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Quantitation of Amphetamines in Urine for
SAMHSA Mandated Workplace Drug Testing
Using a Triple Stage Quadrupole LC-MS
System
Kristine Van Natta, Marta Kozak; Thermo Fisher Scientific, San Jose, CA
Application
Note: 561
Key Words
• TSQ Quantum
Ultra
• Hypersil Gold
• NIDA
• Methampheta-
mine
• Phentermine
Introduction
Federal employees and public transportation workers are
required to pass a pre-employment drug screen known as
the NIDA5, which refers to the five drugs of abuse that
are required to be tested for by the National Institute of
Drug Abuse (NIDA), or the Substance Abuse and Mental
Health Services Administration (SAMHSA) panel. The
assays are divided into 5 groups: opiates, amphetamines,
cocaine (benzoylecgonine), cannabis (THCA) and PCP.
In the past, these five groups have been screened by
immunoassay and confirmed by gas chromatography-
mass spectrometry (GC/MS). In October 2010, SAMHSA
approved the use of liquid chromatography-mass
spectrometry (LC/MS) for confirmation of workplace
drug testing samples. Here we will focus on the
amphetamine group which consists of amphetamine,
methamphetamine, 3,4-methylenedioxyamphetamine
(MDA), 3,4-methylenedioxymethamphetamine (MDMA
or Ecstasy) and methylenedioxyethylamphetamine
(MDEA).
Goal
To develop a specific and robust dilute-and-shoot
quantitative method for the confirmation of amphetamine,
methamphetamine, MDA, MDMA, MDEA in urine that
meets SAMHSA cutoffs. Additionally, the method should
be able to discriminate between the structural isomers
methamphetamine and phentermine.
Methods
Sample Preparation
Urine was spiked with internal standards and hydrolyzed
with
β
-glucuronidase. While amphetamines do not require
hydrolysis, other compounds in the SAMHSA panel such
as the opiates and THC do require hydrolysis. Adding
this step enables all SAMHSA panel compounds to be
processed with one method. Methanol was added to the
hydrolysis mixture and the resulting mixture was centri-
fuged. The supernatant was further diluted and subjected
to LC-MS analysis.
HPLC Conditions
Chromatographic analysis was performed using Thermo
Scientific Accela 600 HPLC pumps and a Thermo Scientific
Hypersil GOLD aQ column (50 x 4.6 mm, 1.9 µm particle
size). The mobile phase consisted of 5 mM ammonium
formate with 0.1% formic acid in both water and metha-
nol. The flow rate was 1.5 mL/min and the column was
maintained at 30 °C. The total run time was 4.5 minutes.
MS Conditions
MS analysis was carried out on a Thermo Scientific TSQ
Quantum Ultra triple stage quadrupole mass spectrometer
equipped with a heated electrospray ionization (HESI-II)
probe. Two selected reaction monitoring (SRM) transitions
were monitored for each compound to provide ion ratio
confirmations (IRC).
Validation
Standard curves were prepared by fortifying pooled blank
human urine with analytes. Quality control (QC) samples
were prepared in a similar manner at concentrations
corresponding to the low (LQC), middle (MQC) and high
(HQC) end of the calibration range. Intra-run variability
and robustness were determined by analyzing six replicates
of each QC level with a calibration curve on three different
days. Matrix effects were investigated by comparing peak
area of analytes prepared in multiple lots of urine to those
of a sample prepared in water.
Results and Discussion
The limits of quantitation (LOQs) for all compounds meet
the SAMHSA confirmation requirements. (Table 1). The
method is linear up to 5,000 ng/mL with R2 values > 0.99
for all compounds. Figure 1 shows representative calibra-
tion curves for all compounds. Quality control results for
the validation are shown in Table 2. Figure 2 shows an
SRM chromatogram at LOQ. Peak areas of analytes in
samples prepared from seven different lots of blank human
urine compared to that of a sample prepared in water
were all within 15% for amphetamine, methamphetamine,
MDMA and MDEA. The peak areas were within 30% for
MDA.