Origin and Interpretation of Low Methamphetamine Levels Found in Amphetamine‐Positive Urine Samples: Support for Methylation of Amphetamine as a Minor Metabolic Pathway

ABSTRACT

Methamphetamine is relatively uncommon on the European drug market, but Swedish drug test laboratories repeatedly detect low methamphetamine concentrations in urine samples containing high amphetamine levels, warranting clinical evaluation for suspected polydrug use. Of 12,062 routine samples screened positive for amphetamines, 86% were confirmed positive (≥ 200 μg/L) for amphetamine, 2.1% for methamphetamine, and 4.0% for 3,4‐methylenedioxymethamphetamine (ecstasy) by liquid chromatography–tandem mass spectrometry. Of the 259 methamphetamine‐positive samples, 98% contained amphetamine concentrations above the reporting limit, consistent with the metabolic conversion of methamphetamine to amphetamine in the body. However, in most (69%) of these samples, the methamphetamine concentration was only < 2% of the amphetamine level, suggesting methamphetamine was not the primary drug taken. Chiral analysis of selected samples showed that after use of illicit street amphetamine with a racemic content of the l‐ and d‐enantiomers, a similar l/d proportion was observed for methamphetamine. Similarly, in samples from patients receiving d‐amphetamine‐based ADHD medication, low d‐methamphetamine levels were detected, even though the pharmaceutical products contained no methamphetamine. This observation, together with the parallel l/d‐enantiomer distributions, supports amphetamine N‐methylation as a trace, albeit quantitatively insignificant, metabolic pathway in humans. From both a clinical and forensic perspective, a low urinary methamphetamine concentration of less than a few percent of the amphetamine level therefore does not warrant further clinical evaluation for suspected polydrug use. The present findings further demonstrate that chiral analysis of both amphetamine and methamphetamine is an effective approach for distinguishing between illicit and therapeutic sources in positive screening drug tests for the amphetamines.

A low urinary concentration of methamphetamine less than 1% of the amphetamine level may not necessarily indicate polydrug use. Instead, it could result from N‐methylation of amphetamine. Chiral analysis of both amphetamines is an effective method for distinguishing between illicit use and therapeutic sources in positive drug screening results.

1. Introduction

Amphetamine and methamphetamine are synthetic central nervous system stimulants that constitute a major illicit drug and health problem worldwide [1]. First synthesized over a century ago, the amphetamines gained widespread use during World War II due to their fatigue‐reducing, appetite‐suppressing, and performance‐enhancing effects [2]. After the war, they were widely available as over‐the‐counter stimulants, weight‐loss medications, and treatments for various other conditions. However, by the 1960s, after frequent reports of substance abuse and addiction, their production and availability were restricted, and several amphetamine‐type stimulants were subsequently controlled under the 1971 United Nations Convention on Psychotropic Substances [3].

Despite the regulatory efforts, the abuse of illicitly manufactured amphetamines has persisted with some geographical differences. Methamphetamine use is particularly prevalent in North America, East and Southeast Asia, and Oceania, while amphetamine use is more common in Europe [4]; although methamphetamine use seems to be increasing also there [5]. In addition to these two, large numbers of substituted amphetamines have emerged over the years on the recreational drug market, including 3,4‐methylenedioxymethamphetamine (MDMA, or ecstasy) and other structurally related new psychoactive substances (“designer drugs”) [6].

Due to their widespread abuse, amphetamine and methamphetamine are commonly measured in urine, oral fluid, or blood samples as part of a drug test [7]; they can be detected for up to several days after the last intake [8]. The time window primarily depends on the dose ingested, but the measured substance concentration is also affected by urine dilution and pH [9]. When assessing the results of a drug test, the origin of detected substances is important to consider [10, 11]. While both substances are used separately as recreational drugs, amphetamine also occurs in samples as a demethylated metabolite of methamphetamine [12, 13]. Furthermore, a positive drug test for amphetamine and/or methamphetamine may result from some therapeutically used precursor medications [14, 15], although most of these have been withdrawn due to the abuse potential.

Given the amphetamines exist as two stereoisomers, determining their enantiomeric composition in a sample by chiral analysis could facilitate clinical decision‐making to identify the likely source of the drug. For example, amphetamine medications used for the treatment of attention‐deficit hyperactivity disorder (ADHD) are predominantly based on dextroamphetamine (also called d‐amphetamine, or S‐(+)‐amphetamine), whereas illicit “street” amphetamine is usually a racemate with near equal parts of d‐amphetamine and levoamphetamine (l‐amphetamine, or R‐(−)‐amphetamine) [16]. Likewise, selegiline (N‐propargyl‐l‐methamphetamine, or l‐deprenyl) used in the treatment of Parkinson's disease is metabolized to l‐methamphetamine and further to l‐amphetamine in the body [17].

Although methamphetamine is relatively uncommon on the European drug market [18, 19], drug testing laboratories repeatedly detect low concentrations of methamphetamine in urine samples containing high amphetamine levels, warranting clinical evaluation for suspected polydrug use. This study investigated the occurrence and potential sources of methamphetamine found in amphetamine‐positive urine drug test samples in Sweden and assessed the clinical relevance of the results.

2. Methods

2.1. Clinical Samples, Materials, and Routine Drug Testing

The study was based on urine samples and test results from routine drug testing for amphetamines at the Department of Clinical Pharmacology, Karolinska University Laboratory in Stockholm (Sweden). Reference materials for d‐ and l‐amphetamine, (±)‐amphetamine, and internal standard (IS; racemic (±)‐amphetamine‐d₅), and the corresponding materials for methamphetamine were obtained from Cerilliant Co. (Round Rock, TX, USA) and Chiron AS (Trondheim, Norway). All reference materials were > 98.7% pure, and methamphetamine contamination in the amphetamine reference materials was < 0.05%. They were obtained as methanol stock solutions, and dilutions were made in 0.1% formic acid in water. The IS working solution was prepared by diluting the stock solutions in water to a final concentration of 500 μg/L for each compound. All other chemicals were of analytical grade, and the water was of HPLC grade.

The urine samples first underwent immunoassay screening for amphetamines with the CEDIA Amphetamine/Ecstasy assay (Thermo Fisher Scientific, Fremont, CA, USA), targeting d‐amphetamine, methamphetamine (d‐form > l‐form), MDMA, and 3,4‐methylenedioxyamphetamine. Those screening preliminary positive were confirmed with a validated and accredited in‐house liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method (screening cutoff/confirmation reporting limit/detection limit 500/200/≥ ~ 10 μg/L). The urinary reporting limits (lower quantification limits; LLOQ) are nationally harmonized [20].

2.2. Chiral Analysis of Amphetamine and Methamphetamine

Chiral analysis of amphetamine is routinely used in the laboratory to monitor compliance with d‐amphetamine‐based ADHD treatment and to identify illicit racemic amphetamine use, but the method also partly separates the enantiomers of methamphetamine [16]. For the present study, an improved chiral method allowing baseline separation of both the amphetamine and methamphetamine enantiomers was used, essentially as described [21]. The LC–MS/MS system consisted of an AQUITY UPLC coupled to a Xevo TQ‐S micro MS (Waters, Milford, MA, USA).

Selected patient urine samples, and calibrators, controls and blanks prepared in drug‐negative urine and included in each analysis, were diluted 10‐fold with the IS working solution, mixed for 30 s, and 2 μL were injected. Chromatographic baseline separation of l‐amphetamine (RT ~ 9.8 min) and d‐amphetamine (RT ~ 10.4 min), and of l‐methamphetamine (RT ~ 10.5 min) and d‐methamphetamine (RT ~ 10.7 min), was achieved on a Lux 3‐μm AMP column (150 × 3.0 mm, 3‐μm particle size; Phenomenex, Torrance, CA, USA) using gradient elution. Mobile phase A consisted of 5‐mmol/L ammonium bicarbonate (pH 11) and mobile phase B of methanol (gradient: 0–7 min, 40% A; 7–9 min, 40 → 5% A; 9–13 min, 5% A; 13–14 min, 5 → 40% A) and the flow rate was 0.35 mL/min.

The MS instrument was operated in positive electrospray ionization mode, and the ion transitions monitored for l‐ and d‐amphetamine were m/z 136.2 > 91.0 (quantifier), m/z 136.2 > 119.1 (qualifier), and m/z 141.11 > 123.80 for amphetamine‐d₅ (IS). The corresponding ion transitions used for l‐ and d‐methamphetamine were m/z 150.0 > 119.1 (quantifier), m/z 150.0 > 91.0 (qualifier), and m/z 155.09 > 121.08 for methamphetamine‐d₅ (IS) (Figure 1A).

FIGURE 1.

Chromatograms from the chiral LC–MS/MS analysis of l‐ and d‐amphetamine (Amph) and l‐ and d‐methamphetamine (Meth) in selected routine urine drug test samples. Results are shown for: (A) a calibrator (75 μg/L) prepared in drug‐negative urine; and for: (B) a patient sample collected after intake of illicit racemic street amphetamine (17,800 μg/L; 62% l‐Amph) that contained a low methamphetamine level (381 μg/L; 57% l‐Meth); (C) a sample after intake of methamphetamine (d‐enantiomer) that contained d‐amphetamine as the metabolite; (D) a sample after intake of racemic amphetamine (53,100 μg/L l/d‐Amph) and essentially enantiopure l‐methamphetamine (2750 μg/L); and (E) a sample collected after intake of d‐amphetamine‐based ADHD medication (16,500 μg/L) that contained 0.13% d‐Meth as a metabolite (signal/noise ratio ≥ 34 for all eight such samples examined; Table 1). The MS instrument was operated in positive electrospray ionization mode and the ion transitions monitored for l‐ and d‐amphetamine were m/z 136.2 > 91.0 (quantifier), m/z 136.2 > 119.1 (qualifier), and m/z 141.11 > 123.80 for amphetamine‐d₅ (internal standard, IS). The corresponding ion transitions monitored for l‐ and d‐methamphetamine were m/z 150.0 > 119.1 (quantifier), m/z 150.0 > 91.0 (qualifier), and m/z 155.09 > 121.08 for methamphetamine‐d₅ (IS).

The detection limit was < 0.5 μg/L; the routine LLOQ was 10 μg/L, and the measuring range was 0–25,000 μg/L (r ² > 0.99). Samples with substance concentrations exceeding the measuring range were reanalyzed after dilution. Acceptance criteria for a positive identification were a relative retention time (RRT) versus calibrator within ±0.5% and ion ratios within ±20%.

3. Results

3.1. Routine Analysis of Amphetamine and Methamphetamine

Of 12,062 consecutive urine test results from the routine LC–MS/MS confirmation analysis following a positive screening for amphetamines during the period November 2021 through February 2025, 10,371 (86%) tested positive (i.e., ≥ 200 μg/L) for amphetamine and 259 (2.1%) for methamphetamine, respectively. The proportion of samples that tested positive for MDMA was 4.0%.

Of the 259 methamphetamine‐positive samples (concentration range 200–238,000, median 448 μg/L), 253 (98%) also contained amphetamine above the reporting limit (range 248–827,000, median 103,000 μg/L). The methamphetamine concentrations in the remaining 6 cases were low, ranging 200–912 (median 450) μg/L, and all also contained a measurable (i.e., > LOD) amphetamine level (range 15–192, median 143 μg/L).

In 56 (22%) of the 259 methamphetamine‐positive urine samples, the methamphetamine concentration (range 390–238,000, median 5012 μg/L) exceeded the amphetamine level, indicating primary ingestion of methamphetamine. In most cases (69%), however, the methamphetamine concentration (range 200–3810, median 327 μg/L) was < 2% (in 58% < 1%) of the amphetamine level. Among all 10,371 amphetamine‐positive samples (range 201–827,000; median 5330 μg/L), a methamphetamine concentration above the LOD was found in 3210 (31%), but the concentration constituted < 1% of the amphetamine level in most (92%) (Figure 2). Overall, there was a statistically significant positive association between the amphetamine and methamphetamine concentrations (r = 0.56, p < 0.0001; MedCalc software), albeit with a considerable scatter (Figure 2).

FIGURE 2.

Methamphetamine to amphetamine concentration ratios in 3089 urine samples from routine drug testing in Sweden. Samples with > 2% methamphetamine were excluded in the graph. In 92% of the samples, methamphetamine accounted for < 1% of the amphetamine concentration. Inset: Correlation between the urinary methamphetamine and amphetamine concentrations (r = 0.562, p < 0.0001; MedCalc software).

3.2. Enantiomeric Distributions of Amphetamine and Methamphetamine

Chiral analysis to determine the enantiomeric distributions of both amphetamines was performed in a selection of urine samples chosen to illustrate variable outcomes. In samples containing a high amphetamine concentration and a measurable methamphetamine level, a similar near racemic distribution (i.e., ≥ ~ 50% of the l‐forms) was observed for both substances (Table 1, Figure 1B). In contrast, in samples that primarily contained methamphetamine, the d‐form predominated for both the parent substance and for amphetamine as a metabolite, but sometimes enantiopure l‐methamphetamine was detected instead (Figure 1C,D).

TABLE 1.

Results from chiral analysis of amphetamine and methamphetamine in human urine drug test samples.

Sample	N	Total amphetamine (μg/L; mean, median, range)	l–Amphetamine (% of total)	Total methamphetamine (μg/L; mean, median, range)	Methamphetamine (% of total amphetamine)	l–Methamphetamine (% of total methamphetamine)
Routine chiral amphetamine analysis
Illicit amphetamine use (> 40% l–amphetamine)	9	60,696, 34,300, 3251–334,000	46.6–73.8	< 100	0.14–1.40 a	– b
Elvanse/Attentin medication (< 1% l–amphetamine)	17	18,356, 17,550, 10,300–37,200	0 c –0.63	< 100	0.04–0.20 a	– b
Chiral amphetamine and methamphetamine analysis
Illicit amphetamine use (> 40% l–amphetamine) and > 100‐μg/L methamphetamine	7	65,240, 69,100, 18,680–133,300	53.6–69.6	239, 226, 111–423	0.13–2.12	53.5–74.8
Illicit amphetamine use (> 40% l–amphetamine)	4	38,258, 41,300, 3330–67,100	45.0–72.4	34.5, 23.5 7.0–84.2	0.03–0.21	33.6–80.5
Methamphetamine use (mainly methamphetamine)	4	2054, 1338, 342–5200	0.00–2.68	5558, 3997, 701–13,537	76.6–769	0.00–11.8
Illicit amphetamine and methamphetamine use	1	59,600	58.6	3032	5.09	96.3
Elvanse/attentin medication	8	30,525, 23,400, 7200–80,400	0 c	8.0, 4.7, 2.2–22.9	0.01–0.06	0
Elvanse (capsule) d	1	900	0.01	0 c	0	0
Attentin (tablet)	1	15,000	0.03	0 c	0	0

^a The calculations were based on absolute area response.

^b – = Not determined.

^c 0 = Not detected.

^d Lisdexamfetamine was hydrolyzed to d‐amphetamine by incubation for 4 h with human red blood cells at 37°C.

In samples originating from patients undergoing ADHD treatment with d‐amphetamine‐based medications (e.g., Elvanse or Attentin), as indicated by the presence of less than 1% l‐amphetamine [16], virtually only d‐amphetamine was detected, but also small amounts of d‐methamphetamine (Table 1, Figure 1E). In contrast, chiral analysis of the pharmaceutical products (for Elvanse, the lisdexamfetamine was first hydrolyzed by incubation with human red blood cells for 4 h at 37°C) [16] showed no presence of methamphetamine.

4. Discussion

This investigation based on routine urine drug tests in Sweden confirmed that amphetamine is by far the most common amphetamine‐type stimulant on the illicit drug market [19], while methamphetamine and MDMA (ecstasy) each accounted for only a small percentage. A complicating factor in the interpretation of such drug test results is that amphetamine also occurs as a main metabolite of methamphetamine, along with parahydroxymethamphetamine [13]. At relatively high urinary methamphetamine concentrations, the amphetamine metabolite level typically constitutes less than 10%; but as elimination progresses, the proportion gradually increases and may eventually surpass that of methamphetamine [13, 22]. However, at relatively high substance concentrations where the methamphetamine level exceeds that of amphetamine, this suggests primary methamphetamine use [23]. Moreover, the possibility of polydrug use [24, 25], intentionally or unintentionally, cannot be excluded. A methamphetamine proportion exceeding 10% may indicate use of both drugs [26].

An additional finding was that about one‐third (31%) of the amphetamine‐positive urine samples also contained low amounts of methamphetamine. The relative proportion was typically less than 1%; but even so, due to the often very high amphetamine concentrations, the methamphetamine level occasionally exceeded the reporting limit of 200 μg/L, requiring clinical evaluation. However, low levels of methamphetamine may not necessarily indicate intentional polydrug use but could result from impurities in illicit amphetamine produced in clandestine laboratories [27].

While illegal street amphetamine is typically found as a racemic mixture, methamphetamine is usually encountered as enantiopure materials [28]. Analysis of methamphetamine seizures in Sweden revealed the presence of either the l‐ or d‐enantiomer (each ~97%–100% pure) but not a racemic mixture (personal communication; Swedish National Forensic Centre, Linköping, Sweden). This is consistent with the current findings from the chiral analysis, and it also provides further evidence that the amphetamines do not undergo racemization [28, 29, 30]. Furthermore, if methamphetamine is not analyzed alongside amphetamine, the ingestion of d‐methamphetamine, which then metabolizes into d‐amphetamine, can be mistakenly confused with the use of d‐amphetamine‐based ADHD medications. This issue may become more pronounced in Europe if methamphetamine use continues to rise, in parallel with the increasing ADHD diagnoses and a growing prescription of amphetamine‐based medications [31].

The findings of low levels of methamphetamine in many amphetamine‐positive samples may be attributed to a minor metabolic pathway involving N‐methylation of amphetamine [32, 33]; although this has not been unequivocally confirmed in more recent studies [27]. Nevertheless, the present findings supported this possibility, as urine samples from ADHD patients treated with d‐amphetamine‐based medications (e.g., Elvanse or Attentin) [16] contained low levels of d‐methamphetamine, despite the absence of any detectable methamphetamine in the pharmaceutical products. Similarly, the findings of low levels of racemic methamphetamine following racemic amphetamine use may also be attributed to an endogenous N‐methylation process.

5. Conclusion

The findings of the present study confirmed that amphetamine remains the predominant amphetamine‐type substance in Sweden, with methamphetamine constituting only a small proportion. Nonetheless, low levels of methamphetamine are regularly detected in amphetamine‐positive drug test samples. In complementary chiral analysis, the enantiomeric distribution of methamphetamine was demonstrated to parallel that of amphetamine after the use of racemic street amphetamine. Additionally, only d‐methamphetamine was detected in urine samples from ADHD patients treated with d‐amphetamine, despite no methamphetamine being found in the pharmaceutical products. These results supported that N‐methylation of amphetamine represents a minor, albeit quantitatively insignificant, metabolic pathway in the human body (Figure 3).

FIGURE 3.

The main metabolic pathway for N‐demethylation of methamphetamine to amphetamine, and the proposed minor pathway for N‐methylation of amphetamine to methamphetamine in the human body.

Consequently, the presence of methamphetamine concentrations above the reporting limit, but constituting only a small proportion (e.g., < 1%) of the amphetamine level, does not necessarily reflect polydrug use. Instead, trace levels of methamphetamine may result from contamination during illicit amphetamine production, and as demonstrated here from the metabolic methylation of amphetamine. Therefore, the presence of a trace methamphetamine concentration relative to the amphetamine level does not warrant clinical or forensic concern. These findings further support that chiral analysis of both amphetamine and methamphetamine is an effective approach for distinguishing between illicit and therapeutic sources in positive drug screening tests for the amphetamines.

Conflicts of Interest

The authors declare no conflicts of interest.

Helander A., Andersson A., and Villén T., “Origin and Interpretation of Low Methamphetamine Levels Found in Amphetamine‐Positive Urine Samples: Support for Methylation of Amphetamine as a Minor Metabolic Pathway,” Drug Testing and Analysis 17, no. 11 (2025): 2276–2282, 10.1002/dta.3940.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.