Important notes:
(i) This work contributes to the assessment for this course and must be completed individually.
(ii) You must submit an identical hard-copy AND electronic copy (via TurnitinUK on Blackboard) of this assessment
otherwise you may receive a mark of zero.
(iii) Written answers must be in your own words and should be clear and concise.
(iv) Show your working at all times. The boxes provided should be sufficient but use additional paper if you want to.
(v) Cite all references used according to the guidelines.
MOLECULAR ANALYSIS
Part One – Sewage Epidemiology
Introduction
The widespread use of illicit drugs by humans and their consequent excretion in urine and faeces results in the presence of
these compounds and their metabolites in the aquatic environment including waste-, surface and drinking water. Comparable
with therapeutic drugs, illicit drugs can have adverse effects on aquatic organisms and to evaluate these effects, it is
important to monitor the presence of these compounds in the environment.
After use, illicit drugs are (partly) metabolized and eliminated from the human body through faecal or urinary excretion.
These excretion products (parent compounds and metabolites) will enter the sewage system and they can be detected in influent
wastewater. If the removal of these compounds in wastewater treatment plants (WWTPs) is insufficient, illicit drugs and their
metabolites may enter surface water through effluents. A number of controlled substances can be detected in wastewater.
However this worksheet will focus only on cocaine.
Measurements can be used to monitor trends in community drug use over time. Differences in concentrations of various drugs
indicate shifts in usage patterns and drug preferences. For example, daily measurements of cocaine and its metabolite
benzoylecgonine in wastewater have shown a significant increase in cocaine, amphetamines and ecstasy during weekend periods
when compared to weekdays and this reflects the known increment in usage at weekends. Determinations have been undertaken in
several European countries and results compared in the literature.
The general idea is that the quantity of a cocaine metabolite in wastewater can inform on usage patterns of cocaine across
different populations and at different times. Analysis is carried out after extraction by LC-MS.
The mass spectrum of cocaine
Using graph paper use the following fragment ion and abundance data to draw the mass spectrum of cocaine (two marks).
Fragment ion (m/z) Abundance
42 10
82 70
83 30
94 25
105 25
122 5
182 100
198 15
272 20
303 30
Label the molecular ion using the correct convention (one mark). Label the base peak (one mark).
Work out the masses of the following three ion fragments (three marks each).
Use C = 12; O = 16; N = 14; H = 1
Fragment A =
Fragment B =
Fragment C =
Back calculations
Cocaine is, in part, metabolised in the liver to benzoylecgonine (BZE) and ecgonine and these molecules are excreted in
urine. Only 1–9% of a cocaine dose is excreted in urine unchanged, while 35–54% and 32–49% is excreted as the metabolites BZE
and ecgonine methyl ester, respectively. Hence a mid-range value of 45% is identified for BZE and it is scaled up to give a
figure for the amount of cocaine consumed.
Describe the differences between the chemical structures of cocaine and benzoylecgonine, noting which molecule is the more
polar (five marks)?
Different methods can be used to back-calculate the amount of consumed cocaine from concentrations of cocaine and its
metabolites in influent wastewater. The following is based on the concentration of BZE, the major metabolite of cocaine,
which is present in wastewater at relatively high concentrations. From concentrations of BZE (ng/L) and with the flow rate
(L/day) of the wastewater stream, BZE loads (g/day) can be calculated. These loads are then further transformed into an
amount of cocaine consumed (g/day) taking into account the molecular mass ratio of cocaine to BZE (1.05) and the mid-range
excretion percentage of 45% as BZE. These two factors are combined to give a value of 2.33.
This leads to the following formula:
Cocaine (g day-1) = concentration BZE x flow rate/106 x 2.33
From the data below calculate the cocaine consumption for each city in g day-1 per 1000 inhabitants (two marks each).
Inhabitants BZE
ng L-1 Flow Rate
L day-1 Cocaine consumption
g day-1 per 1000 inhabitants
Gotham City 850000 1306 233303
Springfield 89847 410 29768
Sin City 157268 2130 57932
Sunnydale 63333 37 38712
South Park 80000 132 15703
If a line (single dose) of cocaine is c. 50 ± 10% mg of the drug, how many doses are there per day in total in the five
cities (five marks)? Include the error estimation.
Based on the data above how much cocaine is consumed in total in all five cities in one year (six marks)?
Identify any advantages and disadvantages of monitoring the metabolite, benzoylecgonine, rather than cocaine itself (six
marks).
Suggest possible uncertainties in the analysis of drugs in wastewaters that may invalidate attempts to understand patterns of
drug consumption in human populations. For example, some studies have suggested that the ratio of cocaine/BZE measured in
wastewater does not fully agree with the expected urinary ratios resulting from human metabolism (five marks). It might be
helpful to refer to Figure 1.
Briefly outline one experiment that you would undertake to test whether there is any loss or degradation of cocaine or
formation of cocaine metabolites during the residence time in the sewer before the water is sampled at the wastewater
treatment plant (five marks).
What is the implication, in terms of calculated cocaine consumption patterns, if hydrolysis of cocaine to BZE is found to be
a significant problem in wastewater prior to sampling (five marks)?
Part 2 – Faecal sterols and bile acids
Two main groups of organic compounds found in faecal matter can be used to monitor sewage, namely: 5ß-stanols and bile acids.
These marker compounds have been used as a proxy indicator of sewage pollution both ancient and modern. Both 5ß-cholestan-3ß
-ol and 5a-cholestan-3ß-ol are alteration products of cholesterol. The former molecule is also known as coprostanol.
Conversion to coprostanol is microbially mediated by enteric bacteria in the guts of mammals and effects a biohydrogenation
of the ?5 double bond yielding 5ß(H) rather than 5a(H) stereoisomers, the latter being the usual product of biohydrogenation
under aerobic conditions in the natural environment. Coprostanol is the major 5ß-stanol in human faeces constituting about
60% of the total sterol content and as such is the main component of interest in studies of domestic sewage pollution.
Derivatization of bile acids
The following figure shows the structures of four common bile acids. Prior to GC-MS bile acids must be derivatized.
Give possible reasons for the need to derivatize these molecules prior to GC-MS (five marks).
In one study of bile acids the carboxylic acid group was methylated using diazomethane, and the hydroxyl groups
trimethylsilylated. What is the mass of the molecular ion of deoxycholic acid derivatized in this way (eight marks)? Use C =
12; O = 16; H = 1; Si = 28.
Cholesterol
5ß-cholestan-3ß-ol and 5a-cholestan-3ß-ol give virtually identical mass spectra but have different chromatographic
properties. That noted how is it possible to distinguish these two molecules in a soil sample (six marks)?