Compliance testing of patients in ADHD treatment with lisdexamphetamine (Elvanse®) using oral fluid as specimen

Highlights

• •Using oral fluid as a possible specimen for compliance monitoring during amphetamine treatment of ADHD.
• •Documentation of oral fluid concentrations of amphetamine and the prodrug lisdexamphetamine.
• •Ultrasensitive LC-MS/MS method for lisdexamphetamine.
• •Chiral analysis of amphetamine in oral fluid.

Abstract

Pharmacological treatment of the attention-deficit/hyperactivity disorder (ADHD) includes use of the psychostimulant amphetamine. Non-adherence to medication is a well-documented problem in ADHD treatment and a cause of treatment failure. The study evaluated the possibility of using oral fluid for compliance monitoring during treatment with lisdexamphetamine (Elvanse®). UPLC-MS/MS methods for general oral fluid drug testing, lisdexamphetamine and amphetamine quantification and chiral analysis of amphetamine were used. The applied measuring ranges were 1–500 ng/mL for amphetamine and 0.01–15 ng/mL for lisdexamphetamine. Amphetamine (racemic) was detected and quantified in 98 (96%) of the 102 samples. The concentrations ranged from 2 to 8410 ng/mL. In 17 of these, the chiral analysis demonstrated intake of illicit amphetamine because L-amphetamine was present. The median D- + L-amphetamine concentration in the compliant group was 280 ng/mL, while the median concentration in the non-compliant group was statistically higher, 1677 ng/mL. In the non-compliant cases where L-amphetamine was detected, the L/D-amphetamine ratios ranged from 0.75 to 13.1 with a median of 1.0. Lisdexamphetamine was detected and quantified in 76 of the 102 cases, which represent 79% of the 98 cases with detected oral fluid amphetamine. The concentrations ranged from 0.01 to 6895 ng/mL. Drug testing had a positive rate of 23% in patients not taking illicit amphetamine and 82% among non-compliant patients with detected L-amphetamine. In conclusion, the study demonstrated the value of measuring amphetamine with a chiral method to detect intake of illicit amphetamine and to perform drug testing in oral fluid as a mean for compliance monitoring.

1. Introduction

Pharmacological treatment of the attention-deficit/hyperactivity disorder (ADHD) includes the use of psychostimulants, e.g. amphetamine [1]. The prevalence of this disorder in adults is close to 5% [2], [3] and it has received attention as a global health burden [4]. The etiology of ADHD has been suggested to involve a need for increased brain dopamine and norepinephrine neurotransmitter activity, which underlies the rationale for treating the condition with a psychostimulant [1]. As most psychostimulants have addictive properties it is important to closely monitor the pharmacological treatment. Non-adherence to medication is a well-documented problem in ADHD treatment and is a major cause of treatment failure [5]. When treating patients with a history of a substance use disorder with psychostimulants it is important not only to control compliance to the prescribed medication, but also to monitor abstention of using illicit amphetamine and other illicit or non-prescribed drugs.

Laboratory based testing of ADHD patients treated with a psychostimulant has, therefore, the dual purpose of confirming compliance to prescribed medication and the abstention from taking medically non-motivated psychoactive drugs. Therapeutic drug monitoring, which is usually performed in blood, plasma or serum, is used for guiding individualized dosing, but this is not recommended at the moment and not in practice for ADHD treatment [6]. Urine has remained the standard matrix for drug testing in most clinical settings [7], but oral fluid (OF) is an established alternative [8], [9]. In psychiatric patients both venous blood sampling and supervised urine sampling might be contraindicated due to the invasive and indiscreet nature of the procedures. Recently, OF has, therefore, attracted interest as an alternative and less intrusive matrix [8].

It is generally assumed that OF drug concentrations are closely related to blood concentrations and reflect the nonprotein bound (“free”) fraction of the drug in serum [10]. It has recently been concluded, however, that OF cannot simply replace blood/serum/plasma for estimating drug concentration levels because the mechanism for a substance to appear in the oral cavity is more complex than a close relationship [11]. OF is rather a specimen suitable for compliance testing [12].

Lisdexamfetamine mesylate/mesilate (LDX, Elvanse®, Venvanse®, Vyvanse®, Samexid®, Tyvense®) is a long acting oral prodrug stimulant indicated for the treatment of ADHD. Conversion of LDX mesylate into the free LDX form in the circulation and, subsequently, into the therapeutically active D-amphetamine and lysine, occurs primarily in red blood cells [13], and might be influenced by inter-individual differences, e.g. the hematocrit value. Compliance testing of LDX patients should target illicit amphetamine use by chiral analysis and, ideally, also the prodrug LDX. However, plasma/serum concentrations of LDX are known to be low, <1 ng/mL, and the elimination rate is fast, making it analytically challenging to detect at the end of the dose interval [14], [15], [16]. Because of the low serum concentrations of LDX, the LDX concentrations in OF were expected to be in a low range despite its alkaline pKa value of 10.2 [17]. Substances with alkaline pKa are known to have OF to plasma concentration ratios above 1 [18], [19].

The aim of the present work was to evaluate the possibility of using oral fluid for compliance monitoring during treatment with Elvanse® in an adult patient population with combined ADHD and substance use disorder. In addition to established methods for OF drug testing and amphetamine quantification, we, therefore, developed both a sensitive LDX and a chiral amphetamine ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS) method for this purpose.

2. Materials and methods

2.1. Chemicals

Amphetamine and amphetamine-d5 were from LGC Standards GmbH (Wesel, Germany); D-amphetamine was from Lipomed GmbH (Weil am Rhein, Germany); LDX and LDX-d4 were from Cerriliant Co (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)

Methanol (MeOH), acetonitrile (AcCN), ammonium hydroxide (32%), formic acid, and water of LC-MS grade were from Biosolve BV (Valkenswaard, Holland). Ammonium trifluoroacetate and ammonium acetate of LC-MS grade were from Sigma-Aldrich Chemie; 2-propanol and dichloroethane of p.a. grade were from Carl Roth GmbH (Karlsruhe, Germany); methylene chloride, ethyl acetate, heptane, sodium carbonate and sodium bicarbonate of p.a. grade were from Merck KGaA (Darmstadt, Germany).

2.2. Preparation of oral fluid standards and controls

Standards for calibration were prepared in a matrix made of 50% artificial oral fluid in Greiner SES (saliva extraction solution) buffer (Greiner Bio-One GmbH, Kremsmünster, Austria). Controls were prepared in a matrix made of ∼50% authentic pooled blank saliva collected from staff in Greiner SES buffer. For amphetamine, analytical standards were prepared at 20 concentration levels in the range of 0.1–500 ng/mL. Controls were prepared at 3.5, 6.1 and 15.7 ng/mL. For LDX, the standards were prepared at 24 concentration levels in the range of 0.1–15 ng/mL. In addition, in the lower concentration range, 10 concentration levels between 5 and 50 pg/mL were used.

Calibration curves were constructed using linear regression analysis, with weighting factor 1/x.

2.3. Clinical samples

The study comprised 102 OF samples from 70 patients on Elvanse® therapy, which were sent to the laboratory with a request for routine chiral analysis of amphetamine, which comprised quantification of amphetamine and chiral analysis to estimate the relative proportion of L- and D-amphetamine. Most of the requests also comprised drug testing with a method with 66 analytes (Supplemental Table 1). The LDX dose was reported in 29 cases and ranged from 30 to 140 mg/day, with a mean value of 68 mg. In seven cases the daily dose was >70 mg and in six cases the dose was taken two times per day. The highest recommended daily dose is 70 mg. Following the requested service, a surplus second aliquot of the sample was used for LDX analysis after anonymizing the samples.

2.4. Sampling of oral fluid

The oral fluid specimen was collected using the Greiner-Bio-One SCS device described in detail at the company website [20]. In this sampling system, a 4 mL SES buffer solution containing a food dye is kept in the mouth for at least 2 min. The resulting sample is collected in the collection cup and finally transferred into two vacuum tubes [20], [21]. The SES solution is buffered to a pH of 4.2, which is intended to stimulate saliva production. The samples were sent to the laboratory via the postal service and immediately analysed or stored at +4 °C.

The degree of OF dilution and fraction of neat saliva, %saliva, was determined spectrophotometrically on an Olympus AU680 instrument (Beckman Coulter Inc, Krefeld, Germany) using the Saliva Quantification kit from Greiner Bio-One. Analyte concentrations in neat saliva were determined using the measured OF dilution factor. The neat saliva content was between 7% and 79%, with a mean of 54%. The uncertainty in measuring %saliva was <6%, with a bias of <1%.

2.5. Analysis of amphetamine in oral fluid (Method 1)

A 20 μL aliquot of OF sample was mixed with 400 μL acetonitrile containing internal standard (50 pg amphetamine-d5) and 15 μL of 10 M ammonium acetate, followed by centrifugation for phase separation. The upper acetonitrile layer was transferred to a 96 deep well plate and evaporated to dryness by vacuum centrifugation (at 25 °C for 30 min), and the residue was finally re-dissolved in 100 μL mobile phase A. The injection volume into the LC-MS/MS system was 5 μL.

The analytical column was a Waters (Waters Co, Milford, MA, USA) HSS C18 1.7 μm, 2.1 × 150 mm column kept at 50 °C with a flow rate of 0.4 mL/min. The chromatography system used an 8.5 min gradient elution program with mobile phase A being 20 mM ammonium formate and 0.1% formic acid, pH 3 and mobile phase B being MeOH with 0.1% formic acid). Gradient program: 0% B from 0 to 0.50 min, 15–23% B from 0.51 to 5.20 min, 23–55% B from 5.21 to 6.50 min, 100% B from 6.51 to 8.00 min, 0% B 8.01–8.50 min.

This method is accredited according to ISO17025 for forensic drug testing.

2.6. Analysis of lisdexamphetamine in oral fluid (Method 2)

A 100 μL aliquot of OF sample was mixed with 10 μL MeOH containing internal standard (1.0 ng LDX-d4), 10 μL NH4 OH (32%), 400 μL AcCN and 50 μL of 10 M ammonium acetate, followed by centrifugation for phase separation. The upper AcCN layer was transferred to a 96 well plate with glass inserts and evaporated to dryness by vacuum centrifugation, and the residue was finally redissolved in 100 μL mobile phase A. The injection volume into the LC-MS/MS system was 1 μL. The vendor lists the precision of this injection volume to be <1%. The use of an internal standard compensates for minor variations in injection volume.

The chromatography system was the same as for Method 1, but with shorter analysis time. Gradient program: 0% B from 0 to 0.50 min, 15–23% B from 0.51 to 3.50 min, 100% B from 3.51 to 4.50 min, 0% B 4.51–5.0 min. The total time between injections was 20 min.

2.7. Chiral analysis of amphetamine in oral fluid samples (Method 3)

A 200 μL aliquot of OF was mixed with 5 μL MeOH containing internal standard (10 ng amphetamine-d5), 200 μL of 0.06 M phosphate buffer, 40 mg sodium carbonate and 40 mg sodium bicarbonate (pH 9). The mixture was shaken with 1 mL of an extraction solvent (consisting of 240 mL 2-propanol, 360 mL methylene chloride, 315 mL dichlorethylene, 570 mL n-heptane, 248 mL acetonitrile and 248 mL ethyl acetate). After mixing and centrifugation for phase separation, the organic phase (∼0.9 mL) was transferred to a glass vial and evaporated to dryness by vacuum centrifugation. The residue was redissolved in 100 μL mobile phase A. The injection volume into the LC-MS/MS system was 1 μL.

The analytical column was a SUPELCO Astec CHIROBIOTIC® V2 5 μm, 4.6 × 250 mm (Sigma-Aldrich Chemie) kept at 25 °C with a flow rate of 0.3 mL/min. The chromatography system used isocratic elution with the mobile phase being 95% MeOH with 3.05 mM ammonium trifluoroacetate.

2.8. Testing for other drugs in OF

Supplemental Table 1 presents the other substances tested for when requested and the applied reporting limits. The method is in routine use under ISO 15189 accreditation and has been presented at the SOFT 2012 and TIAFT 2013 meetings.

2.9. Instrument

A Waters Acquity/Xevo® TQ-S UPLC-MS/MS system was used, which operated in positive electrospray ionization and SRM mode with 3 transitions monitored per analyte and internal standard. Capillary voltage was set to 0.5 kV, ion source temperature was 150 °C, and desolvation gas was heated to 650 °C and delivered at a flow rate of 1000 L/h. Cone gas flow (N₂) was 150 L/h and the collision gas flow (Ar) was 0.22 mL/min. The monitored ions, collision energies and dwell times are given in Table 1. The system was operated using the MassLynx™ Software version 4.1. The two qualifier ions were to secure the identification of the analyte. Both peak area ratios of qualifiers to quantifier had to be within ±20% of the target value. Needle wash was done with methanol, 1 mL pre-injection and 2 mL post-injection.

Table 1.

Mass spectrometry parameters.

Analyte	Precursor ion m/z	Product ions m/z	Collision energy eV	Dwell time ms
	Method 1
Amphetamine	136.1	91.0	14	4
		119.0	8	4
		65.0	25	4
Amphetamine-d5	141.1	92.8	14	4
		124.0	8	4
		66.2	30	4
	Method 2
LDX	264.2	84.1	20	47
		129.1	12	47
		119.1	22	47
LDX-d4	268.2	88.1	20	47
		133.1	12	47
		119.1	22	47
	Method 3
Amphetamine	136.1	91.0	14	30
		119.0	8	30
		65.0	25	30
Amphetamine-d5	141.1	92.8	14	30
		124.0	8	30
		66.2	30	30

2.10. Method validation

The linearity of analyte concentration to the analyte/internal standard peak area ratio was evaluated in the concentration ranges 0.5–500 ng/mL for amphetamine and 0.005–15 ng/mL for LDX. Because of the wide ranges, the calculations were done in two parts, 0.5–50 and 5–500 ng/mL, for amphetamine, and three parts, 0.005–0.05, 0.1–1 and 1–15 ng/mL, for LDX. Quantification uncertainty and accuracy was documented for amphetamine using the quality controls in 3 concentrations in each batch (between days). For LDX an experiment was performed, with triplicate determinations over 10 days, with controls made at 1.0 and 10.0 ng/mL using a blank OF sample made from authentic saliva (64%). Uncertainty in measuring L/D-amphetamine peak area ratio was documented by analysing a test solution containing 100 ng/mL racemic amphetamine in each batch. Limits of detection (LOD) and limits of lower quantification (LLOQ) were documented following the guidelines of the German Society of Toxicological and Forensic Chemistry (www.gftch.org, [22], [23]). For the chiral amphetamine method, LOD was estimated by serial dilution of a 100 ng/mL OF extract. Matrix effects were studied using infusion of analytes and evaluating the response at the expected retention time of the analyte and by addition experiments following recommendations of the German Society of Toxicological and Forensic Chemistry [23]. Stability was documented for amphetamine in authentic and prepared OF matrix samples stored at +4 °C for 28 d and for LDX with controls made at 1.0 and 10.0 ng/mL using 5 different blank OF samples with saliva concentrations between 23 and 75% stored at +4 °C for 14 days. The method for amphetamine was part of the BTMF proficiency program from GTFCh and the DOF program from LGC. No interference was observed for 64 other drugs of abuse and cortisol (Supplemental Table 1), which are part of the routine method.

3. Results

3.1. Method validation

Two of the analytical methods were used and validated for quantification of amphetamine and LDX in OF. Both methods had linear relations between concentration and peak area ratios of analyte to internal standards in both low and high ranges with r² values > 0.999. The LOD and LLOQ for amphetamine were 0.7 and 0.9 ng/mL, respectively; and 5.9 and 7.2 pg/mL for LDX. The applied measuring ranges were 1–500 ng/mL for amphetamine and 0.01–15 ng/mL for LDX. Samples with higher concentrations were reanalysed after dilution with blank standards. Between day measuring uncertainties were estimated to be below 5% for amphetamine (bias < 1%) and within day to be <10% for LDX (Table 2).

Table 2.

Measuring uncertainties in the quantifications of amphetamine and >LDX in OF.

	Assigned value ng/mL	Mean	CV%	Bias%	N
Amphetamine	3.52	3.54	4.0	0.5	24
	6.11	6.14	3.4	0.4	24
	15.7	15.6	2.4	−0.6	24
LDX	1.0	0.99	4.2	−1.3	33
	10.0	9.90	3.2	−1.0	33

The chiral method was only used to determine the relative proportion of L- and D-amphetamine based on peak areas with reference to the corresponding internal standards. The validation of LOD showed that each enantiomer was detectable at the 2.5 ng/mL OF concentration level.

No matrix effects on amphetamine and LDX response was observed when injection OF extracts from different individuals while infusing the compounds. However, in the addition experiment, using standards prepared from six different OF specimens, a reduction in response of 24% was seen for LDX at the 2 ng/mL concentration and 18% at 10 ng/mL. For amphetamine, the matrix effect using the same experiment was a 17% increase at 50 ng/mL concentration and a 5% increase at 400 ng/mL.

Representative chromatograms are shown in Fig. 1a–c. The chromatograms were free from interfering peaks. Analyte identities were always supported by correct product ion ratios.

Fig. 1.

Chromatograms from the analysis of amphetamine and LDX in oral fluid; (a) analysis of an authentic specimen measured to contain 385 ng/mL of amphetamine, (b) analysis of an authentic specimen measured to contain 0.28 ng/mL of LDX (lisdexamphetamine), (c) chiral analysis of amphetamine in an authentic specimen measured to contain 4122 ng/mL of D-amphetamine and 4291 ng/mL of L-amphetamine.

Stability of amphetamine was documented by reanalyses of 45 specimen after 4 weeks storage at 4 °C and found to be 98 ± 10%. Stability for LDX stored at +4 °C for 14 days was found to be 97 ± 3%, n = 20.

3.2. Clinical data

Amphetamine was detected and quantified in 98 (96%) of the 102 samples (Fig. 2). The concentrations ranged from 2 to 8410 ng/mL. In 17 of these, the chiral analysis demonstrated intake of illicit amphetamine since L-amphetamine was present and, therefore, these were considered as non-compliant. The amphetamine concentration in four different subgroups, compliant, LDX positives, non-compliant and non-compliant but LDX positives, are also shown in Fig. 2. The trend was that compliant patients, defined as not having L-amphetamine detected had lower median amphetamine concentrations than those indicated to be non-compliant (Fig. 2). A statistical analysis using the Mann Whitney test showed that the non-compliant subgroup had higher amphetamine concentrations (p < 0.01).

Fig. 2.

Concentrations of amphetamine in 5 different subgroups.

In the non-compliant cases with L-amphetamine detected, the D/L-amphetamine concentration ratios ranged from 0.75 to 13.1 with a median value of 1.0 (Fig. 3). In the 12 cases with detected LDX the median D/L ratio was 1.4. In 81 samples from compliant patients no L-amphetamine was detected. An examination of the samples with high amphetamine concentrations demonstrated the L/D ratio to be <0.1%.

Fig. 3.

The D/L amphetamine concentration ratio in the 17 non-compliant cases.

LDX was detected and quantified in 76 of the 102 cases (Fig. 4). This represents 79% of the 96 cases with detected OF amphetamine. The concentrations ranged from 0.01 to 6895 ng/mL. Sixty-four out of 81 (79%) compliant patients had detected LDX in OF and 12 out of 17 (71%) non-compliant patients had detected LDX in OF. Median LDX concentrations in the different subgroups were similar and are shown in Fig. 4. Several of the measured values were identified to be statistical outliers (Fig. 4).

Fig. 4.

Concentrations of LDX (lisdexamphetamine) in 3 different subgroups.

An attempt was made to investigate a possible correlation between amphetamine and LDX OF concentrations by regression analysis. A plot of all individual paired data demonstrated no indication of such a correlation, but a very scattered graph with most points having low LDX concentrations with scattered amphetamine concentrations. When evaluating the data from the 64 compliant cases, and excluding outliers, a similar scattered graph was obtained.

Among the 98 cases with detected amphetamine, drug results for other drugs of abuse (see Supplemental Table 1) were available in 88 cases. Among the compliant cases, 23% were positive for other illicit or non-prescribed drugs, whereas in the non-compliant subgroup 82% were positive for other drugs. Table 3 lists the detected drugs in the two subgroups. The positive rate for detecting drugs in the non-compliant group was statistically higher (p < 0.01, Mann Whitney)

Table 3.

Detected drugs in the comprehensive drug screening method.

Drug	Detected cases
	Compliant group (N = 71)	Non-compliant group (N = 17)
	Number of cases (%)	Number of cases (%)
Methylphenidate	4 (6)	6 (35)
Cocaine	0	2 (12)
MDMA	0	2 (12)
Benzodiazepine*	4 (6)	12 (71)
Zopiclone	4 (6)	2 (12)
Zolpidem	0	2 (12)
THC	2 (3)	4 (24)
Codeine	2 (3)	0
Morphine	0	2 (12)
6-Acetylmorphine	0	1 (6)
Fentanyl	0	1 (6)
Methadone	0	1 (6)
Oxycodone	1 (1)	0
Pregabalin	1 (1)	0
Gabapentin	1 (1)	1 (6)
Lidocaine	2 (3)	0

*Alprazolam, clonazepam, diazepam, oxazepam, bromazepam.

4. Discussion

The present study successfully used three LC-MS/MS methods to study LDX and amphetamine concentrations in OF from patients undergoing Elvanse® therapy. The results showed that some patients were not compliant with the therapy, since in 4 samples from 4 patients no amphetamine was detected in OF, and in another 13 patients (17 samples) the results demonstrated intake of illicit amphetamine. In 12 of these 17 samples, LDX was detected indicating that illicit amphetamine was used in addition to the prescribed medication. Thus, in 17 out of 70 patients (24%) the results from the amphetamine determinations demonstrated non-compliance.

Amphetamine was measured in OF at a median concentration value of 280 ng/mL in the 81 compliant cases (i.e., no L-amphetamine was detected). The lowest value in this group was 2 ng/mL, which was only slightly above the LLOQ of the method. In the subgroup with 64 LDX positive compliant cases, the median concentration was 328 ng/mL with a lowest value of 7.5 ng/mL. In the case with 7.5 ng/mL amphetamine, the LDX concentration was 4 times higher than the median value for the subgroup. The reason for the tendency of this subgroup to have a higher amphetamine concentration could be that the sampling was closer to the drug intake. One reason for the wide range of concentrations for both amphetamine and LDX could be that they do not always represent trough levels, since the samples may have been collected at different time points during the dose interval.

Elvanse® is taken orally, normally once daily in the morning to avoid insomnia. D-amphetamine is formed from LDX by hydrolytic enzymes in the circulation [13]. The two compounds have very different pharmacokinetics. LDX plasma concentration peaks with a T_max at about one hour and declines with an elimination half-life of about one hour. D-amphetamine concentration on the other hand peak with a T_max at about 3 h and has an elimination half-life of about 10 h [14], [15], [16]. The kinetics of these substances in OF has not been studied. However, it is well established that amphetamine saliva concentrations are much higher than in plasma. The saliva to serum ratio was been reported to be ∼20 [24] and the oral fluid to whole blood ratio to be 19 and 15 in separate studies [25], [26]. This high ratio is due to ion trapping of amphetamine in OF due to the pKa of amphetamine and the acidic pH of saliva. However, as has been shown, the ratio is influenced by the pH of saliva [27]. The sampling device used in our study offers a solution to this problem, as the liquid used in the collection process is buffered to an acidic pH.

After an Elvanse® dose of 70 mg (mean dose in this study was 68 mg), D-amphetamine plasma concentrations peak at about 80 ng/mL after 3 h, and are about 15 ng/mL at 24 h after dose intake. Higher concentrations are to be expected in OF. The median value observed for the compliant subgroup (280 ng/mL) agrees well with the reported D-amphetamine concentrations during the dose interval and an OF to plasma ratio of ∼20, when most samplings were completed before the next dose. Another conclusion from our results is that the measuring range of our method with an LLOQ of 1 ng/mL is adequate.

In 17 cases L-amphetamine was detected and quantified, which demonstrated intake of illicit amphetamine since it is a racemic mixture of amphetamine enantiomers. The median D/L ratio between enantiomers was 1.0. This is somewhat surprising since in most of these cases LDX was also detected, which should result in an expected ratio above 1. The mean ratio in the 12 LDX positive cases was 1.4. This suggests that the intake of illicit amphetamine in these cases is more determinant for the concentrations than the medication. This conclusion is supported by the higher amphetamine concentrations observed in these cases (Fig. 2). The results from the drug testing demonstrated that use of other illicit drugs were much more common in the non-compliant group, and comprised stimulants, benzodiazepines and opioids (Table 3). One source of amphetamine is as a metabolite from methamphetamine. Methamphetamine was part of the general screening method, but was not detected in any case. However, the proportion of non-compliant cases in the study population may not be representative for the entire group of patients receiving Elvanse® treatment, as sampling could have been initiated by suspicion.

The LDX median concentrations were below 1 ng/mL. Following a dose of 70 mg the C_max for LDX was 58 ng/mL [16]. With a half-life of 1 h it would take about 6 h to reach this concentration level and 12 h to reach levels close to the LDX LLOQ of the method used in our study, which was 100-fold more sensitive than the method used for plasma determinations [14], [15]. Since the LDX dose interval is 24 h it is not surprising that 21% of the cases considered compliant had no LDX detected. LDX measurement in OF (or in plasma) may, therefore, not be the best way to monitor compliance to prescribed medication. If LDX measurements are used, sampling time should be close after dose intake.

In conclusion, the study demonstrated the value of measuring amphetamine in OF with a chiral method to detect intake of illicit amphetamine and to perform drug testing in oral fluid as a mean for compliance monitoring. In order to control intake of lisdexamphetamine by analytical investigations, a very sensitive method is needed.

Appendix A. Supplementary data

The following are the Supplementary data to this article: