The document discusses quantitative and qualitative analysis of toxicology compounds. In the qualitative analysis, four compounds were extracted from rabbit serum at different pH levels using liquid-liquid extraction and GC/MS. Two compounds were extracted at basic pH and one at acidic pH, while one compound was not extracted. In the quantitative analysis, reverse phase HPLC was used to analyze malathion concentrations in water samples. Calibration curves were made using standards and an internal standard. The sample analysis found a higher concentration than expected due to loss of product, which was corrected using the internal standard data. Improper pipetting was identified as the source of error.

1. Quantitative and Qualitative Analysis
of Toxicology
Justas Lauzadis

2. Introduction
Toxicology has been making an impact globally since the dawn of time. What
first began as a tool for hunting and capturing prey, soon evolved to political arenas and
the possible cause of the fall of Rome (HernBerg 2000). Toxicology has rapidly evoked
the use of highly advanced technological and groundbreaking science to better the quality
of life as well. Whether it is from lead poisoning diagnosis or to filtering known toxins
with the use of Cyanobacteria (Westrick 2008). Modern day equipment can sense just
micrograms of trace elements and even separate them based on their isotropic character.
But Toxicology is not only limited to environmental and agricultural science. Forensics
has grasped the technology and has since shined light upon drug related deaths. It was
calculated that in 2013, there were 43,982 drug overdose-related deaths in the US alone
(CDC 2015). Although it may sound easy to just take a sample and run it through a GC or
HPLC machine, the reality is much harsher. Strategies that are used to identify unknown
substance revolve around separating the compounds from the biological fluid by using
different chemical and physical characteristics.
In these two series of experiments, the goal was to use Quantitative and
Qualitative procedures to analyze compounds of interest. For the Qualitative portion of
the experiment, the goal was to do a liquid/liquid extraction of four different compounds
with different characteristics such as basicity and hydrophobicity from blood serum,
followed by identification with the use of GC/Mass Spectrometry. The Quantitative
analysis focused on concentration of Malathion in drinking water. This analysis would be
done with the use of high-pressure liquid chromatography, which would separate
compounds based on their hydrophobic interactions with a solid phase.

3. Methods
The first portion of this toxicology experiment was qualitative and involved the
use of the GC and Mass Spectrometer to observe what drugs were present in the 2mL of
Rabbit serum. In order to separate the individual drugs from the homogenous mixture, a
liquid-liquid extraction was done. The drugs were isolated in an organic layer of
methylene chloride by varying pH and then collecting organic layer, while the serum
stayed in the aqueous layer. The pH was measures by taking 3 drops of the serum and
placing it on pH paper, this was done opposed to just dipping to avoid contaminating the
sample. Collection was done for pHs of 3 and 10. Before the extraction and at the
desired pH, the serum and methylene chloride were vortexed for five minutes followed
by a two-minute centrifuge. The organic layer was then extracted and later evaporated
under nitrogen. After the evaporation of methylene chloride was complete, a few drops of
methylene chloride were added back into the sample and 1 µL was added into the GC
using a Hamilton syringe. The GC insertion site was at a temperature of 2500
Celsius. The GC oven temperature was monitored by a program, which increased the
temperature from 1000
Celsius to 2500
Celsius. The Stationary phase of the GC was a
silica gel based oil that coated tube inside the machine.
The next portion of the toxicological study was a quantitative analysis of
Malathion, a pesticide that was once widely used throughout much of Long Island and
the United States. Reverse Phase HPLC with a column containing C18as the stationary
phase and a mixture of 65/35 Methanol water was used for the mobile phase. The
Mixture of methanol and water was all pre made into one bottle versus the common
procedure of the machine extracting the exact amount from each tube separately to make

4. the desired ratio. HPLC instrument was calibrated by using known concentrations of
malathion at 10, 50 and 100 µg/ml. Methylparathion was used as the internal standard, of
which 50 µg/ml was added to the calibration standards and the unknown sample. The
calibration standards were passed through at an increasing concentration rate in order to
avoid repeated washing. The washing was only done prior to the addition of the unknown
water sample. 15 µl of the calibration standards as well as the unknown were eluted at a
flow rate of 2-4 ml/min. The results obtained would show differences in retention time
due to hydrophobic interactions.
Results/ Discussion
The qualitative portion of the analysis was based heavily on acid base extractions
in order to obtain the four different substances that had been mixed into the serum
sample. In Sample C the four components that were added were Phentermine,
Chlorpromazine HCL, Caffeine, and Pentobarbital. The initial first extraction was done
with NaOH and the two resulting compounds that were extracted were Pentobarbital
(figure 3) and Chlorpromazine (figure 2). The next extraction was then done with HCl to
yield Caffeine (figure 4) as the other substance in the serum. It was concluded after the
two GS analysis that there was in fact one substance missing. In order to figure out why it
was missing it was important to understand the chemistry behind the technique. When
doing organic layer extractions, the main principle is charge. If a substance were basic,
the best organic layer would be basic. In the basic layer the compound would be allowed
to freely float in its natural state, while the acidic molecules would be deprotonated and
brought up to the aqueous layer. When the other compounds are deprotonated, polarity
also becomes a driving force thus pushing the molecule into the water or aqueous layer.

5. The extractions were first started with base in order to prevent any disassociations of
nitrile or chloride groups. If the process was reversed compounds like Chlorpromazine
and Phentermine would degrade and alternative substances like Hydrochloric acid and
Ammonia would be detected. This however is not the case for the absence of
Phentermine. It is hypothesized that Phentermine was never effectively trapped in an
organic layer due to the pH’s that were examined. The Pka for Phentermine is 10.25
effectively (Drug Bank 2013). The compound is also not very soluble in water with only
18.6 grams only making it into 1 liter of solution of water. The use of excess mixing was
advised in order to create clear separation between peaks and insure full acid base
interactions. This information is a perfect representation of why the extractions needed to
be centrifuged. The original protocol did not call for this step, but after vortexing for fix
minutes we had noticed that there was no complete separation of the layers with a milky
white layer faintly separating the organic and aqueous layers. This observation was made
during both the basic and acidic extractions, but it should be noted that this phenomenon
was much more present in the basic extraction. This was also the layer and extraction in
which the missing phentermine compound should have been.
Although there were successful peaks for the drugs that we were looking for,
there were also long chain fatty acids that ended up in the analysis. These impurities are
very hard to avoid because the GC can pick up extremely minute quantities. The source
of these fatty acids is inevitably from the oils on a laboratory worker’s skin. No
significant levels of such impurities were discovered to create a content figure. The
percent match in the peaks from the Mass Spec can determine the effectiveness of the
extractions. Although this is not a direct representation of pureness, because of the

6. violent process of knocking electrons off a complex, it is a good starting point to
determine if the product was successfully isolated. Out of the three discovered
substances, Pentobarbital in figure 3, showed the smallest percent match of only 61. The
result could be do to possible deportations of the nitrile groups in the benzene ring core.
The Pka for Pentobarbital is 8.48 (Drug Bank 2013), which has the option to be
pragmatic if the pH of the organic layer is highly basic at 10-11 but not significantly
enough to cause the formation of another ring at a carbonyl oxygen and the penta-carbon
tail. The most probable cause for the lost is simple resonance, if accounted for, the
percentage match can be brought up to the high 90’s due to the addition of all similar
groups. Additional peaks in the Mass Spec, at similar abundances are isotopes of the
compound. An example of this can be seen in figure 2 with Chlorpromazine. Since
chloride is naturally present in both Cl2 and singular Cl forms, the mass spec has two
relatively significant peaks at 272.04m/z and 318.10m/z, the difference between them
being molecular weight of chloride.
Quantitative assays require much more accuracy, and this for this reason they
require a few fail-safe techniques. In this experiment we used a compound called
methylparathion. This compound has similar chemical characteristics to Marathon and
would help correct errors in loss of product extraction, inaccurate final dilutions, as well
as inaccurate injections volumes. In figure 5 and 6, the internal standard is the first peak
on the left hand side. In each figure, which represents the internal standard, it was
estimated that there was 50µg of it present. Figure 6 had disproved that hypothesis and
showed the importance of the internal standard first hand. The calculated loss of product
was 58.3 percent. Such a dramatic loss would need to be adjusted to obtain the correct

7. value. Since we only obtained 20.89µg of the internal standard, all the values were
required to be multiplied by a factor of 2.39 to reach the hypothetical 100% yield. After
the corrections, the concentration for Malathion was increased to 109.5µg instead of the
initial 45.79µg. If this sample were taken from a larger body of water, the concentration
would be above the line for what the EPA has established as “expected to be not
harmful”. Such concentrations could be toxic, leading to neuromuscular damage, to
athletes during exhibition events or during times of training. Increased exposure to
malathion has been linked to cause cognitive impairment as well (Dos Santos 2015).
This concentration of drug could also affect livestock, eventually infiltrating the body of
cattle, which could accumulate until the same cattle is found on our plate. This cycle is
commonly known as Bioaccumulation and is more prominent with sea found and levels
of lead and mercury.
The possible errors in this experiment can all be traced down to pipetting. The
water sample was run through the solid phase extraction at a brutally slow pace of
approximately 1 drop every 2-3 minutes. But this would prove to go to waste when one
crucial step was missed. Immediately after the extraction with the sample water was
done, 1ml of regular water was to be passed through to make sure that the all of the
Malathion was bound. This immediate step was missed and thus resulted in a loss of 60%
product. This was 40% more than the adjacent group that served as the “control” for this
unfortunate accidental experiment. The use of 50µg of methylparathion was the saving
factor in this experiment. Since the concentration was known, the results could be
corrected without falsifying the data. Possible improvements are hard to judge with
miscellaneous human errors interfering with the data. The best course of action would be

8. a repetition of the experiment with the same sample size. Since the 1ml of water added
the extraction showed to be crucial, the concentration of washing water and collecting
methanol can safely be doubled without the loss of product. The highly hydrophobic
interactions that the Malathion has with the solid phase are key behind this solid phase
extraction.

9. References
Centers for Disease Control and Prevention (2015). National Vital Statistics System
mortality data. http://www.cdc.gov/nchs/deaths.htm.
Dos Santos AA, Naime AA, de Oliveira J, Colle D, Dos Santos DB, Hort MA, Moreira
EL, Suñol C, de Bem AF, Farina M. Long-term and low-dose malathion
exposure causes cognitive impairment in adult mice: evidence of hippocampal
mitochondrial dysfunction, astrogliosis and apoptotic events. Arch Toxicol.
2015 Jan 25. [Epub ahead of print] PubMed PMID: 25618550.
Drug Bank (2013). Pentobarbital.
http://http://www.drugbank.ca/drugs/DB00312#properties
Drug Bank (2013). Phentermine.
http:// http://www.drugbank.ca/drugs/DB00191#properties
Hernberg Sven. (2000). Lead Poisoning in a Historical Perspective. AMERICAN
JOURNAL OF INDUSTRIAL MEDICINE 38:244±254 (2000)
Westrick JA. (2008) Cyanobacterial toxin removal in drinking water treatment processes
and recreational waters. Adv Exp Med Biol. 2008;619:275-90. doi: 10.1007/978-
0-387-75865-7_13. PubMed PMID: 18461774.

10. Figure1: Malathion Standard Curve. A increasing value of Malathion was used in order to create a standard
curve in which the unknown could be directly compared to. In order to reduce errors in the experimentaldata,a
internalstandard was also added at 50µg for each value. When comparing retrieved concentrations, the
concentration would need to be adjusted to the appropriate value for 100% yield. An R squared value of .99978
the data was deemed to be reliable and safe to extrapolate data from.

11. Figure2: The correspondingMass spectrometry and structure/retention of Chlorpromazine. this compound was
measured in the basic extraction of the serum sample. The purity of the sample is deemed to be quiet high due to the
93.62 percent chance probability of a correct structure match. The most prominent peaks for the mass spec are 318.10
m/z. There were two prominent retention times of 5.25 min and 8.28 min. these are all accounted for with different
hydrophobicinteractions of the substance.

12. Figure3: The correspondingMass spectrometry and structure/retention of pentobarbital. this compound was
measured in the basic extraction of the serum sample exactly like Chlorpromazine. The purity of the sample is not ideal with
only a 61.56 percent chance probability of a correct structure match. This could be attributed to the pHat which the
extraction was done. The most prominent peaks for the mass spec are 140.98 and 156.03 m/z.There were two prominent
retention times of 5.25 min and 8.28 min. these are all accounted for with different hydrophobicinteractions of the
substance.

13. Figure4: The correspondingMass spectrometry and structure/retention of Caffeine. the compound was
measured in the acidic extraction of the serum.The purity of the sample is extremely high with 98.23 percent
chance probability of a correct structure match. The most prominent peaks for the mass specare 109.02 and
194.08 m/z. There were two prominent retention times of 4.85 min and 5.23 min. these are all accounted for with
different hydrophobicinteractions of the substance

14. Std 10 Name
Retention
Time Area % Area Height
1 9.106 6893026 87.76 408920
2 9.881 961469 12.24 53621
Std 50 Name
Retention
Time Area % Area Height
1 9.228 5756957 50.51 360557
2 10.009 5640650 49.49 309525
Std
100 Name
Retention
Time Area % Area Height
1 9.088 6212065 35.68 372339
2 9.871 = 64.32 591714
AU
0.00
0.20
0.40
0.60
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00
9.088
9.871
AU
0.00
0.10
0.20
0.30
0.40
Minutes
2.00 4.00 6.00 8.00 10.00 12.00
9.106
9.881
AU
0.00
0.10
0.20
0.30
Minutes
2.00 4.00 6.00 8.00 10.00 12.00
9.228
10.009
Figure5: HPLC retention time and areas for Standards. Results were obtained by increasing the concentration
from 10µg-100µg in order tocalibrate the apparatus.From these values figure 1 was complied and a standard curve
was made.The first peak( left to right) observed is the internalstandard methylparathion. Diagrams go from left to
right in increasing concentration.

15. sample Name
Retention
Time Area % Area Height
1 9.108 2246984 35.54 141450
2 9.888 4075650 64.46 219649
AU 0.00
0.10
0.20
Minutes
2.00 4.00 6.00 8.00 10.00 12.00
9.108
9.888
Figure6: HPLC results for Malathion Sample. The resulting data gave us a finalconcentration of 45.789 µg of
malathion collected and only 20.89 µg of the internalstandard.Compensations were done to account for the
tremendous amount of error. Approximately 60 percent of the product was lost. These results were obtained
from using the standard curve in figure 1.