1. CT3 Educational Material
John Mackey
Oct 2016
Clinical toxicology III is a lab at ARUP that performs various qualitative toxicology analysis for
clients nationwide. There are two major categories of patient samples that clinical toxicology III
(CT3) performs their chemistry assay’s and analysis’s on: pain medication management and
drug abuse during pregnancy. Pain management assays are performed using serum plasma or
urine. Drug abuse during pregnancy is qualitatively evaluated with meconium stool and
umbilical cord tissue. The purpose of this paper is to briefly explain basic concepts of the
chemistry behind CT3 as well as serve as introductory and teaching material for employees.
In the science industry, especially healthcare and medical lab testing, it is important to note the
extreme importance of ethics. There are several large bodies of legislation and regulation that
govern CT3. The most notable are HIPPA regulations and what constitutes PHI. HIPPA stands for
Health Insurance Portability and Accountability Act, and is a law designed to provide privacy
standards for medical records of patients. PHI stands for Protected Health Information. PHI
includes, but is not limited to specific information that can identify a patient such as names,
telephone numbers, email, and geographic locations of the patient smaller than a state.
ELISA
Meconium and umbilical cords first screened with a biochemistry technique called ELISA. A low
and high positive for each screening is first required to establish a linear trend (figure 1). Data
can receive a qualitative pass/fail result based on the interpretation by the data bench
technologists on their TOF instruments.
Figure 1
ELISA stands for Enzyme-Linked Immunosorbent Assay. All
ELISA assay’s use plastic coated wells and operate on the
basis of antibody/antigen affinity. Sandwich ELISA is added
and the antigen binds to capture antibody. Substrate is
added and converted to a detectable form, usually light.
In addition, ELISA assays can be indirect or direct. Indirect
capture is where the ‘primary’ antibody is directly detected
by a secondary antibody (can be more than 1). Our lab only
uses direct, which detects with the primary antibody only.
Direct ELISA is quick, and has low cross reactivity. However, the signal amplification is smaller
and it can be expensive.
In addition CT3 only uses competitive ELISA. In competitive ELISA labelled antigens compete for
the primary antibody binding sites with the sample antigen. The patient antigen does not
contain a detectable marker (light in our case) and competes for binding sites with the antigen

2. the technologist adds. Patient samples that give off less light, and appear more clear, contain
the metabolite’s and analytes of interest, while the colored samples (yellow) do not. (Figure 2)
Figure 2
In CT3, this process starts with pre-fabricated 96 well plates. The plate is blocked with
detergent in order to prevent nonspecific binding. This makes it so the only the antibody’s are
available for binding, and makes the antigen/antibody interaction even more specific increasing
accuracy. The sample is then mixed with an enzyme conjugate, which is the enzyme or
metabolite that ‘lights up’ when there is substrate and enzyme conjugate interaction. The
patient sample and enzyme conjugate are then added together, and the specific metabolite of
interest competes with the color changing enzyme conjugate. Since there is a limited number of
specific antibodies and binding locations, the color is proportionately equal to the number of
metabolites/analytes found in the patient in proportion to the enzyme conjugate. In other
words, this means a high level of drug analyte’s ‘drowns’ out the signal of the added color
changing enzyme conjugate.
The sample is then washed and a colorless substrate specifically binds to the conjugated
enzyme. In our lab, a strong acid changes the color from blue to yellow. Colorless means
metabolites competed for the limited antibody spots on the ELISA plate well. Each analyte
needs a different plate well. The MEC9 test uses nine different plate wells to determine which
drugs (if any) the patient has in their system.
Patient samples that receive an initial positive screen are then analyzed more accurately by the
TOF mass-spectrometer.
PAIN HYB U
Pain management testing (PAIN HYB U) is done using urine or serum plasma patient samples.
Again, data is controlled for using a linearized graph established from positive and negative
spikes. In the PAIN HYB U test however, the analytes of interest have already undergone a
xenobiotic metabolic pathy called glucuronidation. Glucuronidation is the biochemical process
of attaching glycosidic bonds to toxic and foreign substances. This makes them less toxic
(usually) and makes it more water soluble. This happens in the liver, and the process leads the
metabolites to the kidneys and eventually urine.

3. This metabolic pathway is important because it is difficult to detect drug metabolites of interest
in the glucuronidated form. In our lab, the glyosidic bond is broken by B-Glucuronidase which
cleave glucuronic acid bonds. We test to see if the glyosidic bond is gone by controlling each
sample with resorufin-glucuronide. If resorufin in it’s pure form is missing, then it means that
hydrolysis did not occur.
After the JANUS (pipetting robot arm) has pipetted each patient sample with the TOF mass-
spectrometer controls, positive/negative, and resorufin-glucuronide the plate is then passed
through an SLE column. Supported Liquid Extraction (SLE) is done automatically, and is a
column designed to extract analytes from biological fluids to allow high analyte concentration
recoveries. Larger and heavier bio material is trapped (proteins, phospholipids) using diatoms
(figure 3). The organic layer is then eluted using ethyl acetate, dried, reconstituted, and sent
through a TOF instrument to analyze.
Figure 3
TOF MASS SPECTROMETRY
TOF mass spectrometry operates on several simple
principles of physics that are perfected the point of
extreme accuracy that allow us to identify individual
molecules, and match its’ ‘fingerprint’ to a database
to find out exactly what the molecule is.
First the sample is run through a liquid chromatography phase. The column material is made of
silica gel pores that are similar to sand grains. This allows separation of polar and nonpolar
molecules, since polar or hydrophilic groups are more attracted to the silica gel (figure 4). This
means polar compounds, like water, take longer to elute through the column.
Figure 4
After passing
through the
column, the analyte
of interest is
fragmented into
smaller pieces by
passing a small
stream of the
solution through a
high voltage. After passing through a quadrapole screen, and multiple slit’s for additional
filtering, the ions are brought above an oppositely charged plate that ‘launches’ the fragmented
ions high into a vacuum tube (figure 5). Based on the time it takes to be detected, a charge to
mass ratio can be deduced, which allows comparison to well established mass spectrometry
databases. Our TOF mass spectrometer’s can identify compounds based on polarity, and the
unique ‘fingerprint’ found in mass spectrometry databases.

4. Figure 5
Reference’s
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http://www.interchim.fr/cat/Article%20LCGC_SupportedLiquidExtraction.pdf