Alternative routine for reporting chiral amphetamine test results in assessment of attention‐deficit/hyperactivity disorder medication: experiences from 2013 to 2023

Abstract

This study evaluated an alternative routine for reporting urinary chiral amphetamine results in assessment of attention‐deficit/hyperactivity disorder (ADHD) treatment with amphetamine medications and for detecting side‐use of illicit racemic amphetamine. Currently in Sweden, only enantiopure d‐amphetamine‐based ADHD medications (lisdexamphetamine dimesylate and dexamphetamine sulfate) are approved. It is therefore unsuitable to express the chiral result as the l/d‐ratio, as before, because l‐amphetamine should not be present provided treatment compliance. A new routine for LC‐MS/MS chiral amphetamine testing was therefore introduced in 2020, whereby the relative proportion (%) of l‐amphetamine and the total amphetamine and creatinine concentrations are reported. Evaluation of the new routine on 24,354 results from 2013 to 2023 revealed that it was useful to distinguish ADHD medication adherence from illicit drug use as the source for a positive test. The l‐amphetamine proportion also reflected the enantiomeric content of the medications used. Overall, most results confirmed adherence to ADHD medication, as the l‐amphetamine percentage was <1% in 76% of samples (2023) which is the recommended cutoff with enantiopure d‐amphetamine medications. However, in all years, illicit drug use was indicated (>40% l‐amphetamine) in 8.3%–14.5% of cases. In conclusion, this study demonstrated the clinical value and utility of a new routine for reporting urinary chiral amphetamine results to differentiate adherence to ADHD medication from illicit drug use. Unlike the l/d‐amphetamine ratio, it considers differences in total amphetamine concentration and urine dilution, factors that can affect the interpretation.

The study describes experiences with a new routine for reporting chiral amphetamine test results that is adapted to the currently used d‐amphetamine‐based ADHD drugs. Instead of the l/d‐ratio, which was previously used, the percentage (%) of l‐amphetamine and the total amphetamine and creatinine concentrations are now reported.

1. INTRODUCTION

Diagnoses and prescriptions for attention‐deficit/hyperactivity disorder (ADHD) have shown a constant increase in recent decades, which can be linked to a broadened clinical diagnosis and a general increased knowledge and awareness of the condition. ¹ , ² , ³ The first‐line agents in ADHD pharmacotherapy are central nervous system stimulants that work by increasing dopamine and norepinephrine levels, leading to increased attention, improved concentration, and reduced impulsivity and hyperactivity. Methylphenidate is the most common agent, ⁴ but amphetamine‐based medications are also frequently used. ⁵

Having a stimulant prescription does not eliminate the risk for side use of illicit drugs, and medication nonadherence and diversion are also problems that need to be controlled. ⁶ , ⁷ , ⁸ Laboratory tests on urine, blood, or oral fluid samples are useful to rule out substance abuse before starting ADHD medication and for dose adjustment and assessment of compliance during treatment. With methylphenidate, the parent compound or its metabolite ritalinic acid can be monitored. ⁹ With amphetamine, testing is more challenging because it exists as two enantiomers, dextroamphetamine (also called d‐ or S‐(+)‐amphetamine) and levoamphetamine (l‐ or R‐(−)‐amphetamine). Amphetamine medications for ADHD are predominantly based on d‐amphetamine, which has higher stimulant effect, ¹⁰ , ¹¹ either in the form of short‐acting salts or slow‐release lisdexamphetamine prodrug. ⁶ , ¹¹ Illicit “street” amphetamine, on the other hand, is a racemate containing equal parts of each enantiomer. Stereoselective (chiral) amphetamine analysis can therefore be used to distinguish between adherence to the prescribed ADHD medication or recreational illicit drug use as the source for a positive amphetamine drug test. ¹² , ¹³ , ¹⁴ , ¹⁵

The test results from chiral amphetamine analysis were originally reported as the l/d‐amphetamine ratio, since the medications in question at the time contained both enantiomers. ¹⁶ , ¹⁷ Today in Sweden, only enantiopure d‐amphetamine‐based formulations are approved as prescription drugs for ADHD, whereas mixtures of d‐ and l‐amphetamine are available under license but hence rarely used. ³ Consequently, it is no longer suitable to express the chiral test result as the enantiomer ratio since the numerator (l‐amphetamine) is mostly not expected to be present. In addition, depending on the l/d‐ratio cutoff used, there is a possibility of masking a previous side intake of illicit amphetamine by intake of d‐amphetamine‐based medication shortly before testing. The maximum amphetamine concentration in blood is reached within ~3–4 h, even for slow‐release formulations, and with a half‐life of ~10–12 h, the urinary concentration remains high in the following day. ¹⁶ , ¹⁸ , ¹⁹ , ²⁰ Furthermore, if the urine is diluted through excess fluid intake before testing, ²¹ the l‐amphetamine concentration may fall below the quantification limit.

To overcome the disadvantages of using the enantiomer ratio to monitor patient compliance during treatment with enantiopure d‐amphetamine‐based medications, a new routine for chiral amphetamine analysis that focused on the absence, or presence, of l‐amphetamine was introduced in Sweden in 2020. ²² The recommendation was to report the relative amount (%) of l‐amphetamine, along with the total amphetamine and the creatinine concentration. An external quality assessment scheme with the same analytical targets was also started (Equalis, Uppsala, Sweden).

This report presents results and experiences from chiral amphetamine analysis in urine, based on the new analytical recommendation which was applied on results for patient samples submitted for routine testing over the past decade at the Karolinska University Laboratory (Stockholm, Sweden).

2. METHODS

2.1. Clinical samples and chemicals

The study is based on test results from routine urinary chiral amphetamine analysis, since the start in March 2013 until December 2023, at the Department of Clinical Pharmacology, Karolinska University Laboratory (Stockholm, Sweden). The origin of the samples and the reason for ordering a chiral analysis were unknown.

Until 2019, all urine samples first underwent immunoassay screening with the CEDIA Amphetamine/Ecstasy assay (Thermo Fisher Scientific, Fremont, CA, USA) that targets d‐amphetamine. Since 2020, the samples are tested directly with a chiral liquid chromatographic–tandem mass spectrometric method (LC‐MS/MS).

Reference materials for d‐ and l‐amphetamine (i.e., S‐(+)‐amphetamine and R‐(−)‐amphetamine, respectively) and the internal standard (IS; racemic (±)‐amphetamine‐d₅) and the corresponding materials for methamphetamine were obtained from Cerilliant Co. (Round Rock, TX, USA). All other chemicals were of analytical grade and the water was of HPLC grade. The reference materials were obtained as methanol solutions, and dilutions were made in 0.1% formic acid in water.

Statistical calculations were made with the MedCalc software.

2.2. Chiral LC‐MS/MS analysis of l‐ and d‐amphetamine

The patient urine samples, as well as calibrators, controls and blanks prepared in drug‐negative urine, were mixed for 30 s with an equal volume of IS solution in water, centrifuged at ~2000 g for 5 min, and 2 μL of the supernatant was injected. Chromatographic baseline separation of l‐ and d‐amphetamine (Figure 1) was achieved on a Chiralpak CBH column (2.1 × 100 mm, particle size 5 μm; Sigma‐Aldrich, Saint Louis, MO, USA) using isocratic elution with a mobile phase of 95% 10 mmol/L ammonium acetate in water and 5% acetonitrile at 0.2 mL/min, on an AQUITY UPLC system (Waters, Milford, MA, USA) coupled to a Xevo TQ‐S micro MS (Waters).

FIGURE 1.

Selected chromatograms from the routine urinary chiral amphetamine (Amph; top) analysis. The ion transitions monitored for l‐ and d‐amphetamine were m/z 136.05 > 91.00 (quantifier), m/z 136.05 > 119.10 (qualifier), and m/z 141.10 > 93.05 for racemic amphetamine‐d5 (IS). The method also allowed for partial separation and identification of l‐ and d‐methamphetamine (Meth; bottom), using m/z 150.20 > 91.00 (quantifier), m/z 150.20 > 119.10 (qualifier), and m/z 155.09 > 121.08 for racemic methamphetamine‐d5 (IS). (a) A urine sample containing ~8000 μg/L d‐amphetamine and no l‐amphetamine collected from an ADHD patient treated with lisdexamphetamine (Elvanse); (b) A urine sample containing ~6000 μg/L l‐amphetamine and ~3000 μg/L d‐amphetamine indicating use of illicit racemic amphetamine (the sample also contained traces, ~1.4%, of racemic methamphetamine). (c) A urine sample containing ~4200 μg/L d‐methamphetamine, ~120 μg/L l‐amphetamine, and ~720 μg/L d‐amphetamine, indicating use of illicit d‐methamphetamine and illicit racemic amphetamine. The methamphetamine positive sample (c) derived from a different run, which explains the different separation between enantiomers.

The MS instrument was operated in positive mode and the ion transitions monitored for l‐ and d‐amphetamine were m/z 136.05 > 91.00 (quantifier), m/z 136.05 > 119.10 (qualifier), and m/z 141.10 > 93.05 for amphetamine‐d₅ (IS). The method also enabled partial separation and identification of l‐ and d‐methamphetamine, using the ion transitions m/z 150.20 > 91.00 (quantifier), m/z 150.20 > 119.10 (qualifier), and m/z 155.09 > 121.08 for methamphetamine‐d5 (IS). The methamphetamine enantiomers were not baseline separated (Figure 1), but peaks were always separable (Δ ~ 0.15–0.20 min) and results were reproducible within each run.

The detection limit for each enantiomer was ~1 μg/L, the applied lower quantification limit (LLOQ) 10 μg/L, and the measuring range 10–25,000 μg/L. If the substance concentration in the sample exceeded the measuring range, it was reanalyzed after dilution. In the system suitability test, acceptance criteria for a positive identification were a relative retention time (RRT) versus calibrator within ±0.5% and ion ratios within ±20%. The method is validated and accredited for the measurement of amphetamine enantiomers.

2.3. New routine for analysis and reporting of chiral amphetamine

With adherence to the currently approved d‐amphetamine‐based ADHD medications, that is, lisdexamphetamine dimesylate (Elvanse in Europe; Vyvanse in North America) and dexamphetamine sulfate (Attentin), l‐amphetamine should not be found in the sample. The recommended routine cutoff for a passing test result was set at <1% l‐amphetamine to exclude the risk of trace levels being present in the medication. ²² Consequently, with an LLOQ at 10 μg/L, the <1% cutoff is applicable down to 1000 μg/L total amphetamine concentration, whereas a slightly higher cut‐off must be employed at lower total concentrations. However, use of illegal racemic amphetamine can be detected at concentrations down to around the LLOQ.

3. RESULTS

During the study period of 2013–2023, a total of 24,354 routine patient urine samples were analyzed with the LC‐MS/MS chiral amphetamine method at the Karolinska University Laboratory. The yearly number of tests has shown a steady increase from 242 in 2013, the first year the analysis was provided, to 4677 in 2023 (i.e., almost 20‐fold).

Selected yearly results from the chiral amphetamine analysis in urine samples, applying the new analytical and reporting routine, are shown in Figure 2. In the first year (2013), most samples (~75%) contained <10% (median 6.0%) l‐amphetamine (Figure 3). The accumulation if results near ~5% l‐amphetamine resulted from ADHD treatment with a predominantly d‐amphetamine sulfate‐based formulation (Metamina) that was prescribed on license, while a few contained <1% l‐amphetamine which resulted from enantiopure d‐amphetamine medications, which then started to be used. The other samples contained 10%–77% l‐amphetamine indicating use of illicit racemic amphetamine or mixed intake of ADHD medication and illicit drug. In 2014 (Figure 2), in addition to a continued use of Metamina, the frequency of samples that contained only d‐amphetamine started to increase. Since 2016 (Figure 2) and onwards, the chiral test results have confirmed a main use of the approved enantiopure d‐amphetamine‐based medications, that is, either short‐acting d‐amphetamine sulfate (Attentin) or, mainly, slow‐release lisdexamphetamine dimesylate (Elvanse). In 2023 (Figures 2 and 3), 76% of the urine samples contained <1% l‐amphetamine, whereas the other samples contained 1%–88% l‐amphetamine, which indicated illicit drug use or mixed medication and illicit drug use. In all years, illicit amphetamine use was indicated (i.e., >40% l‐amphetamine) in ~10% (mean 10.4%, range 8.3%–14.5%) of the urine samples.

FIGURE 2.

Distribution of the relative proportion (%) of l‐amphetamine to total amphetamine (l + d) concentration in urine samples submitted for routine LC‐MS/MS chiral amphetamine analysis in the years 2013 (N = 242), 2014 (N = 249), 2016 (N = 1133), and 2023 (N = 4677; data from the Karolinska University Laboratory, Stockholm, Sweden). In 2013, the accumulation of samples containing ~5% l‐amphetamine was due to ADHD treatment with a licensed drug with the corresponding enantiomeric content (Metamina). From 2013 to 2014 onwards, ADHD treatment switched to enantiopure d‐amphetamine‐based medications (Elvanse and Attentin), which are currently the only approved ones. During all years, ~10% (range 8.3%–14.5%) of the test results indicated illicit racemic drug use (i.e., >40% l‐amphetamine) and others a mix of ADHD medication and illicit drug use.

FIGURE 3.

Comparison of the relative proportion (%) of l‐amphetamine in urine samples submitted for routine LC‐MS/MS chiral analysis in 2013 (N = 242; data from the Karolinska University Laboratory, Stockholm, Sweden), when most ADHD patients in Sweden were using a licensed medication containing ~5% l‐amphetamine (Metamina) and in 2023 (N = 4677) when only enantiopure d‐amphetamine‐based medications (Elvanse and Attentin) were approved.

A tendency for a negative association between the relative proportion of l‐amphetamine and the total amphetamine concentration was noted in Figure 2 for test values appearing to result from illicit amphetamine use (i.e., containing >40% of the l‐enantiomer). This was further evident when plotting the results for all such samples from 2013 to 2023 (Figure 4). The median proportion of l‐amphetamine was gradually lower in the subgroups containing <5000 μg/L (61.2% l‐amphetamine) versus 5000–10,000 μg/L (57.2%), 10,000–50,000 μg/L (54.3%), and >50,000 μg/L (53.5%) total amphetamine (p < 0.0001 for all; Mann–Whitney test). Furthermore, in the urine samples with the highest proportions of l‐amphetamine (>70%; N = 192), the total amphetamine concentration was considerably lower (median 1527 μg/L) than for those with a near‐equal enantiomer proportion (i.e., 40%–60% of each; median 28,956 μg/L, N = 1645; p < 0.0001). For comparison, in the urine samples containing <1% l‐amphetamine, which supported compliance with ADHD treatment, the total amphetamine median concentration was 4820 μg/L (N = 18,235).

FIGURE 4.

Negative association between the relative proportion (%) of l‐amphetamine and the total amphetamine (l + d) concentration in urine samples submitted for routine LC‐MS/MS chiral amphetamine analysis and indicating use of illicit racemic amphetamine (i.e., >40% l‐amphetamine).

4. DISCUSSION

Parallel to the steady increase in diagnoses and pharmacological treatment of ADHD, the demand for chiral analysis of amphetamine, which is used as an alternative second‐line medication alongside methylphenidate, has increased considerably, as demonstrated in this Swedish study covering data from 2013 to 2023. In addition to being valuable in clinical decision‐making to investigate treatment compliance, the chiral analysis provides the opportunity to also detect recreational side use of illicit racemic amphetamine.

In Sweden, the transition to approving only enantiopure d‐amphetamine‐based ADHD medications meant that it was no longer suitable to report the chiral test result as the l/d‐enantiomer ratio. This is because the numerator (l‐amphetamine) is no longer expected to be present in the samples at treatment compliance, but its presence rather confirms illicit racemic amphetamine use. It was therefore recommended that the routine for reporting chiral test results should instead focus on the absence, or presence, of l‐amphetamine and on factors that can affect test interpretation, that is, the total amphetamine concentration and the dilution of the urine sample as determined from the creatinine concentration. ²² Dilution of the urine and, hence, of substance levels in the sample is a common way of trying to deceive drug testing. However, the l/d‐enantiomer ratio takes neither the urine dilution nor the total amphetamine concentration into account, as long as both analytes stay within the measuring range, which is an important disadvantage of this routine.

Reporting the total amphetamine concentration is valuable for the interpretation of chiral test results, as the enantiomer distribution varies over time after last intake. This occurs because the elimination is stereoselective with d‐amphetamine being metabolized somewhat faster (the half‐life is typically ~1–2 h shorter) than l‐amphetamine. ¹¹ , ²³ Consequently, after using racemic amphetamine, the relative proportion of l‐amphetamine will gradually increase until the substance is no longer detectable. This was particularly evident in one sample in the present study that only contained l‐amphetamine at a low concentration (7 μg/L) below the routine quantification limit. The time‐dependent shift in enantiomer elimination was also apparent from the observation that the samples with the highest relative proportions of l‐amphetamine (>70%) coincided with a lower total amphetamine concentration (Figure 4). Furthermore, for all urine samples indicated to result from illicit amphetamine use (i.e., containing >40% l‐amphetamine), the median proportion of l‐amphetamine was gradually lower in the subgroups with gradually higher total amphetamine concentrations, albeit with considerable overlaps, eventually approaching the expected racemic (~1:1) composition at the highest concentrations that indicated recent use.

It should be noted that a near even enantiomeric composition despite a low total amphetamine concentration may be due to urine dilution through excess fluid intake prior to sampling. However, in 50 randomly selected samples with a very low total amphetamine concentration (<100 μg/L), only one showed a creatinine concentration <2 mmol/L (range 1.0–29.6, median 6.8 mmol/L), the routine cutoff to indicate urine dilution, which is often used to try to avoid detection in drug testing. ²¹ Another factor to be considered in the assessment of a chiral test result is that the amphetamine excretion rate is dramatically influenced by urinary pH, with very little excretion occurring in highly alkaline urine and vice versa in acidic urine. ¹¹ , ²⁴

The urinary relative proportion of l‐amphetamine corresponds to the enantiomeric content in the medication used, which facilitates clinical interpretation. Overall, most chiral test results from 2013 to 2023 have supported compliance with the ADHD medication in use, and the shift from a medication containing ~5% l‐amphetamine (Metamina) to the currently approved enantiopure d‐amphetamine‐based formulations, which started in 2013–2014 (Figure 2), was evident from the yearly results. However, side use of illegal racemic amphetamine has also been demonstrated to be relatively common (~10%) in all years.

When chiral amphetamine analysis is used to identify the likely source of a positive amphetamine drug test, it should be considered that there is still some use in Sweden of licensed formulations containing different proportions (~5%–50%) of l‐amphetamine. ¹¹ , ²³ Starting new treatments with the licensed formulations is not recommended, ²⁵ as assessment of treatment compliance by chiral analysis becomes more problematic, and their use has therefore gradually declined. In 2013, ~2500 licenses for Metamina were granted, while in 2022, the licensed amphetamine formulations together accounted for only 0.6% of all ADHD stimulant prescriptions (there were 708 licenses for a racemic formulation named “Amfetamin 5 mg,” 212 for Metamina containing ~5% l‐amphetamine, and 46 for Adderall containing ~25% l‐amphetamine).

The LC‐MS/MS method for chiral amphetamine analysis in urine also enabled partial separation and identification of the methamphetamine enantiomers. This is a clinical advantage when evaluating the origin of amphetamine in a sample, since methamphetamine is to some extent (<10%) demethylated to amphetamine in the body and eventually excreted in the urine. ²⁶ , ²⁷ Illicit methamphetamine, although currently very rarely used in Sweden, ²⁸ is primarily produced from ephedrine or pseudoephedrine, which yields the d‐enantiomer, and because the enantiomer composition will not change during metabolism, only d‐amphetamine will be found (Figure 1).

It should also be noted that some prescribed drugs can be converted to amphetamine or methamphetamine in the body (e.g., amfetaminil, benzphetamine, clobenzorex, famprofazone, fenethylline, fenproporex, and prenylamine), eventually resulting in urinary excretion of amphetamine, which may complicate interpretation of a chiral test result. ²⁶ , ²⁹ However, most of these are no longer approved but have been withdrawn from the market due to the abuse potential. An exception is selegiline (deprenyl), used in the treatment of Parkinson's disease, which is metabolized to l‐methamphetamine and further to l‐amphetamine. ³⁰

5. CONCLUSION

In conclusion, the results of this study demonstrated the clinical value and utility of a new test routine for chiral amphetamine analysis in urine to differentiate adherence to ADHD medication with enantiopure d‐amphetamine‐based formulations from recreational illicit racemic drug use. Unlike the l/d‐amphetamine ratio, which was previously used, the new routine takes into account differences in the total amphetamine concentration and in urine dilution, both of which are factors that can affect the interpretation of the test result.

Helander A, Andersson A, Villén T. Alternative routine for reporting chiral amphetamine test results in assessment of attention‐deficit/hyperactivity disorder medication: experiences from 2013 to 2023. Drug Test Anal. 2025;17(1):163‐169. doi: 10.1002/dta.3690