little when the drug was administered as a slow-release formulation. The use of diuretics had a minor effect on the percentages, but as expected, caused dilution of the samples; thus decreasing the concentrations seen and detection times for each of the components.

Noteworthy differences were noted in the metabolism of ethyl-amphetamine enantiomers. Under restricted urine pH conditions the d-enantiomer was metabolized much more rapidly than the l-enantiomer. In all cases, the amount of ethyl-amphetamine exceeded the amount of amphetamine excreted. In the first 24 h the ratio of ethyl-amphetamine to amphetamine was approximately 1.3:1 after administration of d-ethyl-amphetamine, 5:1 after administration of racemic ethyl-amphetamine, and approximately 12:1 following administration of the 1-enantiomer.

Nagai et al. studied the metabolism of racemic ethyl-amphetamine and showed the total excretion of l-ethyl-amphetamine to be greater than d; however, the amphetamine results showed d-amphetamine to be excreted in the greatest amounts. Another study showed a maximum of slightly less than 30% of the d-enantiomer was excreted intact, while as much as 78.9% of the l-enantiomer was excreted. Amphetamine accounted for up to 18.4% d- and 7.1% I-enantiomers. This information can be very useful in the evaluation of results. Detection of the parent drug and 4hydroxyethylamphetamine at appropriate concentrations, together with the presence of both enantiomers, is clear evidence for the involvement of this drug. However, it should be noted that 4-hydroethylamphetamine is also a metabolite of mebeverine; therefore, its presence alone does not demonstrate the use of ethyl-amphetamine. The presence of the parent drug and the even higher concentration of the hydroxylated metabolite make determination of the use of ethyl-amphetamine relatively easy compared to some of the other precursor compounds.

Extraction of ethylamphetamine, amphetamine, and 4-hydroxyethylamphetamine has been accomplished from hydrolyzed urine at pH 11 with ethyl acetate/methylene chloride (1:1) giving recoveries for all 3 compounds between 75% and 86%. The pK of ethyl-amphetamine was reported to be 10.23 requiring the sample pH to be adjusted higher than ordinarily required for amphetamine or methamphetamine to allow efficient extraction into an organic solvent. The extract was then evaporated and derivatized with heptafluorobutyric anhydride. GC-MS analysis was accomplished on an OV-17 packed column or 25 m, 5% phenyl-methyl silicone capillary column using either electron ionization or iso-butane chemical ionization fit. Other analytical procedures for ethyl-amphetamine include use of GC-MS, including analysis of the derivatized and underivatized drug. Another procedure used SPME followed by MS analysis of the drug from urine samples. Liquid chromatography (LC-MS) has also been used for the analysis of multiple drugs, including ethyl-amphetamine. This procedure had a limit of detection of 10 ng/mL.

Fenethylline (Refer to Figure 9-7). Losing radio-labeled fenethylline, 24.5% of the dose was shown to be excreted as amphetamine, 27.2% as hippuric acid, and 6.6% as 4-hydroxyamphetamine. A relatively small amount of the drug (3 6%) was excreted intact. There was a statistically significant difference in the amount of hippuric acid formed from fenethylline compared to an equivalent dose of amphetamine. Although this is metabolically interesting, it is of less practical value in assessing the use of this drug versus amphetamine in a single biological sample because hippuric acid is a breakdown product of a number of different compounds.

Fenethylline is racemic for the amphetamine portion of the molecule; therefore enantiomer analysis can be a significant aid in interpretation of amphetamine positive data. In addition, another major metabolite formed after cleavage of the fenethylline molecule is theophylline.

Theophylline and several of its metabolites are also found in urine following administration of this drug. Ellison et al. measured theophylline to be 13.7% of the dose in the first 24 h. Use of thin-Layer chromatography (TLC) to detect the drug and its metabolites is described and was facilitated by the use of radio-labeled 3H-fenethylline. HPLC was used with ultraviolet (UV) and fluorescent detection following a simple solvent extraction to identify many of the different metabolites of this drug. Theophylline is readily measured and can be useful in the interpretation of the metabolism of fenethylline. More recent applications have been developed to identify fenethylline as one of a number of compounds using GC-MS, LC-MS-MS, and SPME.

There is some disagreement whether the pharmacology of fenethylline is the result of the drug itself or its metabolic product amphetamine. Fenethylline does not show the same pharmacological activity as amphetamine, most notably with respect to the effects on the heart. Analysis showed that both fenethylline and amphetamine were deposited in hair and could be detected following a single dose of fenethylline. In 4 of 5 subjects the amount of fenethylline in the hair exceeded the amount of amphetamine. The combination of racemic amphetamine and the presence of theophylline help in the identification of this precursor drug from the use of amphetamine alone, although concurrent use should not be ignored because of the availability of theophylline. Also, although much abused amphetamine is d- only racemic amphetamine is also commonly found depending on starting materials.

Fenproporex (Refer to Figure 9.8). A controlled study  involving the administration of a single 10-mg dose of fenproporex showed peak concentrations of approximately 2,100 ng/mL of amphetamine reached from about 6 to 20 h post-dose. The amphetamine was shown to be both the d and I-enantiomers. The percentage of fenproporex metabolized to amphetamine ranged from 27 to 34%, consistent with a previous study. Fenproporex was also seen in many of the samples reaching a maximum of slightly greater than 700ng/mL but was not detectable in all samples where amphetamine was still present. In an early study of fenproporex metabolism it was established that under acid urine conditions, between 5.4% and 8.7% of the drug was excreted intact. However, without control of the urine pH, only about 3% of the dose was excreted intact. Following multiple doses (10 mg/day) for 1 week of the drug were reported in another study, peak concentrations ranged from 2,851 to 4,150 for amphetamine and from 263 to 3,032ng/mL for fenproporex. Amphetamine-positive samples were seen for approximately 40 h after the last dose. The percentage of each amphetamine enantiomer remained between 40% and 60% for the period of time the drug was administered and for an additional approximately 48 h after administration was stopped. It is interesting to note that all samples from the single and multi-dose study that was positive for amphetamine (2500ng/mL) also contained detectable fenproporex, although it is possible this might not hold true for all users of fenproporex.

An extensive Amphetamine evaluation of fenproporex metabolism identified different isomers of hydroxyfenproporex, dihydroxyfenproporex, hydroxy-methoxy-fenproporex, 2 different hydrology isomers of amphetamine, dihydroxyamphetamine, hydroxymethoxy-amphetamine, norephedrine, desamino-oxo-fenproporex, desamino-oxo-hydroxyfenproporex, desamino-oxo-dihydroxyfenproporex and desamino-oxohydroxy-methoxy-fenproporex in addition to amphetamine. One of the isomers of hydroxyfenproporex was detected for up to 28 h, but amphetamine (limit of detection [LOD] 100 ng/mL) was detected for up to 60 h post dose. A recent study evaluated the metabolism of fenproporex and showed that CYP2D6, CYP1A2, CYP2BG, and CYP3A4 catalyzed the de-alkylation of both enantiomers with slight preference for the d-enantiomer. This is also shown in Figure 9.8.

The drug has been extracted with ether from alkalinized urine followed by derivatization with acetic anhydride and analysis by GC. Analysis of fenproporex was also accomplished with minor modification to a procedure for analysis of amphetamine and methamphetamine. The procedure was used for identification and quantification of samples from single and multiple-dose controlled studies as well as samples from individuals claiming use of fenproporex as the source of amphetamine. Fenproporex was also analyzed using SPME with MS detection by several groups. Hair analysis for drugs including fenproporex has also been described.

Lisdexamfetamine (Refer to Figure 9-9) Lisdexamfetamine is the most recent entry into this group of compounds. The drug was developed to be a pro-drug requiring the conversion of the pro-drug to the active drug by metabolism in the body. The advantage of this is that the drug cannot be abused by crushing the pill into powder followed by snorting to get the rapid absorption and associated rush, a common form of abuse of ADHD drugs.

As a result of this drug being a new entrant to the marketplace, there are few studies describing the drug or its metabolic profile beyond the early studies associated with its approval. Krishnan and Stark describe the pharmacokinetics of the drug in healthy adults indicating the use of an LC-MS-MS procedure, however, specific details of the procedure used were not delineated.

Mefenorex (Refer to Figure 9-10). Metabolic studies of mefenorex have been reported by a number of investigators and have identified several metabolites. These included hydroxymefenorex, together with amphetamine and hydroxyamphetamine. The hydroxylation of mefenorex at the 4 position was reported to be 37% of the initial dose fang). Another study, showed peak concentrations of amphetamine ranged from 1.58 to 30.33 g/mL, and mefenorex from 0.38 to 4.7 g/mL. Amphetamine could be detected for as long as 71 h post-dose, and mefenorex for up to 29 h post-dose. Excretion of mefenorex and amphetamine accounted for 0.63% and 5.5% of the 40-mg dose, and 1.5% and 10.4% of the 80 mg dose, respectively. This study also identified hydroxy-methoxy-mefenorex as a metabolite. Another study identified several metabolites in addition to amphetamine: 2 different isomers of hydroxymefenorex, dihydroxymefenorex, hydroxy-methoxy-mefenorex, hydroxyamphetarrune~ desaminooxo-hydroxymefenorex, desaminooxo-hydroxymefenorex, desaminooxo-hydroxy-methoxy-mefenorex, dechloro-hydroxymefenorex, dechloro-dihydroxymefenorex, dechloro-dihydroxy-mefenorex, and dechloro-trihydroxymefenorex, Hydroxymethoxymefenorex, hydroxymefenorex, and the dechloro compounds are unique to mefenorex and can be used to assess the involvement of this drug The parent drug mefenorex (LOD, 50 ng/ml) was found only 16 to 20 h post-dose. One of the isomers of hydroxymefenorex was detected for up to 32 h, but amphetamine (LOD, 100 ng/mL) was detected up to 68 h post-dose (Figures 9.10 and 9.11).

Nazarali et al. described a procedure for the analysis of mefenorex in brain tissue using GC win electron capture detection of the pentafluorobenzoyl derivative.

GC-MS analysis of mefenorex along with other compounds has been described by several researchers. Mefenorex was also analyzed using SPME win MS detection. Hair analysis for drugs including mefenorex has also been described. Another multidrug procedure for the analysis of various drugs including mefenorex used LC-MS. This procedure used an ion trap and reported a detection limit of 10 ng/mL for mefenorex.

Mesocarb (Refer to Figure 9-12). Mesocarb is excreted in only trace amounts, but both mono (Refer to Figure 9-13) and dihydroxy (Refer to figure 9-14) metabolites were found in the urine. Efficient detection of these hydroxylated metabolizes requires hydrolysis before extraction. One study reported finding conjugated hydroxymesocarb in samples following administration of mesocarb to 2 human subjects. Between 27% and 29% of the dose was excreted as this conjugated metabolite following a 10-mg dose. The metabolite was detected for 48 h in I subject, and 72 h in the other, following administration. This study did not report the presence of intact mesocarb m the urine samples collected. Analysis of mesocarb and metabolizes using LC-MS-MS has been described by a number of authors in urine and plasma. GC-MS has also been used for analysis of mesocarb. SPME with MS detection has also been used as an analytical tool for the determination of mesocarb in biological samples.

Prenylamine (Refer to Figure 9-15). Prenylamine is a racemic mixture consisting of both enantiomers. Its metabolism and disposition has been shown to be enantio-selective. Since Prenylamine is a racemic mixture, both the d- and l-enantiomers of amphetamine would be found in urine. This can be of significant assistance in the Interpretation of the origin of amphetamines. Determination of Prenylamine in plasma showed that the amount of l-prenylamine exceeded the amount of the d-enantiomer by 4 to 1. Another study also indicated the d-enantiomer is metabolized more rapidly than the l-form. Although doubled, the difference was not as great. It is reasonable to assume, therefore, that more of the product amphetamine would be d-enantiomer than l-form initially. This is similar to what is seen with the metabolism of amphetamine and methamphetamine. This was confirmed in a study that looked at 5 subjects administered 1280 mg of prenylamine. The ratio of l:d started less than 1 but increased to greater than 1 over the period of several days. The same study measured the concentration of amphetamine and found the maximum to be 1280 ng/mL. It is interesting to note that only 3 samples from a single subject contained amphetamine above 500 ng/mL, all other samples from that and the other subjects were below 500 ng/mL All samples collected at 48 h, the end of collection time, were reported as less than limit of quantification (LOQ) 10 ng/mL. It was also noted that the metabolite 3,3-diphenylpropaneamine was seen in ail amphetamine-positive samples. In addition to amphetamine, 4-hydroxyamphetamine, norephedrine, 4-hydroxynorephedrine, and diphenylpropylamine were identified as metabolites. One study showed approximately 2.5% of the dose 180 mg was excreted as amphetamine following administration of the drug for 5 days. This was reported to be equivalent to the amount of amphetamine excreted following a 2-mg dose of amphetamine. Excretion of amphetamine was doubled using ammonium chloride because of the changes in urine pH. One study showed legs than 1% of a 200 mg dose was excreted unchanged. Prenylamine was in both free and conjugated forms, with slightly more than half conjugated.

A number of different methods have been developed for the analysis of prenylamine. These methods include TLC, HPLC  and GC-MS. Schmidt et al. described a procedure for the extraction and analysis of the drug from acidified plasma by taking a 1-mL sample and extracting the drug with .5 mL methylene chloride followed by washing with 2 mL 0.1 M NaOH solution. The dried extract was then derivatized with pentafluoroprooprionic anhydride (PFPA) then analyzed by GC-MS with a 25-m 5% phenylmethyl silicone column. Electron ionization at 20 eV was used and the method demonstrated a detection limit of 0.2ng/mL, which was 10 times more sensitive than a previously reported HPLC assay.

A GC-MS procedure for the analysis of prenylamine and several other drugs from urine using solid-phase extraction on a C-18 column has been reported. The drug was eluted from the column using chloroform/ isopropanol followed by derivatization and GC-MS analysis. The quantification limits for the drugs in this study were 200ng/mL for derivatized compounds and 500ng/mL for underivatizable compounds.

HPLC analysis of prenylamine from plasma and urine was conducted using a C-18 column following either alkaline or ion-pair extraction. Derivatization of the compound with R-napthylethyl isocyanate allowed separation of the enantiomers and, due to the strong fluorescence of the derivative, allowed detection of as little as 2 ng/mL of either enantiomer of prenylamine in urine.

Methamphetamine- and Amphetamine-Producing Drugs

Benzphetamine (Refer to Figure 9-16). The metabolism of benzphetamine is nearly complete with little, if any, of the drug excreted in the urine unchanged. The intact drug has been reported in some studies. A reasonable explanation for the difference in these reports is the method used by Budd and Jain By, which had a detection limit for benzphetamine of 150 ng/mL while the other methods had a much lower detection limit In addition, the study by Budd and Jain involved only the administration of a single 20-mg dose of benzphetamine while prescriptions are typically for either 25 mg or 50 mg tablets taken 1 to 3 times daily for up to several weeks. Since the pH of benzphetamine has been determined to be 6.55, it is Likely that little of the drug would be excreted intact. Even in those studies that reported benzphetamine, it was found at low concentrations and for only short periods of time.

In addition to amphetamine and methamphetamine, several metabolites have been identified from samples collected following benzphetamine administration 4-hydroxyamphetamine, 4-hydromethamphetamine, benzylamphetamine, 1-(3-hydroxyphenyl)-2-(N-benzlamino)propane, 1-(3-methoxy-4-hydroxyphenyl)-2-(N-benzylamino)propane, and 1-(hydroxyphenyl)-2-(N-methyl-N-benzylamino) propane were identified. Several of these metabolites are characteristic of Benzphetamine use. The 1-(hydroxyphenyl}-2-(N-methyl-N-benzylamino) propane was excreted at concentrations equal to or m many cases higher than those of methamphetamine or amphetamine. Up to 6% of the dose was excreted as free metabolite, and as much as 28% as the glucuronide conjugate. In the same subjects, amphetamine represented 7.6 to 8.9%, and methamphetamine 2.2 to 3.1% of the dose.

Other studies identified 4-hydroxybenzphetamine and 4-hydroxynorbenzphetamine as conjugated metabolites of Benzphetamine. These metabolites were excreted as conjugates, accounted for 10 to 15% of the initial 10-mg dose, and could be detected for 24 host-dose. Samples collected from subjects 0 to 3 h post-dose showed benzphetamine, but the parent drug was not detected in subsequent samples. Norbenzphetamine (N-desmethylbenzphetamine) and hydroxynorbenzphetamine, along with amphetamine and methamphetamine, were identified in urine following administration of 30 mg of Benzphetamine per day for 5 days (n = 2). Norbenzphetamine was detected for up to 12 h after administration of the last dose, whereas the other metabolites were detected for longer periods. Benzphetamine was detected only in the sample collected 1 h after administration in 1 of 2 subjects. Analysis of hair samples from these subjects showed that Benzphetamine and norberzphetamine were incorporated, but 4-hydroxynorbenzphetamine was not.

A number of procedures are reported for the analysis of benzphetamine. Benzphetamine is easily analyzed using procedures developed for analysis for the amphetamines. In fact, the signal seen for Benzphetamine is proportionately higher than for many other compounds including amphetamine and methamphetamine leading to low detection limits for most assays. Because Benzphetamine is detected at very low concentrations and for very short periods of time even after multiple dosing, looking for the parent is unlikely to be of much assistance in determination of its involvement of this drug in a methamphetamine containing sample. Studies of metabolism of Benzphetamine have identified metabolites that are characteristic and detected for longer periods of time than the parent drug.

Of the various metabolites identified, 1-(4-hydroxyphenyl)-2-(Nmethyl-N-benzylamino)propane (Refer to Figure 9.17) was found to be unique and characteristic of the use of benzphetamine.

It was also excreted at concentrations equal to, and in most cases, higher than, those of methamphetamine or amphetamine. Much of the 1-(hydroxyphenyl)-2-(N-methyl-N-benzylamino)propane was excreted as the glucuronide cord e. Between 5% and 6% of the dose was excreted as the free metabolite, while another 14 to 28% was excreted as the glucuronide conjugate, thus requiring hydrolysis before analysis. The benzphetamine metabolites were incubated at pH 5.0 for 48 h at 37 °C with ,β glucuronidase to hydrolyze the conjugates. The sample was then made alkaline (pH 9.0) by the addition of Na2CO3, then extracted into chloroform/isopropanol (3:1), which gave recoveries of 96 to 100%.

Other methods for the analysis for benzphetamine include SPME with MS detection, which gave a detection limit of 20ng/mL. Several LG procedures using MS and MS-MS  have been described. Analysis of the drug in hair has also been documented.

Deprenyl (Refer to Figure 9-18). Deprenyl has been used for many different medical indications, although it is approved for use in the treatment of Parkinson disease. From a forensic standpoint the most significant characteristic of deprenyl is that it is the l-enantiomer, therefore all metabolizes are also the l-enantiomer. There is no indication that any racemization occurs with deprenyl during metabolism. As a result, methamphetamine and amphetamine resulting from the metabolism of deprenyl would not likely screen positive by most immunoassay systems. Even if it were to screen positives the l-enantiomer of methamphetamine has little CNS stimulant activity. Under workplace drug-testing guidelines samples that are less than  1-methamphetamine are canceled during Medical Review Officer Review as consistent with over-the-counter nasal inhalers containing l-methamphetamine. Deprenyl is metabolized to methamphetamine, amphetamine, desmethyldeprenyl (Figure 9-19), and typical amphetamine metabolites.

Desmethyldeprenyl is a unique metabolite, and its presence can help demonstrate the use of deprenyl. The metabolism of deprenyl has been studied by a number of investigators either as controlled studies or analysis of patients prescribed deprenyl. Doses for the controlled studies ranged from 5 to 45 mg. The ratio of amphetamine to methamphetamine can be used in evaluation of the involvement of this drug v/s methamphetamine. In 4 deprenyl users, the ratio ranged from 0.37 to 0.42. Plasma ratios were determined to be approximately 0.33 during the 36 h after administration of a 10-mg dose. Desmethyldeprenyl can be detected in urine and its presence can clearly demonstrate the use of deprenyl. The metabolism of deprenyl to Desmethyldeprenyl has been reported in a number of different studies. Following administration of 15 midday for 5 days, one study reported the results of urine samples collected for 96 h after the last dose. Amphetamine and methamphetamine were found in all samples collected 96 h after the last dose. Deprenyl could be detected for 36 h and Desmethyldeprenyl for 72 h.

Analysis of desmethyldeprenyl has been described by a number of different methods, including packed and capillary GC and GC-MS. Salonen found 0.03% and 0.01% of 3 mg/kg and 10 mg/kg doses administered to dogs as Desmethyldeprenyl. The percentages of amphetamine 10.1% and 8.1%, and methamphetamine 2.4% and 4.5%, for the same doses indicate the amount of desmethyldeprenyl excreted is small compared to the other metabolites. Measuring plasma concentrations of amphetamine, methamphetamine, and desmethyldeprenyl showed the desmethyldeprenyl concentration dropped below those of amphetamine and methamphetamine in fewer than 10 h after administration. In a postmortem case involving deprenyl, femoral blood concentrations for amphetamine and methamphetamine were 70 and 170 ng/mL, respectively, which was noted as being 4 times the concentration listed by the manufacturer for serum concentrations of the drug during clinical trials. Administration of a 10-mg dose to a healthy male volunteer led to peak plasma concentrations of approximately 4 ng/mL of amphetamine and 12 ng/mL of methamphetamine.

Analysis of desmethyldeprenyl has been accomplished by extraction of the drug from plasma by addition of NaOH and Triton X-100 (to release the bound fraction of the drug) followed by 6 ml. of toluene.

After shaking, the organic layer was removed and the drug back extracted into 1 mL of 1.0 M HC1 solution. Sodium chloride and 0.5 mL 1.0 M NaOH were then added and the drug was re-extracted into 0.1 mL toluene followed by derivatization with HFBA^A Negative ion chemical ionization GC-MS analysis gave detection limits of 0.25 ng/mL of desmethyldeprenyl. Another method used electron capture detection of the trichloroacetyl derivative following extraction of 0.5 mL of plasma or urine with hexane. This procedure gave a detection limit of 3 ng/mL (S/N 3:1). Another procedure used SPME followed by MS analysis of the drug from urine samples. Another procedure used capillary electrophoresis with direct injection of the urine. This is one of the many capillary electrophoresis methods that determine enantiomeric composition, a common application of this technique.

Dimethylamphetamine (Figure 9-20). Dimethylamphetamine is not available as a prescription drub and has no recognized medical use. Therefore, it would be reasonably easy to determine that its presence was from illegal use. Reportedly, it has less pharmacological effect than methamphetamine. It does, however, make interpretation of the results relatively easy when considering the illegal nature of use. If it is present, its use is illegal. If only the metabolic product methamphetamine is measured, absent valid medical prescription, the use is illegal. Metabolism of dimethylamphetamine has been studied both in rats and humans. The metabolic pattern is very similar for both species, with the expected difference in aromatic hydroxylation being more pronounced in the rat. The major excretion products of dimethylamphetamine in humans are dimethylamphetamine and dimethylamphetamine-N-oxide, making up an average of 15.6% and 21.7% of the initial dose, respectively, during the first 24 h as compared to 7.5%, for methamphetamine and 0.65% for amphetamine. From 24 to 72 h, methamphetamine was the most prominent metabolite found, although dimethylamphetamine, dimethylamphetamine-N-oxide, and the 4-hydroxy forms of dimethylamphetamine and methamphetamine were also seen.

Extraction of dimethylamphetamine has been accomplished from alkaline urine using diethyl ether. Following back-extraction into 0.2 M HCI, the solution was again made basic and the drug re-extracted into diethyl ether. The N-oxide form of the drug was measured following reduction to dimethylamphetamine and subsequent extraction and analysis. To determine the hydroxylated metabolizes, the samples were incubated with -glucuronidase before extraction.

Efficiencies of extraction were 97 to 100% and analysis was accomplished on a GC column of 2% apiezon/5% KOH for the free base and 3% OV-17 for the trifluoroacetyl and trimethylsilyl derivatives. Mass spectral analysis was accomplished with isobutane chemical ionization.

Analysis of dimethylamphetamine, dimethylamphetamine-N-oxide, methamphetamine, and amphetamine was accomplished directly from urine using LC-MS for both quantitative and enantiomeric determination. Analysis of 10 samples from dimethylamphetamine users showed all of the samples to be d-enantiomer only and the amount of dimethylamphetamine-N-oxide exceeded the dimethylamphetamine in each of the samples. Other reports have described the analysis of dimethylamphetamine, and metabolites, using a variety of analytical procedures including LC-MS, GC-MS, SPME, and capillary electrophoresis.

Famprofazone (Figure 9-21). Famprofazone is a component of a multi-ingredient drug. Famprofazone metabolism yields a number of metabolites that can be used in the interpretation of analytical results. Unfortunately, amphetamine and methamphetamine could be detected in urine after these unique metabolites were no longer detectable; therefore, their use is limited to when they are present. The proportion of amphetamine and methamphetamine enantiomers following Famprofazone administration has been described in several reports. Both enantiomers of amphetamine and methamphetamine were found in urine. Famprofazone is manufactured in Switzerland, and recently also in Taiwan.

Among the metabolites of Famprofazone identified are norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, p-hydroxyamphetamine, and p-hydroxymethamphetamine, which arise from several different compounds and are not unique to famprofazone. 3-hydroxymethylpyrazolone, another metabolite of famprofazone, is also found in urine and represents a significant percentage of the parent drug.

A unique metabolite of famprofazone, 3-hydroxydesmethylfamprofazone, was found following use of the drug. Peak concentrations of famprofazone and p-hydroxydesmethylfamprofazone were 0.179 and 8.84 g/mL, respectively with both reaching peak concentrations at 4 h post-dose. Unfortunately, methamphetamine and amphetamine could be detected at concentrations that were positive after these were no longer detectable. Similar hut alternative methods have been described in several other studies.

Fencamine (Refer to Figure 9-22). The excretion of fencamine was studied in rats and humans by Mallol et al. 6371- Identification of the drug and metabolites was accomplished using TLC and colorimetry. Intact fencamine could be detected for 48 h following a 50 mg oral dose of the drug to humans. Peak concentrations of the drug in urine were seen less than 3 h following administration. Approximately 32% of the dose was excreted as the intact drug in 48 h and 26.6% in the first 24 h in humans ~ = 8}.

Because fencamine is administered as the cl,l-mixture, the resultant amphetamine would be both the d and l-enantiomers. Evaluation of the amphetamine enantiomer composition would therefore be of substantial assistance in interpretation, effectively eliminating fencamine as the potential source for results that are enantiomerically pure cl- or l-amphetamine.

Furfenorex (Refer to Figure 9-23). The metabolism of furfenorex is nearly complete with very little, if any, of the parent drug excreted in the urine unchanged. The metabolic profile for this drug is less complex in humans than in rats, but still yields a number of different metabolizes. Of the various metabolites identified 1-phenyl-2-(N-methyl-N-γ-valerolactonylamino)propane (Refer to Figure 9-24) was found to be unique and characteristic of the use of furfenorex.

In the first 24 h, the percentage of dose excreted as amphetamine exceeded methamphetamine. Subsequently, in the samples taken up to 72 h, the percentage of methamphetamine exceeded amphetamine. In all cases, the percentage excreted as (1-phenyl-2-Nmethyl-N-γ-valerolactonylamino)propane exceeded both amphetamine and methamphetamine.

To extract this metabolite efficiently from urine, however, it is necessary to use an acid rather than a basic extraction. Extraction at pH 3.5 yielded 10-fold higher recovery of 1-phenyl-2-(N-methyl-N-γ-valerolactonylamino)propane compared to a basic extraction. This metabolite was extracted from acidified urine by washing several times with diethyl ether followed by extraction into chloroform/isopropanol (3:1), giving recoveries of 96 to 100%. Since amphetamine and methamphetamine are typically extracted under basic conditions, it would require a separate procedure to extract the metabolite or use of a solid phase matrix adjusted to the proper pH to tonically extract the compound. Identification of this compound would clearly demonstrate furfenorex as the origin of amphetamine and methamphetamine.

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