Oral Fluid with Three Modes of Collection and Plasma Methamphetamine and Amphetamine Enantiomer Concentrations After Controlled Intranasal l-Methamphetamine Administration

Abstract

Methamphetamine is included in drug testing programs due to its high abuse potential. d-Methamphetamine is a scheduled potent central nervous system stimulant, while l-methamphetamine is the unscheduled active ingredient in the over-the-counter nasal decongestant Vicks® VapoInhaler™. No data are available in oral fluid (OF) and few in plasma after controlled Vicks VapoInhaler administration. We quantified methamphetamine and amphetamine enantiomers in OF collected with two different devices and plasma via a fully validated LC-MS/MS method. Additionally, OF were analyzed with an on-site screening device. Sixteen participants received 7 Vicks VapoInhaler doses according to manufacturer's recommendations. Specimens were collected before and up to 32h after the first dose. No d-methamphetamine or d-amphetamine was detected in any sample. All participants had measurable OF l-methamphetamine with median maximum concentrations 14.8 and 16.1μg/L in Quantisal™ and Oral-Eze® devices, respectively, after a median of 5 doses. One participant had measurable OF l-amphetamine with maximum concentrations 3.7 and 5.5μg/L after 6 doses with the Quantisal and Oral-Eze devices, respectively. There were no positive DrugTest® 5000 results. In the cutoff range 20-50μg/L methamphetamine with amphetamine ≥limit of detection, 3.1-10.1% of specimens were positive; first positive results were observed after 1-4 doses. Two participants had detectable plasma l-methamphetamine, with maximum observed concentrations 6.3 and 10.0μg/L after 2 and 5 doses, respectively. Positive OF and plasma methamphetamine results are possible after Vicks VapoInhaler administration. Chiral confirmatory analyses are necessary to rule out VapoInhaler intake. Implementing a selective d-methamphetamine screening assay can help eliminate false-positive OF results.

Introduction

Methamphetamine is an important component in federally mandated workplace drug testing and driving under the influence of drugs (DUID) programs because of its high abuse potential. A chiral center is present on the molecule, resulting in two enantiomers; the d-methamphetamine isomer is more potent^[1] and is a Schedule II controlled substance available by prescription. The l-enantiomer is unscheduled and is the active ingredient in the over-the-counter nasal decongestant Vicks® VapoInhaler™.

According to the manufacturer, each inhaler contains 50mg l-methamphetamine (labeled as Levmetamfetamine), with 0.04-0.15mg l-methamphetamine delivered per 800mL dose, with possible trace d-methamphetamine. Previously, Vicks VapoInhaler administration was associated with a positive methamphetamine blood test^[2]. It is therefore essential to resolve methamphetamine and amphetamine enantiomers in order to more effectively interpret a positive test result.

No data are available for methamphetamine oral fluid (OF) concentrations after controlled Vicks VapoInhaler administration, and few data for plasma concentrations^[3]. OF is an alternative testing matrix of increasing importance in workplace drug testing and DUID programs. OF sampling offers several advantages over blood and urine collection: it is less invasive, does not require a same-sex collector, and minimizes sample adulteration^[4]. Disadvantages include sampling time requirements, potential difficulty in collecting adequate sample volume, and addition of preservative buffers that dilute specimens and can pose analytical challenges. In its 2004 Mandatory Guidelines, the US Substance Abuse and Mental Health Services Administration (SAMHSA) proposed a 50μg/L OF methamphetamine cutoff with amphetamine present ≥method limit of detection (LOD)^[5]. The European Union's Driving Under the Influence of Drugs, Alcohol, and Medicines (DRUID) program proposed a 25μg/L OF methamphetamine analytical cutoff for forensic cases and a 410μg/L OF methamphetamine cutoff equivalent to 20μg/L in whole blood for epidemiological prevalence studies^[6], and a meeting of international experts in drugged-driving in Talloires sponsored by six international organizations recommended a 20μg/L cutoff^[7].

In the present study, healthy adults were administered 7 doses of the Vicks VapoInhaler according to manufacturer's recommendations – 2 inhalations per nostril, every 2 hours – with up to 0.60mg l-methamphetamine delivered per dose. Participants provided OF and plasma specimens before and up to 32h after the first dose. OF was collected with two different devices and one on-site screening device. We quantified d,l-methamphetamine and d,l-amphetamine via a fully validated LC-MS/MS method with chiral derivatization, characterized methamphetamine concentrations in OF collected with both devices and plasma, assessed the performance of the on-site OF screening test compared to confirmatory results, and evaluated different OF methamphetamine cutoffs. These data will aid in OF and plasma methamphetamine results interpretation in clinical and forensic settings.

Materials and Methods

Chemicals, Reagents, and Instrumentation

d,l-Methamphetamine and d,l-amphetamine were analyzed according to a previously published method^[8]. Amphetamines derivatization utilized 1-fluoro-2,4-dinitrophenyl-5-l-alanineamide (Marfey's reagent) (Sigma-Aldrich, Allentown, PA, USA). Solid phase extraction was accomplished with Strata™-XC Polymeric Strong Cation columns (3mL/60mg) (Phenomenex®, Torrance, CA, USA). The HPLC system consisted of a DGU-20A3 degasser, LC-20ADXR pumps, SIL-20ACXR autosampler, and a CTO-10AC column oven (Shimadzu Corp., Columbia, MD, USA). Tandem mass spectrometry was performed on a 3200 QTrap® mass spectrometer with a TurboIonSpray source (ABSciex, Foster City, CA, USA). Separations were performed on a Kinetex® 2.6μm C18 column with an identically packed SecurityGuard ULTRA cartridge (Phenomenex).

Clinical Study

Participants were dosed with Vicks VapoInhaler (Proctor & Gamble, Baltimore, MD, USA) according to manufacturer's recommendations. OF was collected with the Quantisal™ (Immunalysis, Pomona, CA, USA) and Oral-Eze® (Quest Diagnostics Inc., Lenexa, KS, USA) devices. OF also was screened with the DrugTest® 5000 (Draeger Safety, Lübeck, Germany). Whole blood was collected from an indwelling venous catheter into grey top Vacutainer® tubes (Becton Dickson, Franklin Lakes, NJ, USA). After collection, whole blood and OF were stored in 3.6mL Nunc™ CryoTubes™ (Thermo Scientific, Waltham, MA).

Participants

Participants provided written informed consent to participate in this National Institute on Drug Abuse Intramural Research Program Institutional Review Board-approved study. Individuals were recruited by television, radio, and newspaper advertisements, flyers and participant referrals. Participants received a comprehensive medical and psychological evaluation to verify compliance with eligibility criteria. Participants were 18-65 years of age with adequate peripheral venous access and able to give valid informed consent. Exclusion criteria included any current medical condition precluding safe study participation, current dependence on any psychoactive substance other than nicotine or caffeine, or inability to tolerate intranasal administration.

VapoInhaler Administration

Participants entered the secure research unit ≥2h before dosing. A urine specimen was collected in a polypropylene container prior to dosing. An aliquot was analyzed for amphetamines with an iScreen and a urine pregnancy test was performed for women with reproductive potential. Subjects with positive results were excluded. In accordance with the manufacturer's recommended dosage, each participant on the first day inhaled deeply from a Vicks VapoInhaler twice in each nostril every two hours between 0900 and 1900h (total of 24 inhalations), and at 0600h on the second day (four inhalations). The inhaler contained 50mg l-methamphetamine, with 0.04-0.15mg administered per inhalation (total of up to 0.60mg l-methamphetamine per dose).

Specimen Collection

Biological specimens were collected approximately 15min before, and 0.5, 1, 2, 2.5, 3, 4, 4.5, 5, 6, 6.5, 7, 8, 8.5, 9, 10, 10.5, 11, 12, 13, 15, 21, 24, 26, 28, 30, and 32h after the first VapoInhaler dose. OF was collected with the Quantisal and Oral-Eze devices by placing the absorptive pad under the tongue (Quantisal) or between the lower check and gum (Oral-Eze) until the volume-adequacy indicator turned blue, indicating approximately 1mL OF was collected or 5 min elapsed, whichever occurred first. The pad was removed and placed in a plastic tube containing the elution/stabilization buffer, and stored for ≥12h at 4°C to allow analyte elution from the pad. For Quantisal, a serum separator was depressed into the tube, the buffer solution decanted into a 3.6mL CryoTube, and stored refrigerated at 4°C until analysis. For Oral-Eze, a weight was inserted in the tube followed by centrifugation at 2,200×g and 4°C for 5min, the buffer solution decanted into a CryoTube and stored refrigerated until analysis. Quantisal specimens were always collected before Oral-Eze specimens throughout the study. Low-volume specimens were recorded at the time of collection and analyzed as collected, without applying weight corrections.

OF also was screened with the DrugTest 5000 on-site device with a 35μg/L d-methamphetamine cutoff. According to the manufacturer l-methamphetamine concentrations >100,000μg/L would be required to produce a positive test, indicating low cross-reactivity. The collector was continuously moved around the participants’ mouth until the indicator turned blue. After inserting the collector and test cartridge into the analyzer and closing the door, positive or negative results were displayed in approximately 8min. Invalid tests (specimen collected correctly but no reading) were recorded at the time of screening.

Whole blood was collected from an indwelling peripheral venous catheter into a grey top Vacutainer tube containing NaF and potassium oxalate and placed on ice for no more than 2h. Tubes were centrifuged at 3,000rpm for 10min and plasma was decanted into a 3.6mL CryoTube and stored frozen at −20°C until analysis.

Amphetamines Chiral Analysis

Individual methamphetamine and amphetamine enantiomers were quantified in OF collected with the Quantisal and Oral-Eze devices and plasma via a fully validated LC-MS/MS method employing a chiral derivatization reagent^[8]. Briefly, racemic d₁₁-methamphetamine and d₁₁-amphetamine internal standards were added to 0.75mL Oral-Eze, 1mL Quantisal specimens, or 0.5mL plasma, followed by protein precipitation with 1% formic acid in water. Supernatants were applied to pre-conditioned Strata-XC Polymeric Strong Cation solid phase extraction columns . After washing, analytes were eluted with 5% ammonium hydroxide in methanol. The eluate was evaporated to dryness and reconstituted in water. Derivatization was performed with Marfey's reagent and heating at 45°C for 1h. Samples were allowed to cool to room temperature followed by addition of 1M HCl in water and vortexing. After evaporation to dryness under nitrogen at 40°C, samples were reconstituted in 200μL water (mobile phase A):methanol (mobile phase B) (40:60 v/v), centrifuged at 2000×g at 4°C for 5min, and supernatant transferred to a 96 well plate. Ten (OF) or 20μL (plasma) were injected into the LC-MS/MS system. Derivatized enantiomer separations were performed under isocratic conditions (water:methanol, 40:60) and analytes were identified and quantified by two MRM transitions and their ratio in ESI negative mode. In all matrices, the method was linear for all enantiomers from 1-500μg/L, with imprecision and accuracy of ≤11.3% and 86.8-108%, respectively. Extraction efficiencies ranged from 67.4-117% and matrix effects from −17.0-468%, with variation always ≤19.1%.

Data Analysis

Data from the enantiomeric separations were acquired and analyzed with Analyst software version 1.5.1 (ABSciex). Calculations for maximum observed concentrations (C_max), time of C_max (t_max), and times of first (t_first) and last detection (t_last) were performed with Microsoft Excel 2007. Statistical analyses were performed with IBM SPSS Statistics 20. OF results were evaluated based on various currently recommended cutoffs. Differences between C_max, t_max, t_first, and t_last observed in the two OF collection devices were evaluated by Wilcoxon signed-rank test; when evaluating differences for t_first and t_last based on proposed cutoffs, only cutoffs where at least three participants produced positive OF specimens were evaluated. Differences between the percent positive OF specimens collected with the two devices was evaluated by Pearson's Chi-squared test. Significant differences were present if the 2-tailed p<0.05.

Results

Sixteen healthy adults aged 19-54 years old (12 men, 4 women; 11 black, 3 white, 2 more than one race) were admitted to the secure clinical research unit. All admission urine amphetamine iScreen and pregnancy tests were negative. Participants provided 430 plasma and 432 OF specimens for each device. Two plasma specimens could not be collected from one participant due to complications with his intravenous catheter. There were 88 and 130 low-volume Quantisal and Oral-Eze OF collections (5 min elapsed before volume-adequacy indicator turned blue), respectively, after dosing. Table 1 summarizes percent positive specimens at different time periods for full and low-volume specimens. Low-volume specimens were observed at every time, but there were always more full-volume specimens. Overall, 62.8 and 67.6% of low-volume specimens collected with the Quantisal and Oral-Eze devices, respectively, were l-methamphetamine positive.

Table 1.

Detection of l-methamphetamine in oral fluid (OF) after intranasal Vicks VapoInhaler administration

Time	Quantisal			Oral-Eze
	# full volume, # low volume OF specimens	% Positive – Full	% Positive – Low	# full volume, # low volume OF specimens	% Positive – Full	% Positive – Low
0.5-1	24, 8	54.2	50.0	22, 10	77.3	50.0
2-3	37, 11	83.8	36.4	34, 14	73.5	57.1
4-5	37, 11	83.8	45.5	29, 19	79.3	57.9
6-8	50, 14	88.0	100	45, 19	93.3	73.7
8.5-9	29, 3	96.6	100	22, 10	100	100
10-12	51, 13	88.2	69.2	42, 22	90.5	90.9
13-15	20, 12	90.0	75.0	19, 13	94.7	69.2
21-26	36, 12	72.2	66.7	32, 16	65.6	68.8
28-32	44, 4	47.7	75.0	41, 7	48.8	57.1

Positivity rates are at the method's limit of quantification (1 μg/L) for full and low-volume Quantisal™ and Oral-Eze® OF specimens collected from 16 healthy adult participants who received 6 Vicks® VapoInhaler™ doses (4 inhalations per dose) 2h apart on Day 1 and 1 more on the morning of Day 2 (n=432 specimens per device).

Figure 1 depicts OF l-methamphetamine positivity rates, with similar rates for OF collected with the Quantisal and Oral-Eze devices throughout the study. All participants produced positive l-methamphetamine OF specimens based on the method's limit of quantification (LOQ, 1μg/L); 316 (76.0%) and 318 (76.4%) specimens were l-methamphetamine positive in Quantisal and Oral-Eze devices, respectively (p=0.878). l-Methamphetamine was detected in Quantisal and Oral-Eze OF after the first dose in 9 and 11 participants, and all participants were positive after 4 and 5 doses, respectively.

Figure 1.

Summary of % oral fluid specimens positive at the method limit of quantification (1 μg/L) at each timepoint collected with the Quantisal™ and Oral-Eze® devices for 16 participants who received 6 Vicks® VapoInhaler™ doses (4 inhalations per dose) 2h apart on Day 1 and 1 more on the morning of Day 2 (n=432 specimens per device).

Figure 2 depicts OF l-methamphetamine concentration-time courses for Quantisal and Oral-Eze. The median (range) t_first in Quantisal and Oral-Eze were 0.5 (0.5-4.5) and 0.5 (0.5-8)h, respectively (p=0.570). Maximum concentrations observed were 14.8 (5.2-380) and 16.1 (4-182)μg/L in Quantisal and Oral-Eze devices, respectively, after a median of 5 doses in both devices (p=0.535). The median time of the last positive specimen after the 7^th dose was 8h for both OF collection devices (p=0.944). At the final collection (11h after the 7^th dose) median concentrations were 2.2 (1.0-18.1) and 2.3(1.3-55.8)μg/L in Quantisal and Oral-Eze, respectively, among those still positive (n=7). OF l-amphetamine was detected in only one participant with both devices. First detection was after 4 doses in both devices. Maximum concentrations were 3.7 and 5.5μg/L in Quantisal and Oral-Eze, respectively, observed after 6 doses. This participant's OF was l-amphetamine positive at 1.1 and 4.5μg/L at the last collection time with Quantisal and Oral-Eze, respectively. No d-methamphetamine or d-amphetamine was detected in any participants’ OF.

Figure 2.

Median (interquartile range) l-methamphetamine concentrations in oral fluid at the method limit of quantification (1μg/L) collected with the Quantisal™ and Oral-Eze® devices for 16 participants who received 6 Vicks® VapoInhaler™ doses (4 inhalations per dose) 2h apart on Day 1 and 1 more on the morning of Day 2 (vertical dashed lines, n=432 specimens per device). No d-methamphetamine or d-amphetamine was detected in any specimen.

All DrugTest 5000 results were negative, despite the presence of l-methamphetamine up to 380μg/L in 432 authentic OF specimens collected with two different devices, indicating that the DrugTest 5000 has no cross reactivity to l-methamphetamine within this concentration range.

OF results were evaluated according to four cutoffs and results are summarized in Table 2. At SAMHSA's currently proposed 50μg/L OF methamphetamine and amphetamine ≥method LOD (0.5μg/L) cutoff, only one participant produced positive specimens. He had 13 (3.1% of all post-dose collections) and 16 (3.8%) positive OF specimens with the Quantisal and Oral-Eze devices, respectively; the first positive was after 4 doses with both devices and his last positive was 3 and >11h after his 7^th (last) dose, respectively. Without the amphetamine criterion three and two participants produced 19 (4.6%) and 24 (5.8%) positive OF specimens with first detection after 2-4 and 1-2 doses with Quantisal and Oral-Eze, respectively; last positives were detected up to 3h after the 7^th dose in Quantisal and >11h after the 7^th dose in Oral-Eze. At the DRUID 25μg/L cutoff, five and three participants produced 7.0 and 7.5% positive OF specimens with first positives after 2-6 and 1-2 doses in Quantisal and Oral-Eze, respectively; last positives were observed up to 9h or >11h after the 7^th dose in Oral-Eze and Quantisal, respectively. Finally, at the Talloires 20μg/L cutoff six and five participants produced 8.2 and 10.1% positive OF specimens with first positives in Quantisal and Oral-Eze after 1-6 and 1-5 doses, respectively; last positive specimens were observed 9h (Quantisal) or >11h (Oral-Eze) after the 7^th dose. No specimen was positive with the DRUID 410μg/L OF cutoff equivalent to 20μg/L whole blood. Overall, in the cutoff range 20-50μg/L OF methamphetamine (without an amphetamine component) 4.6-10.1% of specimens were positive, with a median of 1-4 Vicks doses until the first OF positive specimen. No significant differences between Quantisal and Oral-Eze t_first and t_last with the DRUID (t_first p=0.180, t_last p=0.655) or Talloires (p=0.785 and p=1.00) cutoffs were observed.

Table 2.

Percent positive specimens (number of participants with any positive specimen) and median (range) times of first (t_first) and last (t_last) detection (h), relative to the first dose, for four oral fluid l-methamphetamine cutoffs after Vicks VapoInhaler administration.

Device		SAMHSA 50μg/L + amphetamine ≥LOD	SAMHSA 50μg/L only	DRUID 25μg/L	Talloires 20μg/L
Quantisal	Number of participants	1	3	5	6
	% Specimens Positive	3.1	4.6	7.0	8.2
	Median tfirst (h, range)	6.5	6.5 (2.5-6.5)	2.0 (2.0-10.0)	2.3 (0.5-10.0)
	Median tlast (h, range)	24.0	6.5 (6.5-24.0)	6.5 (4.0-30.0)	7.5 (2.0-30.0)
Oral-Eze	Number of participants	1	2	3	5
	% Specimens Positive	3.8	5.8	7.5	10.1
	Median tfirst (h, range)	7.0	1.5 (1.0-2.0)	1.0 (0.5-2.5)	2.5 (0.5-8.5)
	Median tlast (h, range)	>32.0	17.0 (2.0 - >32.0)	12.0 (2.0 - >32.0)	11.0 (2.0 - >32.0)

Oral fluid was collected with the Quantisal™ and Oral-Eze® devices for 16 participants who received 6 Vicks® VapoInhaler™ doses (4 inhalations per dose) 2h apart on Day 1 and 1 more on the morning of Day 2 (n=432 specimens per device). The method's limit of detection (LOD) was 0.5μg/L.

Figure 3 depicts the concentration-time course for the only two participants with positive plasma l-methamphetamine throughout the study session. First positive specimens occurred after the first dose at 1.4 and 1.1μg/L for these participants. Observed plasma C_max's were 10.0 and 6.3μg/L occurring after 5 and 2 doses, respectively. Both participants were still positive at the last collection time (11h after the 7^th dose) at 2.6 and 3.8μg/L. No d or l-amphetamine or d-methamphetamine was detected in plasma.

Figure 3.

Plasma l-methamphetamine concentrations after 6 Vicks® VapoInhaler™ doses (4 inhalations per dose) 2h apart on Day 1 and 1 more on the morning of Day 2 (vertical dashed lines). Only 2 participants had detectable l-methamphetamine based on the method limit of quantification (1μg/L). No d,l-amphetamine or d-methamphetamine was detected in plasma

Figure 4 compares Quantisal and Oral-Eze OF and plasma concentrations for the two participants who were plasma l-methamphetamine positive. Calculated OF/plasma ratios ranged from 4.8-39.7 for participant A and 0-10.8 for participant H. Participant A OF concentrations were often an order of magnitude greater than plasma for both devices, while the OF/plasma ratio for participant H was >10 only once. These data demonstrate large inter and intra-subject variabilities in OF/P ratios.

Figure 4.

Comparison of oral fluid and plasma l-methamphetamine concentrations after 6 Vicks® VapoInhaler™ doses (4 inhalations per dose) 2h apart on Day 1 and 1 more on the morning of Day 2 (vertical dashed lines). Only 2 participants had detectable plasma l-methamphetamine based on the method limit of quantification (1μg/L). No d,l-amphetamine or d-methamphetamine was detected in plasma. Data are plotted on a logarithmic scale.

Discussion

The present study is the first to report OF amphetamines determination after controlled Vicks VapoInhaler administration. Methamphetamine accumulation in OF was apparent after multiple doses - the percent positive specimens was 56.3-68.8% after the 1^st dose, increasing steadily to 100% after the 4^th-6^th doses, and slowly declined for the remainder of the study. At the last collection – 11h after the last dose – 43.8% of participants were still OF l-methamphetamine positive with both devices. l-Amphetamine was present in only one participant's OF, having the highest plasma and OF l-methamphetamine concentrations (1.4-10.0μg/L plasma, 14.1-131μg/L Quantisal, 17.1-182μg/L Oral-Eze). His first l-methamphetamine-positive OF was detected after the first dose with both devices, and his first l-amphetamine-positive OF specimen was observed after 4 doses; therefore, multiple doses were required before detectable l-amphetamine was present in OF. This could be due to the low administered doses (0.16-0.60mg l-methamphetamine per dose) and that methamphetamine metabolism is enantioselective, with approximately 3 times more d-amphetamine formed after a d-methamphetamine dose than l-amphetamine formed from the same-sized l-methamphetamine dose^[9].

Significant differences in the performance between the two OF collection devices were not observed. l-Methamphetamine concentrations in OF after collection with the Quantisal and Oral-Eze devices were nearly identical: 76.0 and 76.4% of specimens were positive (p=0.878), respectively, with the method LOQ (1μg/L); median observed t_first were both 0.5 (p=0.570), C_max were 14.8 and 16.1μg/L, respectively (p=0.535), and t_last in both devices was 8h after the last dose (p=0.944). In the study timeline, Oral-Eze specimens were always collected after Quantisal, which can account for the greater number of low-volume Oral-Eze specimens (130) compared to Quantisal (88). However, the percentage of positive Quantisal and Oral-Eze low-volume specimens based on the LOQ was similar (62.8 and 67.6%, respectively). The large positivity rate of the low-volume specimens demonstrate that they offer important information, and should be analyzed rather than discarded.

According to the manufacturer, the Vicks VapoInhaler may contain trace amounts of d-methamphetamine, but it was never detected in plasma or OF. No test analyzed on the DrugTest 5000 (with a d-methamphetamine target) was ever positive. In the range 1.0-380μg/L OF l-methamphetamine, there was no cross-reactivity to d-methamphetamine. Previously, the DrugTest 5000 was evaluated for methamphetamine in drivers suspected of DUID; two reports evaluated the device against confirmatory plasma analysis^[10-11], and the third against residual OF-buffer mixtures from two other on-site devices^[12]. In all these reports, specificity was ≥95% for methamphetamine. The authors did not provide methamphetamine concentration ranges in confirmatory results, and chiral analyses were not performed. However, the DrugTest 5000 appears to have ample specificity to avoid triggering a positive d-methamphetamine result after manufacture-recommended doses of Vicks VapoInhaler.

Positive OF results based on multiple proposed OF methamphetamine cutoffs were observed. With cutoffs ≥20μg/L, ≤10.1% of OF specimens would be expected to be positive after 1-4 doses (4 inhalations/dose). Eleven hours after the last dose, ≤6.3% specimens were positive at cutoffs ≥20 μg/L. There was variability among participants in the number of doses administered before positive OF l-methamphetamine results were observed, possibly due to differences in amount of l-methamphetamine delivered intranasally. While all participants were instructed to inhale as deeply as possible during all doses, differences in breathing capacity could account for the observed variability. Additionally, the study monitored participants after 7 doses over 2 administration days; however, the manufacturer states the inhaler can be administered for up to 7 days. It is possible more participants would be positive based on these cutoffs with extended exposure since it appeared l-methamphetamine and l-amphetamine accumulated in the OF with multiple doses.

l-Methamphetamine pharmacokinetics after controlled Vicks VapoInhaler administration were evaluated previously in plasma^[3]. Participants self-administered the inhaler according to the manufacturer's recommendations for 4 doses (16 inhalations total) then at 2 and 4 times (32 and 64 inhalations total) the recommended dose; each dose was administered 2h apart in all three conditions. The investigators reported plasma methamphetamine and amphetamine were often below their LOQ (5μg/L), even at 4 times the recommended dose. They did not report concentrations for specimens that did exceed the LOQ. In the present study, participants self-administered one dose (4 inhalations) every 2h for 6 doses followed by a 7^th dose 11h after the 6^th; plasma l-methamphetamine was detected in only 2 participants with maximum concentrations 6.3 and 10.0μg/L observed after 2 and 5 doses, respectively. Our results agreed with previous findings^[3]. In the present study, no d,l-amphetamine or d-methamphetamine was detected in plasma. OF/plasma ratios calculated with both OF devices showed large inter- and intra-subject variabilities.

Previously, we published urine amphetamines concentrations following controlled Vicks VapoInhaler administration from these participants^[13]. True positive results were those with a positive immunoassay result (d-methamphetamine target) and ≥250μg/L d-methamphetamine by GC-MS confirmation; all urine specimens were analyzed via GC-MS regardless of immunoassay result. Urine specimens were screened with 3 different immunoassays and sensitivities, specificities, and efficiencies were 100%, 97.8-100%, and 97.8-100%, respectively. No urine specimen contained d-methamphetamine after GC-MS analysis. Therefore, there were no true positive urine results. These are important data as the urine methamphetamine cutoff utilized in federally regulated drug testing is 250μg/L methamphetamine plus the presence of 100μg/L amphetamine. Two participants did produce urine specimens with ≥250μg/L l-methamphetamine but they did not contain >100μg/L amphetamine. They also were the only participants with positive plasma l-methamphetamine; no plasma l-amphetamine was detected. Comparing matrices, 1 participant was positive with the SAMHSA 50μg/L OF methamphetamine + amphetamine ≥LOD cutoff, 3 and 2 participants were positive with the SAMHSA 50μg/L OF methamphetamine only cutoff, 5 and 3 were positive with 25μg/L OF methamphetamine cutoffs, and 6 and 5 were positive with 20μg/L OF methamphetamine cutoff in Quantisal and Oral-Eze samples, respectively. However, if d-methamphetamine was the target analyte none of the specimens would be true positives, similar to the urine results.

Our findings showed that positive OF methamphetamine results are possible after administration of Vicks VapoInhaler manufacturer-recommended doses at the currently proposed SAMHSA, DRUID and Talloires methamphetamine OF cut-offs; positive results were first observed after a minimal (1-4) number of doses. Implementing a selective d-methamphetamine initial screen will eliminate false-positive OF specimens. Finally, performing chiral analysis for confirmatory tests will differentiate Vicks VapoInhaler use from illicit methamphetamine abuse. This is possible because illicit methamphetamine typically contains only d-methamphetamine or a combination of d- and l-methamphetamine. Our results document that no d-methamphetamine was detected after Vicks VapoInhaler administration.

Key Findings.

l-Methamphetamine is the unscheduled active ingredient in the over-the-counter nasal decongestant Vicks® VapoInhaler™, however no data are available for oral fluid (OF) l-methamphetamine concentrations and few data are available for plasma concentrations after controlled Vicks VapoInhaler administration. Our data illustrate OF and plasma l-methamphetamine pharmacokinetics, the effectiveness of the Quantisal™ and Oral-Eze® collection devices and the DrugTest® 5000 on-site OF screening device, and OF l-methamphetamine detection windows based on currently proposed guidelines after multiple Vicks VapoInhaler doses.

List of Abbreviations

DUID

driving under the influence of drugs

OF

oral fluid

SAMHSA

Substance Abuse and Mental Health Services Administration

LOD

limit of detection

DRUID

Driving Under the Influence of Drugs, Alcohol, and Medicines

C_max

maximum observed concentration

t_max

time of C_max

t_first

time of first detection

t_last

time of last detection

LOQ

limit of quantification