Cross-reactivity Assessment of Bath salts in different Immunoassays
Thomas
1
1
Kampfrath ,
Angela
1
Sibio ,
Lee
2
Blum ,
Saeed
1
Jortani
2
Department of Pathology and Laboratory Medicine University of Louisville, Louisville, Kentucky; NMS Labs, Willow Grove PA
Here, we studied the cross-reactivity of three different
synthetic cathinones (bathsalts) in urine at varies
• Bath salts are synthetic derivatives of cathinone, the active compound
Urine of confirmed bath salts
positive patients
in Khat (Catha edulis)
Patient number Cathinone Concentration
concentrations in common immunoassays.
Abstract
Background: Bath salts, a class of synthetic molecules known as
cathinones, are the latest drugs of abuse becoming increasingly popular in
the United States. They are very popular among younger abusers trying to
avert detection by the standard drug screening procedures. With little
information known on their risks and effects by the medical community,
frequent overdoses, hallucinations, and even death have been reported.
Currently, the different cathinones are analyzed by gas chromatography –
mass spectrometry by referral and highly specialized laboratories. Since
he structures of cathinones are similar to amphetamines, cross-reactivities
with common immunoassays are expected. Herein, we report on the
cross-reactivity analysis of three different synthetic cathinones in varies
commercial immunoassays used in routine drug screening practice.
Methods: The selected synthetic cathinones for cross-reactivity
assessment were Mephedrone, MDPV (3,4-methylenedioxypyrovalerone),
and Methylone. These substances are currently the most prevalent
members of the bath salts in the United States and were kindly provided
by Utak Laboratories Inc. (Valencia, CA). Those compounds were added
to aliquots of normal human urine at a concentration of 10000 ng/mL a
typical concentration utilized for cross-reactivity studies with unrelated
drugs. In addition, one sample contained a mixture of all three cathinones
at a concentration of 10000 ng/mL each as street drugs are rather a
mixture and rarely pure. The tested immunoassays were the
Roche Integra (Roche Diagnostics GmbH), Triage® 8 Drugs of Abuse
Panel (Inverness Medical, San Diego, CA), Siemens Viva E (Malvern, PA)
and Beckman Coulter Unicel DxC 800 (Brea, CA).
Results: The Roche amphetamine screen was the only assay that we
tested that showed cross-reactivity with bath salts. At first, we tested a
mixture of Mephedrone, MDPV, and Methylone providing a positive result
as indicated by a reaction rate of 1642 while 1000 is set for the cutoff
(equivalent in assay reactivity to 500 ng/mL). Next, we tested each
cathinone separately at a concentration of 10000 ng/mL. Here, only
mephedrone was able to cross-react and provide a positive result (1060),
while MDPV (417) and methylone (651) were well below the cutoff limit.
None of those compounds gave positive results at the 10000 ng/mL cutoff
in the Triage® 8 panel (amphetamine, barbiturate, cocaine, opiate,
benzodiazepine, THC, methadone, PCP), Siemens Viva E and in the
Beckman Coulter Unicel DxC 800 (amphetamine, barbiturate, cocaine,
opiate, benzodiazepine, THC, methadone).
Conclusion: Out of the three popular bath salts tested, only mephedrone
cross-reacted in the Roche’s amphetamine screen. Neither of the bath
salts tested either alone or as a mixture cross-reacted in the Triage or
Beckman amphetamine assays. All other immunoassay screens resulted
in negative results when bath salts were added to the urine. Considering
the long turn-around time for sending samples for testing bath salts, the
observed cross-reactivity in the Roche’s amphetamine assay may be an
advantage since the clinical management of amphetamine and bath salts
overdoses are similar.
Botanischer Garten Ruhr-Universität Bochum http://www.boga.ruhr-uni-bochum.de
• Khat is a plant endemic to the Middle East and Africa
• Mephedrone and MDPV are most common in the US
1
methylone
380 ng/mL
2
mephedrone
14 ng/mL
3
mephedrone
10 ng/mL
4
MDPV
160 ng/mL
5
MDPV
21 ng/mL
6
methylone
450 ng/mL
7
methylone
14 ng/mL
8
mephedrone
51 ng/mL
MDPV
50 ng/mL
methylone
50 ng/mL
mephedrone
510 ng/mL
methylone
Kindly provided by
NMS Labs
17 ng/mL
• DEA placed mephedrone, MDPV and methylone as Schedule 1 drugs
in 2011
9
• Bath salts are amphetamine-type stimulants and believed to be
synthesized from ephedrine or pseudoephedrine
•
• Limited pharmacologic and physiologic data available
• Reported toxic side-effects are strong hallucinations, hypertension and
Discussion
Cross-Reactivity
Cathinones
Objective
Urine was tested on:
• Siemens Viva E and Roche Integra for amphetamine, barbiturate, benzodiazepine, cocaine, THC,
opiates, methadone and oxycodone
• Beckman Coulter Unicel for amphetamine, barbiturate, benzodiazepine, cocaine, THC, opiates and
methadone
Analyte (cut-off)
Siemens Amphetamine (500 ng/mL)
Barbarbiturate (200 ng/mL)
Benzodiazepine (200 ng/mL)
Cocaine (150 ng/mL)
THC (50 ng/mL)
Opiate (300 ng/mL)
Methadone (300 ng/mL)
Oxycodone (100 ng/mL)
Roche
10
MDPV
70 ng/mL
Several patients co-ingested bathsalts
together with other drugs
•
Roche amphetamine assay was the
only assay showing a positive result
other extreme sympathomimetic and behavioral (violent) symptoms
Pt 1
Pt 2
Pt 3
Pt 4
Pt 5
Pt 6
Pt 7
Pt 8
Pt 9
Pt 10
NA
NA
positive
positive
positive
positive
Amphetamine (500 ng/mL)
Barbarbiturate (200 ng/mL)
Benzodiazepine (100 ng/mL)
Cocaine (150 ng/mL)
THC (50 ng/mL)
right below cut-off
Opiate (300 ng/mL)
Methadone (300 ng/mL)
Oxycodone (100 ng/mL)
positive
positive
positive
positive
at a MDPV concentration of 160
ng/mL
•
Structures
Limitation: Due to the low amount of
sample a true positive amphetamine
in Roche #4 could not have been
ruled out by confirmatory testing
Beckman Amphetamine (1000 ng/mL)
Barbarbiturate (200 ng/mL)
Benzodiazepine (200 ng/mL)
Cocaine (300 ng/mL)
THC (50 ng/mL)
Opiate (300 ng/mL)
Methadone (300 ng/mL)
Oxycodone
positive
positive
positive
positive
NA
NA
NA
NA
NA
NA
NA
Conclusions
Analytical Workup
• Patient urine samples with confirmed presence of bath salts only cross-reacted in
NA
•
Roche amphetamine assay was the only screen which showed cross-reactivity
with the tested bath salts
the Roche amphetamine screen
•
A mixture of Mephedrone, MDPV, and Methylone (parent compound) at a
concentration of 10000 ng/mL each spiked in drug free urine were tested in the
Spiked mephedrone in drug free urine was the only analyte that caused confirmed
cross-reactivity at a concentration of 10000 ng/mL
•
MDPV and its metabolites at a concentration of 160 ng/mL from a patient who had
ingested bath salts provided a false positive amphetamine result only in the
same assays
• This mixture only provided a positive result in the Roche assay as indicated by a
Roche assay
•
ingestion
reaction rate of 1642 while 1000 is set for the cutoff (equivalent in assay reactivity
to 500 ng/mL)
• Each cathinone was tested separately at a concentration of 10000 ng/mL
• Only mephedrone cross-reacted in the Roche assay and provided a positive result
Acknowledgement & References
The authors would like to acknowledge UTAK Laboratories, Inc, without their kind
provision of the bath salts this project could not have been pursued.
1.
(1060), while MDPV (417) and methylone (651) were well below the cutoff limit
2.
• None of those compounds gave positive results in the Triage® 8 panel, Siemens
Image derived from: Truscott, Clinical Chemistry, 2013
3.
Viva E and in the Beckman Coulter Unicel DxC 800
Spiking bath salts in a drug free urine sample do not behave the same as after
Truscott SM, Crittenden NE, Shaw MA, Middleberg RA, Jortani SA. Violent behavior and hallucination in a
32-year-old patient. Clin Chem. 2013 Apr;59(4):612-5.
Petrie M, Lynch KL, Ekins S, Chang JS, Goetz RJ, Wu AH, Krasowski MD. Cross-reactivity studies and
predictive modeling of "Bath Salts" and other amphetamine-type stimulants with amphetamine screening
immunoassays. Clin Toxicol (Phila). 2013 Feb;51(2):83-91.
Johnson RD, Botch-Jones SR. The stability of four designer drugs: MDPV, mephedrone, BZP and TFMPP in
three biological matrices under various storage conditions. J Anal Toxicol. 2013 Mar;37(2):51-5.