PHRM2041PracManual Version 20140723

THE UNIVERSITY OF QUEENSLAND
SCHOOL OF PHARMACY
PHRM2041
DRUG DISCOVERY A2
DRUG ANALYSIS
LABORATORY MANUAL
Semester 2, 2014
Copyright 2014
School of Pharmacy, The University of Queensland
Name:
Syndicate number:
PHRM 2041 DRUG ANALYSIS
CONTENTS
Page number
PRACTICAL CLASS SCHEDULE ................................................................. 3
LABORATORY RULES ............................................................................. 4
LEARNING OBJECTIVES ......................................................................... 6
ABSENCES AND ASSESSMENT................................................................ 6
CALIBRATION CURVES & REGRESSION ANALYSIS ................................. 7
Experiment 1
VISIBLE SPECTROPHOTOMETRIC ANALYSIS
OF PROCAINE
10
Experiment 2
SPECTROFLUOROMETRIC ANALYSIS OF QUININE
18
Experiment 3
QUALITY CONTROL OF PARACETAMOL TABLETS:
UNIFORMITY OF CONTENT BY ULTRAVIOLET
SPECTROPHOTOMETRIC ANALYSIS
26
Experiment 4
THERAPEUTIC DRUG MONITORING:
ASSAY OF CARBAMAZEPINE BY REVERSED PHASE HPLC
31
Experiment 5
QUALITY CONTROL OF ACTIVE PHARMACEUTICAL INGREDIENTS
USING INFRARED SPECTROSCOPY
37
Page 2
PHRM2041 PRACTICAL CLASS SCHEDULE

Syndicates
WK DATE
1-5
6-10
11-15
16-20
21-25
26-30
31-35
36-40
41-45
46-50
51-55
56-60
61-65
66-70
71-75
76-80
1 Aug
No Prac
8 Aug
15
Aug
22
Aug
29
Aug
5
Sept
12
Sept
19
Sept
26
Sept
No Prac
3
4
5
6
7
8
9
Mid semester break

10
10
Oct
11
17
Oct
Structure Elucidation Tutorial
A two hour tutorial held at PACE. Check mySI-net for room number and time. The
material covered may be examined in Blackboard Quiz #2: Structure Elucidation &
Drugs in Sport and in the end of semester exam.
Drugs in Sport Self-Directed Learning Activity
12
24
Oct
13
31
Oct
This activity is timetabled here, however, you can complete it any time after youve
covered the Drug Analysis lectures (i.e. any time after Week 7). This activity can be done
online from anywhere with internet access. Please ensure that you finish this activity on
time in preparation for Blackboard Quiz #2: "Structure Elucidation & Drugs in Sport".
Refer to the Blackboard site for further information.
No Prac
It is the responsibility of every student to contribute to all components of the experiments and to
complete the Laboratory Worksheets in their own Laboratory Manual, because the theoretical concepts
may be examined in Blackboard Quiz #1: Concepts from the Lab Worksheets and in the end of
semester exam. If a student is absent from a practical class, it is their own responsibility to obtain a copy
of a completed Laboratory Worksheet from their colleagues for the purpose of revision. Refer to the
ECP and the Blackboard site for additional information.
Page 3
SCHOOL OF PHARMACY
LABORATORY RULES
1. DRUGS MUST NOT BE USED FOR SELF MEDICATION.
2. Students must wear CLEAN, WHITE, INTACT LABORATORY COATS during
practical sessions.
3. Students must wear CLOSED IN PROTECTIVE FOOTWEAR during practical
sessions.
4. Long hair must be tied back during practical sessions.
5. No smoking, eating or drinking in the laboratory.
6. Brief cases and bags, etc., should be left in the allocated places in the
laboratories.
7. Use SAFETY GLASSES and DISPOSABLE GLOVES where necessary for all
procedures.
8. Know the location of the fire exits and location of the fire-fighting appliances in
and near the laboratories.
9. Know the location of the safety showers and eye wash stations.
FIRST AID
10. In the event of chemical spills on skin, thoroughly wash the affected area with
copious quantities of water. Notify demonstrator immediately.
11. Eye injuries are always serious. Treatment requires immediate and prolonged
flushing with water (20 minutes minimum). Medical advice should be obtained
for any eye injury.
12. Sharps injuries Notify demonstrator immediately. Wash the wound and
encourage bleeding. Health Services should be visited.
13. Report all injuries to the tutor. First aid will be administered.
All accidents must be reported to the demonstrator and recorded online using
the Injury, Illness & Injury Reporting System (see UQ OHS website).
WASTE DISPOSAL
14. Instructions regarding disposal of all laboratory wastes must be strictly observed.
Failure to do so will result in fines being levied on the School by the University or
City Council.
15. Organic solvent wastes must not be discarded via the sinks due to their adverse
impact on the plumbing and the environment. Place these wastes into the
marked containers provided.
16. All gloves are to be placed in the yellow pathological bins.
17. Broken glassware must be reported to a demonstrator. Small pieces of broken
glassware can be placed in the sharps containers. Larger pieces should be wrapped in
newspaper, taped securely, and put in the yellow pathological waste bins .Used
pasteur pipettes and coverslips are also placed in the sharps containers.
Page 4
CONDUCT IN LABORATORIES
18. Students are expected to conduct themselves in a professional manner when
performing practicals.
19. Students are expected to arrive at practical sessions PREPARED and ON TIME.
Practical session times will be strictly observed.
20. Students will be excluded from prac classes if more than 10 minutes late for class
21. Students may not work in the laboratories outside normal class times except
with special permission from academic / scientific staff responsible for that
laboratory.
22. The equipment in the laboratories is expensive, precision made and requires
careful treatment. Ask to have all equipment demonstrated before use.
23. Use correct weighing sequence when using analytical balances. Clean all
balances IMMEDIATELY after use.
HOUSEKEEPING
24. All items which are used in the course of conducting an experiment must be left
in the manner in which they were found i.e. CLEAN AND IN THE APPROPRIATE
PLACE. Bench checks will be carried out to ensure this is done.
25. Return all chemicals to their proper place immediately after use. When a
stock-bottle is almost empty draw this to the attention of a staff member so that
the material can be reordered or the bottle refilled.
26. Keep sinks, benches and common user areas CLEAN.
PREGNANCY
27. Students who are pregnant, or are planning to become pregnant, must
contact the course coordinator before starting practical sessions. Some
controls may need to be put into place to reduce the health risks of contact
with certain chemicals.
Page 5

PRACTICALS
Learning Objectives
1.
To become familiar with methods of drug analysis used commonly in:

(i)
(ii)
(iii)
(iv)
Pharmaceutical industry for in-process quality control and quality assurance of

manufactured drug products;
Therapeutic drug monitoring;
Drug metabolism studies and
Pharmacokinetic studies.
2.
To build on experience gained in previous semesters of the pharmacy course in

quantitative methods of analysis using Beer's law, linear regression and inverse
prediction.
3.
To develop a sound foundation in quantitative methods of drug analysis, for application

to quality control testing of the pharmaceutical products that are studied in the fourth
year projects (PHRM4021 and PHRM4022).
Absences and Assessment

Refer to the Electronic Course Profile (ECP) for detailed information about this course including
absences from practical classes and the submission of Laboratory Worksheets.
Page 6
Calibration Curves & Regression Analysis (for experiments 1 & 4)

Calibration curve
You should sequence your data as shown in the example below. Note that the duplicates
are listed in the same columns. Although the concentration is in units of mg/L for this
example, you should use whichever units of concentration are appropriate for your
experiment.
Standard Solutions
Nominal
Concentration (mg/L)
1
2
3
4
5
6
1
2
3
4
5
6
Actual
Concentration (mg/L)
1.033
2.066
3.100
4.133
5.166
6.200
1.025
2.050
3.075
4.100
5.125
6.150
Absorbance
0.12
0.22
0.33
0.43
0.52
0.65
0.09
0.18
0.27
0.37
0.48
0.56
From this data we can produce the calibration curve shown below. Note that all of the
duplicate standard values are plotted as a single series and then a trendline is added. When
possible, the actual concentration is used (based on the accurately weighed amount of
compound used to produce the stock solution, from which the standards were obtained by
dilution), rather than using the nominal concentration. Do NOT average the duplicates. Do
NOT exclude any data points. Do NOT include the unknowns. Do NOT force the curve
through zero.
Page 7
Regression analysis
Regression analysis is required for experiments 1 and 4.
Select Data then Data Analysis, select Regression, input your ranges by dragging the
cursor over the data; select the Confidence Level checkbox and type in 99 (this will give
you both the 95% and 99% confidence levels).
The output of regression analysis is shown on the following page. Some additional text has
been added to explain how you should apply the analysis to the PHRM2041 experiments.
Installation of the data analysis pack (if required)

You need to find Excel "add-ins" in the software.
Excel2010: Click on File, select Options, select Add-Ins, click on the Go button that is
beside Manage: Excel Add-Ins, select the Analysis ToolPak, click on Ok. A new Data
Analysis option should appear within the Data tab.
Excel2007: Click on the Office Button, select Excel Options, select Add-Ins, click on the
Go button that is beside Manage: Excel Add-Ins, select the Analysis ToolPak, click on
Ok. A new Data Analysis option should appear within the Data tab.
Excel2003: You will need to go to the Data menu and select Add-ins, then select
Analysis ToolPak.
Excel for Mac 2011: The data analysis pack is not available. A free third party app,
StatPlus:mac LE from AnalystSoft, is available but is not supported in this course.
Page 8
Page 9
Experiment 1: VISIBLE SPECTROPHOTOMETRIC

ANALYSIS OF PROCAINE
Before coming to the practical class ensure that you are familiar with the content of the
lecture on Ultraviolet/Visible Spectroscopy and read the section in the front of the lab manual
about Calibration Curves & Regression Analysis. Additional learning resources (e.g. videos)
are available in the Practicals section of the PHRM2041 Blackboard site.
Each syndicate must submit one Laboratory Worksheet to the tutor at the end of the
practical class. Reference materials and/or your tutor are available to help you answer the
questions. It is the responsibility of every student to contribute to all components of the
experiment and to complete the Laboratory Worksheet in their own Laboratory Manual,
because the material covered may be examined in Blackboard Quiz #1: Concepts from the Lab
Worksheets. Refer to the ECP for additional information.
Learning Objectives
1. To demonstrate the use of derivatisation (chemical coupling) to alter the light
absorbing characteristics of a given drug in solution.
2. To develop expertise in the accurate preparation of a range of concentrations of dilute
drug solutions.
3. To apply the Beer-Lambert law to a range of dilute solutions of derivatised procaine
and to produce a calibration curve between visible absorbance and procaine
hydrochloride concentration.
4. To use linear regression and inverse prediction to determine the concentration of an
unknown solution of procaine hydrochloride.
5. To critically compare ultra-violet (UV) and visible (VIS) methods of quantitative analysis
of procaine hydrochloride, particularly with respect to sensitivity and specificity of
quantitation, and potential sources of error.
Theory
Spectrophotometric assays make use of the light absorption characteristics of the drug (or drug
derivative) being assayed. Absorption of radiation by a drug in solution can be quantitatively
measured as:
(1)
% transmittance,
T = IT/I0 x 100
or
(2)
absorbance
A = log (I/T) = log I0/IT
Absorbance is usually preferred because most drugs obey the Beer-Lambert law (Beers law),
A = .b.c
Page 10
which describes the simple linear relationship between absorbance (A) and drug concentration
(c). Aside from concentration, the absorbance also depends on two constants: the path length
of the cuvette (b); and the absorptivity coefficient () which is an instrinsic property of the
drug that indicates how strongly it absorbs light at a given wavelength. The absorptivity
coefficient () is constant for a specific wavelength and drug, but the units of can vary
depending on the units used to measure the path length of the cuvette (b; usually in cm)
and the concentration of the drug (c; usually mol L-1 or % w/v). Most commonly, the molar
absorptivity coefficient molar is used with units of L mol-1 cm-1, and sometimes 1% is used
with units of 100 mL g-1 cm-1.
Many deviations from Beers law are known, for example, deviations occur at high
concentrations of drug due to interactions between the drug molecules and alterations to the
refractive index of the solution. The linear relationship between absorbance and concentration
should be verified experimentally by constructing a calibration curve.
Some drugs which do not absorb UV or visible radiation can still be assayed
spectrophotometrically by coupling chemically (derivatizing) with a compound that does. Drugs
that inherently absorb UV radiation can also be chemically derivatised to shift their absorption
spectrum into the visible region because derivatisation can often increase assay sensitivity and
selectivity. In this experiment, procaine which absorbs UV radiation at 290 nm is derivatised to
form an azo dye that absorbs visible radiation.
Page 11
Experiment overview
1. Procaine hydrochloride standard solutions and unknowns will be prepared and derivatised
to form an azo dye that absorbs visible radiation.
2. A calibration curve of visible absorbance of derivatised procaine solution vs procaine
hydrochloride concentration will be prepared using linear regression.
3. The concentration of an unknown procaine hydrochloride solution will be determined by
inverse prediction.
NaNO2
H2N
O
O
CH2CH3
N
O
H2SO4
CH2CH3
CH2CH3
CH2CH3
NH2
NH
HN
H2N
N
N
O
O
CH2CH3
CH2CH3
Derivatisation of procaine to form an azo dye that absorbs visible radiation.
Preparation of Reagents
The reagents required are prepared as follows 1.
Derivatizing reagent: Prepare accurately 100 mL of

0.1% N-(1-naphthyl) ethylenediamine dihydrochloride.
2.
Standard procaine hydrochloride solution: In duplicate, prepare accurately 100 mL of

0.1% procaine hydrochloride (0.1% is the nominal concentration; record the
exact amount weighed and calculate the actual concentration), then accurately
dilute this solution 1 in 20 to obtain 100 mL of ~0.005% procaine hydrochloride
(calculate the actual concentration).
3.
Ammonium sulfamate solution: Prepare 100 mL of approx. 0.5% ammonium

sulfamate.
4.
Sodium nitrite solution: Prepare 100 mL of 0.1% sodium nitrite.
Page 12
Preparation of procaine derivatives

1.
In duplicate, pipette 0 (blank), 1, 2, 3, 4, and 5 mL of stock solution (~0.005% procaine

hydrochloride standard solution) into 50 mL volumetric flasks.
2.
Assuming that the concentration of your unknown solution is approximately 2%, dilute
it to the concentration of your standard solution (~0.005%). In duplicate, pipette 2 mL
of your diluted unknown sample into another 50 mL volumetric flask.
3.
Add 1 mL of 2 M H2SO4 followed by 5 mL of sodium nitrite solution to each of the 14

flasks, shake well and allow to stand for 5 mins. This generates nitrous acid which
reacts with the procaine aryl amine to form a diazonium salt.
4.
Add 5 mL of ammonium sulfamate solution to each flask, shake, and let stand for 2
mins. This destroys the excess nitrous acid which would react with the derivatizing
reagent in the next step. NH4+ H2N-SO3- + HONO N2 + SO42- + H+ + H2O + NH4+
5.
Add 5 mL of derivatizing reagent solution to each flask, shake well, and dilute to volume
with water. The diazonium salt formed in step 3 reacts with the derivatizing reagent to
form an azo dye. The colour is stable for several hours, the blank should be colourless.
Determination of analytical wavelength

Using the most concentrated standard solution, scan the region 460-600 nm to
determine the wavelength of maximum absorbance (max) which will be the analytical
wavelength.
Analysis of unknown
Measure the absorbance of each standard solution and the unknown solution, relative
to the blank, at the analytical wavelength.
Page 13
Expt 1 Worksheet - Syn #____ Names_________________Date_____

One copy per syndicate to be submitted to the tutor at the end of the practical class.
1. Complete the table below (2 marks)
Duplicate 1
Duplicate 2
Mass of procaine HCl in 100 mL:

Concentration (%):
Concentration (%) after 1 in 20
dilution (stock solution):
Volume of stock
Procaine hydrochloride
max = ________ nm
procaine HCl
concentration (%)
soln. added to
Absorbance at max
Duplicate
the 50 mL vol.
Duplicate 2:
1:
Duplicate 1: Duplicate 2:
flask (mL):
Dilution factor:
0 (blank)
n/a
0 (blank)
0 (blank)
1
2
3
4
5
Initial dilution of
procaine HCl
Final dilution of
Absorbance at max
sample of
procaine HCl
Overall dilution factor
unknown
sample of
for the sample of
concentration to
unknown
Unknown
Unknown
~0.005%:
concentration: unknown concentration: duplicate 1: duplicate 2:
Vol. of sample
Vol. of sample
added to the
added to the
flask (mL):
flask (mL):
2
Vol. of the flask
Vol. of the flask
Average
(mL):
(mL):
absorbance:
50
2. Refer to the section in the front of the lab manual about Calibration Curves & Regression
Analysis. Using Microsoft Excel, present the calibration curve to your tutor as a graph of
absorbance vs concentration (%) using linear regression. What is the equation of the linear
trendline (y = mx + c)? (Optional: For you own records, you can also sketch the same graph
using graph paper provided below) (1 mark)
3. Report and comment on the meaning of the R2 value for your calibration curve? (1 mark)
Page 14
4. The plot of absorbance vs concentration is a straight line (y = mx + c). Which terms in

Beers law (A = .b.c) correspond with y, m, and x; and for this experiment, what is the
value of b and what is the value of 1% ? (1 mark)
y is _____ ; m is _____ ; x is _____;
b = _____ cm; 1% = _____ dL g-1 cm-1.
5. If a calibration curve of absorbance vs procaine hydrochloride concentration (mol L-1) is
linear with the equation y = 240000x for the derivatised procaine, what is the value of the
molar absorptivity coefficient (molar)? Assume that a cuvette with a pathlength of 3 cm was
used. (1 mark)
molar = _____ L mol-1 cm-1.

6. Calculate the concentration (%) of procaine hydrochloride in the original unknown sample
using the linear equation? (1 mark)
Analysis. Conduct regression analysis to calculate the 95% confidence limits for the
concentration (%) of your unknown and complete the following statement:
We can be 95% confident that the concentration of the sample lies between _____% and
_____%. (1 mark)
8. In the figure below, circle the chromophores (the light absorbing part of the molecule)
and fill in the blanks. Explain the advantages of chemical derivatisation by comparing: (a) the
E1% values for procaine and the derivative; and (b) the max values for procaine and the
derivative. (2 marks)
Page 15
H 2N
azo dye derivative of procaine
HN
procaine
H 2N
N
O
O
CH2CH3
O
CH2CH3
From this expt:
CH2CH3
CH2CH3
Lambda max = ~290 nm

Lambda max = ________
Region of the electromagnetic spectrum:
Region of the electromagnetic spectrum:
UV or visible or infrared? ___________
UV or visible or infrared? ___________
1% = ~700 dL g-1 cm-1
1%
The advantages of chemical derivatisation are:
Page 16
= ___________
Page 17
Experiment 2: SPECTROFLUOROMETRIC ANALYSIS

ASSAY OF QUININE
lecture on Fluorescence. Additional learning resources (e.g. videos) are available in the
Practicals section of the PHRM2041 Blackboard site.
Learning Objectives
1. To develop expertise in the accurate preparation of dilute drug solutions in a range of
concentrations.
2. To determine the wavelength of maximum emission for the fluorophore quinine bisulfate,
given the excitation wavelength of 350 nm.
3. To determine and critically compare the relationship between fluorescence intensity and
quinine concentration for the concentration ranges 0.2 2 mg/L and 0.2 - 5 mg/L.
4. To use linear regression and inverse prediction to determine the concentration of an
unknown solution of quinine bisulfate.
Theory
The term fluorescence refers to the emission of radiant energy by a molecule following initial
UV absorbance to put the molecule into the excited state.
The relationship between concentration and fluorescence intensity is related to Beer's Law:
Where:
F = K PoELC
F
K
Po
E
L
C
=
=
=
=
=
=
=
fluorescence intensity
constant
quantum efficiency of fluorophore
intensity of incident radiation
absorption coefficient
path length
concentration
Page 18
Fluorometric techniques can readily result in the quantitation of material at very low
concentration, and may be 1 000 to 10 000 times more sensitive than absorbance methods.
Experiment overview
1. The fluorescence (emission) spectra of quinine bisulfate will be obtained to find the
relevant analytical wavelength.
2. A calibration curve of fluorescence intensity (FI) of quinine solution vs quinine
concentration will be prepared using linear regression.
3. The concentration of an unknown quinine solution will be determined by inverse
prediction.
Note: The fluorescence spectrophotometer is to be operated following demonstration by the
tutor.
Preparation of the standard solutions

Using the quinine stock solution (20 mg/L), dilute in duplicate 1, 2, 3, 4, 5, 10, 15, 25 mL
aliquots to 100 mL with 0.1 M sulfuric acid. Label each flask with the concentration of the
standard solution in units of mg/L.
These standards will be used to construct two calibration curves: Curve 1 all 8 standards;
Curve 2 the 6 lowest concentration standards.
Preparation of the unknown quinine samples

1. (a) You have been provided with an unknown quinine sample. The concentration of the
unknown sample is approximately 50 mg/L (between 20 and 80 mg/L). Does this
concentration fall within the range of the standard solutions used for Curve 2 (i.e. within
the range of the 6 lowest concentration standards)?
(b) If it does not fall within the concentration range, proceed to step 2. If you think it does
fall within the concentration range, check your calculations with the tutor.
2. In duplicate, dilute an aliquot of the unknown quinine sample so that the concentration
is within the range of Curve 2. This will ensure that the sample gives a suitable fluorescence
intensity that lies within the ranges of both calibration curves.
Page 19
Determination of Emission Wavelength
Open the Cary Eclipse folder on the desktop

Select Scan
Click on Setup on the left hand side of the screen
Set data mode to Fluorescence
Set Scan Setup mode to Emission
Set the X mode to Wavelength (nm)
Enter 350 nm as the Excitation value
Enter an Excitation slit (nm) value of 5 and an Emission slit (nm) value of 5
Enter a Start (nm) value equal to the excitation (nm) value plus the sum of the slits i.e
360 nm. The Stop (nm) value should be set 200 nm greater than the Start (nm) value
Set the Scan control to medium
Click OK to confirm any changes made
Place the blank in the sample compartment, press Zero, then remove the blank
Place the least concentrated standard in the sample compartment. Click on Start
(traffic light symbol). Enter a sample name (e.g. scan) and select OK. The scan will
commence and the trace will appear in the Graphics area
What is the wavelength of maximum emission? Check the value with the tutor.
Analysis of the standard solutions and the unknown quinine sample

Note: It is important that you record all data values on your worksheet.
Open the Cary Eclipse folder on the desktop

Select Concentration
Click on Setup on the left hand side of the screen
In the Cary tab
Set the excitation to 350 nm
Set the emission to the value determined from your scan
In the Standards tab
- change units to mg/L
- set the number of replicates to 2
- set the number of standards to 8
- enter the concentration of the standards in the table
- check that standard averaging is highlighted
In the Samples tab set the number of samples to 2, replicates to 1, and name your
samples (your unknowns)
Click OK
Page 20
Place the blank in the sample compartment press Zero, then remove the blank
Click on Start (traffic light). The Standard/Sample Selection dialog box will be
displayed by default all standards and samples are selected
Click OK to clear the dialog box
Follow the prompts and place the appropriate standards and unknown samples in
the sample compartment. Each time you press OK, the fluorescence intensity is
reported
Record all data values on your worksheet, including the correlation coefficient - R2
Click on Recalculate and delete the 2 highest standard concentration values by
changing them from Yes to No in the table and click OK
Record the recalculated correlation coefficient- R2
Page 21

1. What is the wavelength of maximum emission (em) for quinine? (1 mark)
Volume of stock
(20 mg/L) quinine
soln. added to
Dilution factor
the 100 mL vol.
flask (mL)
Quinine
concentration
(mg/L)
Fluorescence intensity
Duplicate 1
Duplicate 2
1
2
3
4
5
10
15
25
R2 value for all standards:
Volume of
sample of
unknown
concentration
added to the vol.
flask (mL)
Volume of the
volumetric
flask (mL)
R2 value for six standards of lowest concentration:

Dilution factor for
the sample of
unknown
concentration
Fluorescence intensity
Unknown
duplicate 1
Unknown
duplicate 2
Average FI:
3. Using graph paper, plot the calibration curve of fluorescence intensity (range: 0 to 1200) vs
concentration using all of the data in the table above. Do not average the FI values: plot each of
the duplicate values on the one graph. Sketch the line of best fit on the graph such that
approximately half the data points are on each side of the line. (1 mark)
Page 22
4. Considering only the six standards of lowest concentration, sketch a second line of best fit on
the graph such that approximately half of those data points are on each side of the line, and
using this line what is the concentration (%) of quinine in the original unknown sample? (1
mark)
5. Is the exciting light higher or lower in energy than the emitted light? Explain your answer by
considering the excitation wavelength (ex), the emission wavelength (em), and the equation E
= h c / . (1 mark)
6. Why is there a difference in energy between the exciting light and the emitted light? Explain
your answer by considering the Jablonski diagram and vibrational relaxation. (1 mark)
Page 23
7. What is the meaning of the R2 value of a calibration curve? According to the R2 value, which
dataset (all stds or 6 lowest conc. stds) does the linear model fit best? Why does the calibration
curve deviate from linearity at high concentrations? Explain your answer by considering
quenching and specifically concentration quenching. (2 marks)
8. Examine the cuvettes used for

this expt (fluorescence) and those
used for expt 1 (visible absorption).
Which cuvette (A or B) is used for
fluorescence and which is used for
absorption spectroscopy? Explain
the reason for the difference
between the two cuvettes. (1 mark)
Page 24
Page 25
Experiment 3: QUALITY CONTROL OF PARACETAMOL

TABLETS: UNIFORMITY OF CONTENT BY ULTRAVIOLET
SPECTROPHOTOMETRIC ANALYSIS
lecture on Ultraviolet/Visible Spectroscopy. Additional learning resources (e.g. videos) are
available in the Practicals section of the PHRM2041 Blackboard site.
Learning Objectives
1. To observe and appreciate the role of UV-Vis spectroscopy in routine pharmaceutical
analysis
2. To demonstrate capability with dilution calculations and experimental process as
well as documentation.
3. To dynamically involve the quality control protocols laid down for pharmaceutical
practice into a practical problem.
Theory
An integral role for analytical chemistry in pharmaceutical industry is the quality control of
manufactured items. Strict guidelines are in place which controls the quality of medications
for consumers. An industrial pharmacist is responsible for not only the formulation but also
verifying the product contains the correct amount of active from random sampling of the
tablet batches.
Paracetamol tablets will be assessed for uniformity of content in the following experiment.
Reporting must be detailed and complete (see guidelines on next page). It is important in
industry that all calculations and processes are documented and clear to enable tracking of
errors to assure that released products are reliable.
Experiment overview
1. Before class, study the Uniformity of Content for Formulated Preparations and Assay
guidelines for paracetamol tablets in the British Pharmacopoeia (BP). Note the value of max
which is required to measure the absorbance of the standards and unknowns.
Page 26
2. A calibration curve of UV absorbance of paracetamol solution vs paracetamol concentration

will be prepared using linear regression.
3. The paracetamol content of 10 individual tablets will determined by inverse prediction.
4. The BP will be consulted to determine whether the tablets pass the uniformity of content
guidelines.
Preparation to be done before coming to the practical class

Read the Uniformity of Content guidelines in the British Pharmacopoeia (BP). Printed copies
and an online version of the BP are available via the library. Refer to the following sections
of the BP: Appendix XII C. Consistency of Formulated Preparations. >> 3. Uniformity of
Content; and Paracetamol Tablets >> Assay. What is the value given for max?
Calibration Curve
1. Accurately prepare duplicate solutions of paracetamol (nominal concentration: 100
mg/100 mL) in 0.1 M NaOH. For each duplicate, record the exact amount of
paracetamol that you weighed and calculate the actual concentration (%) of each
solution.
2. Take 1 mL of each paracetamol solution and dilute to 100 mL with 0.1 M NaOH to
create working stock solutions that are used to prepare all of the standards.
3. Using the working stock solutions from step 2 above, accurately prepare a duplicate
calibration curve with six concentrations (10 mL each) ranging from ~0.0001% to
~0.001% by diluting with 0.1 M NaOH.
4. Record the absorbance value for each standard.
5. Do not swap or share UV cells, use the same UV Spec for all measurements.
Uniformity of Content:
1.
2.
3.
4.
Determine the expected amount of paracetamol in each tablet (250mg or 500mg).

In the following steps, all 10 tablets need to be treated separately.
Record the weight of each tablet.
For each tablet, using a mortar and pestle, completely powder each individual tablet
separately and weigh accurately enough powdered material to give 100 mg of
paracetamol (i.e. 100 mg of the active, not 100 mg of tablet) and dissolve in 100 mL
of 0.1 M NaOH. Hint: You know that each tablet should contain 250 mg or 500 mg of
active and you know the weight of each tablet (active + excipients).
5. Dilute each sample in 0.1 M NaOH to obtain a concentration that is close to the
middle of the calibration curve (you can estimate the concentration of your samples,
and you know the concentration range of your calibration curve, therefore calculate
the dilution factor that is required).
6. Read the absorbance of each sample using the same UV cells and UV spec that you
used to prepare your calibration curve.
Page 27

1. Complete the table below (1 mark)
Concentration of
the paracetamol
working stock
solution:
Duplicate 1:
______%
max = _______ nm
Standard solutions:
Paracetamol
concentration (%)
Duplicate 2:
______%
Volume added to
the 10 mL vol.
flask (mL):
Dilution factor:
Working stock soln

not diluted
Working stock
soln not diluted
Duplicate Duplicate
1:
2:
Absorbance at max
Duplicate 1:
Duplicate 2:
2. Using graph paper, plot the calibration curve of absorbance (range: 0 to 1.0) vs
concentration using the data in the table above. Do not average the absorbance values: plot
each of the duplicate values on the one graph. Sketch the line of best fit on the graph such that
approximately half the data points are on each side of the line. (1 mark)
3. Complete the table below (1 mark)
Expected weight (mg) of paracetamol in each tablet (from label):
Tablet Weight Weight of powder to give Tablet Weight
#
(mg)
100mg paracetamol (mg)
#
(mg)
1
10
Page 28
Weight of powder to give

100mg paracetamol (mg)
4. For each tablet you dissolved enough powder in 100mL to obtain 0.1% solutions. What
dilution factor is required for the concentration of each 0.1% solution to be near the middle of
the calibration curve? What is the concentration for which you are aiming? Do the dilution
after checking with your tutor. (1 mark)
Sample Abs. at
Conc.
Conc. paracetamol
max
#
paracetamol
in the undiluted
in cuvette (%)
solution (%)
Weight of
paracetamol in
tablet (mg)
% of the average
weight*** in the
tablet
1
2
3
4
5
6
7
8
9
10
Total weight of paracetamol in 10 tablets (mg)
***Average weight of paracetamol in 10 tablets (mg)
use this weight to

calculate % in the
column above
6. Refer to the following section of the BP: Appendix XII C. Consistency of Formulated
Preparations. >> 3. Uniformity of Content. Fill in the blanks below. (1 mark)
According to the BP guidelines, the preparation complies with the test if each individual
content is between ______% and ______% of the average content.*** The preparation fails
to comply with the test if more than one individual content is outside these limits or if one
individual content is outside the limits of ______% to ______% of the average content.
7. By applying the BP guidelines, on the basis of Uniformity of Content only, would you pass
the product for release to the consumer? Justify your answer. (1 mark)
Page 29
Page 30
Experiment 4: THERAPEUTIC DRUG MONITORING:

ASSAY OF CARBAMAZEPINE BY REVERSED PHASE HPLC
lecture on HPLC and read the section in the front of the lab manual about Calibration Curves
& Regression Analysis. Additional learning resources (e.g. videos) are available in the
Practicals section of the PHRM2041 Blackboard site.
Learning Objectives
1. To gain an understanding of the methods commonly used for separation and quantification
of therapeutically important drugs such as anticonvulsants.
2. To demonstrate the separation of the anticonvulsant, carbamazepine, from endogenous
components of serum and its quantification using reversed phase high performance liquid
chromatography (HPLC) with UV detection and naphthol as the internal standard.
3. Produce a calibration curve of the ratio of the peak areas of carbamazepine to the internal
standard, naphthol plotted against the carbamazepine serum concentration.
4. To use linear regression and inverse prediction to determine the concentration of
carbamazepine in the unknown serum samples.
Theory
During the last 40 years, the development of sensitive and specific methods for the
determination of drug concentrations in biological fluids, such as serum, has enabled
relationships to be established between the intensity of the pharmacological effects of many
drugs and their concentration in serum. This has led to the determination of usual therapeutic
serum concentration ranges of such drugs and to awareness that these concentration ranges
can be very useful in individualising drug dosage.
Individual adjustment of drug dosage on the basis of a targeted drug concentration in serum
may prevent or minimise adverse effects due to inadvertent overdose and it may facilitate
treatment particularly for diseases which are episodic in nature, such as epilepsy. The goal of
antiepileptic (anticonvulsant) drug therapy is the best possible seizure control with minimal
side-effects. Rational drug dosing adjustment can be carried out only through the use of
Page 31
appropriate drug serum level monitoring and known pharmacokinetic and pharmacodynamic
information. Knowledge of the therapeutic range is used to assess the likelihood of a particular
response to a dosage change. Serum drug concentrations are also measured to assist in
deciding the magnitude of a dose change in patients whose seizures are not optimally
controlled.
In many hospitals, clinical pharmacists are actively involved in interpreting patient serum drug
concentrations and advising on the appropriate course of action with regard to any required
dosage adjustments. Although not directly performing the drug assays themselves, clinical
pharmacists should understand the principles involved in the common analytical procedures,
particularly with regard to assay specificity and possible assay interferences.
Certainty in measurement of drugs in biological fluids is absolutely essential in therapeutic drug
monitoring. Guidelines for quality assurance in laboratory analysis have been published by the
American Chemical Society. At the very least, it is essential to report the linearity of the assay,
the coefficient of variation at low and high drug concentrations, the minimum level of
detection and the procedures used to assure specificity and stability, especially in the presence
of drug metabolites, other co-administered drugs and in biological samples from diseased
patients.
Carbamazepine is an anticonvulsant drug used to treat epilepsy and its serum concentration is
routinely monitored. The accepted therapeutic range for carbamazepine can be found in the
literature. Carbamazepine undergoes partial thermal degradation during gas-liquid
chromatography and hence the best approach to measuring the intact drug is by reversedphase high-performance-liquid-chromatography (RP-HPLC).
Experiment overview
1. Carbamazepine standard solutions and an unknown serum sample will be prepared for
injection into the HPLC.
2. A calibration curve of peak area ratio (carbamazepine : internal standard naphthol) vs
concentration of carbamazepine will be prepared using linear regression.
3. The concentration of carbamazepine in the unknown serum sample will be determined by
inverse prediction.
Preparation of the standard solutions and the unknown serum

samples
To prepare an accurate calibration curve for the monitoring of a drug, at least six different
concentrations are required in triplicate. Unfortunately this is not convenient for a 3 hour
laboratory session; therefore only duplicates will be used. You are provided with a set of tubes
already containing the carbamazepine in a range of concentrations, accurately dispensed and
evaporated to dryness.
Page 32
1. To the duplicate tubes containing the standard concentrations of carbamazepine,

accurately add 1000 L of the internal standard naphthol solution and then 500 L of
serum. Prepare a blank serum sample in the same manner. Ensure that you first add the
naphthol solution and then add the serum second.
2. You have been provided with two tubes each containing 500 L of the unknown serum
sample. To each tube add 1000 L of the internal standard naphthol solution.
3. Cap all of the tubes tightly, shake them vigorously, and label the top of the lid with your
syndicate number. With the help of the tutor, centrifuge the tubes at 4000 rpm (2900 g) for
5 min.
4. Being careful to ensure that no particles are transferred, accurately transfer 600 L of the
supernatant of each sample into another clean labelled tube, then repeat step 3.
5. Being careful to ensure that no particles are transferred, accurately transfer 300 L of the
supernatant of each sample into another clean labelled tube. The samples are now ready
for injection into the HPLC. (If particles are accidently transferred, see your tutor and
repeat step 3 again.)
Analysis of the standard solutions and the unknown serum samples

THE HPLC IS ONLY TO BE USED AFTER INSTRUCTION FROM A TUTOR
1. Inject 40 L of each sample into the injection port, as demonstrated. Note: Inject the
standards in order of increasing concentration. Inject the unknowns after injecting all of the
standards.
2. Ensure that you obtain copies of the chromatograms and any data analysis for your report.
3. Confirm with your tutor that the following conditions were used for your HPLC assay.
Experiment 4 - HPLC conditions
Temperature
Room temperature (approx. 22C)
Stationary phase
(column)
C18 (octadecylsilane)
Mobile phase
(solvent)
40% acetonitrile, 5% butanol, 45% water, 10%

phosphate buffer (1.1M, pH 6.0), 0.1% TEOA
Flow rate
1.25 mL/min
Detection
UV absorbance at 280 nm
Page 33
Expt 4 Worksheet - Syn #_____Names_________________Date_____

1. Answer the following question after obtaining the first four chromatograms (2 blanks and 2
lowest concentration standards). What are the retention times of carbamazepine and
naphthol? Check your answer with the tutor before proceeding. (1 mark)
Concentration of
Carbamazepine
carbamazepine (mg/L)
peak area
Naphthol peak area
Peak area ratio:

(Acarb/Anaphthol)
Average peak area ratio for the unknown sample

Analysis. Using Microsoft Excel, present the calibration curve to your tutor as a graph of peak
area ratio vs concentration using linear regression. What is the equation of the linear trendline
(y = mx + c)? (Optional: For you own records, you can also sketch the same graph using graph
paper provided below) (1 mark)
Page 34
4. Using the linear equation, calculate the concentration (mg/L) of carbamazepine in the
unknown sample? (1 mark)
Analysis. Conduct regression analysis to calculate the 95% confidence limits for the
concentration (mg/L) of your unknown and complete the following statement:
We can be 95% confident that the concentration of the sample lies between _____mg/L and
_____mg/L. (1 mark)
6. By reference to the books provided, what is the accepted therapeutic range for
carbamazepine? Does the patients sample fall within the accepted range? What advice would
you provide regarding this patient? (1 mark)
7. Why are the samples centrifuged? What is the supernatant? What would happen if you
injected particles into the HPLC system? (1 mark)
8. Name parts A-F in the figure below. What processes occur within the column and within the
detector? (1 mark)
9. In reversed phase HPLC, is the stationary phase relatively polar or non-polar, and is the
mobile phase relatively polar or non-polar? Based on their order of elution, which is more
polar, carbamazepine or naphthol? (1 mark)
Page 35
Page 36
Experiment 5: QUALITY CONTROL OF ACTIVE

PHARMACEUTICAL INGREDIENTS USING
INFRARED SPECTROSCOPY
lecture on Infrared Spectroscopy. Additional learning resources (e.g. videos) are available in
the Practicals section of the PHRM2041 Blackboard site.
Learning Objectives
1.
To develop basic expertise in the acquisition and interpretation of IR spectra.
2.
To gain an appreciation of the use of IR spectroscopy for quality control purposes in

pharmaceutical drug product manufacture.
Theory
Infrared (IR) spectroscopy is an essential and vitally important component of any quality
control program in the manufacture of pharmaceutical products. After delivery of all raw
materials to the pharmaceutical manufacturing facility, they must be quarantined until their
identity and purity can be verified. As individual IR spectra are unique to individual
compounds, just as fingerprints are unique to individual people, IR spectroscopy provides a
powerful tool for the verification of the identity and purity of raw materials used in drug
product manufacture. This is done by matching a spectrum of the sample to that of the
'reference' compound. IR spectroscopy may also be used in conjunction with NMR (nuclear
magnetic resonance) and MS (mass spectrometry) for the identification of unknown
compounds, whether they are new 'drugs' or novel metabolites of existing drugs.
The constituent atoms of molecules vibrate with respect to each other and the bonds act like
spring connections. IR spectroscopy involves examination of the twisting, bending, rotating and
vibrational motions of atoms in a molecule. Each different molecule has its own set of
vibrational frequencies which are in the same range as the infrared frequencies of the
electromagnetic spectrum. Infrared radiation occurs between the visible and the microwave
regions of the electromagnetic spectrum. The portion of the infrared radiation that is of
greatest practical use to us, termed the "fundamental" region, occurs from 4000 to
approximately 400 cm-1.
Page 37
Experiment overview
Two unknown samples will be provided one liquid and one solid. The IR spectrum of each
compound will be acquired, and the identity determined by comparison with reference
spectra. A detailed analysis of the spectra will ensue.
Acquisition of IR spectra and identification of the unknown

1. You are provided with two unknown samples one liquid and one solid. The tutor will
demonstrate how to use the instrument. Acquire the infrared spectrum of the unknown solid
then proceed to step 2.
2. Using the entire spectrum (functional group and fingerprint regions), compare the positions
and amplitude of the major bands in the IR spectrum of the unknown with those of the
reference spectra. On this basis, match the spectrum of the unknown with a reference
spectrum and therefore determine the identity of the unknown sample. Confirm with the tutor
that you have chosen the correct reference spectrum.
3. Using the list provided, lookup the structure of the compound and draw that structure onto
the printout of your IR spectrum.
4. Complete the worksheet on the following pages. Correlation tables are provided to assist
you.
Hint: Examine the structure of your molecule and identify the key functional groups. Then use
the provided correlation tables to determine the typical frequency of that group and locate it in
the spectrum. E.g. The structure contains a ketone C=O; your correlation table tells you that it
typically appears at 1725-1705 cm-1 as a strong peak; look at that region of your acquired
spectrum and you find a strong peak at 1718 cm-1 which must be the C=O stretch; write that
information in the worksheet table.
5. When every syndicate has obtained the spectrum of their unknown solid, repeat the
process using your unknown liquid.
Page 38

Solid sample number: _______
1. What is the name and structure of the unknown solid? (1 mark)
2. For the unknown solid, complete the table below (3 marks)
Intensity
Peak
(label
your
spectrum
with
these
letters)
Frequency of
(strong,
vibration (cm-1)
medium,
Functional group
weak)
Type of
vibration
(stretch,
bend)
Page 39
Liquid sample number: _______

3. What is the name and structure of the unknown liquid? (1 mark)
4. For the unknown liquid, complete the table below (3 marks)
Intensity
Peak
(label
your
spectrum
with
these
letters)
Frequency of
(strong,
vibration (cm-1)
medium,
Functional group
weak)
Type of
vibration
(stretch,
bend)
Include your labelled spectra when you submit your worksheet.
Page 40
5. Summarise the factors that determine the frequency of infrared radiation that is absorbed
by a molecule? (1 mark)
6. Name the four types of bending vibration that may be observed in an infrared spectrum? (1
mark)
Page 41

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THE UNIVERSITY OF QUEENSLAND

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

PHRM2041 PRACTICAL CLASS SCHEDULE

Mid semester break

Structure Elucidation Tutorial

Drugs in Sport Self-Directed Learning Activity

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

To become familiar with methods of drug analysis used commonly in:

Pharmaceutical industry for in-process quality control and quality assurance of

To build on experience gained in previous semesters of the pharmacy course in

To develop a sound foundation in quantitative methods of drug analysis, for application

Absences and Assessment

PHRM 2041 DRUG ANALYSIS

Calibration Curves & Regression Analysis (for experiments 1 & 4)

PHRM 2041 DRUG ANALYSIS

Installation of the data analysis pack (if required)

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

Experiment 1: VISIBLE SPECTROPHOTOMETRIC

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

Derivatisation of procaine to form an azo dye that absorbs visible radiation.

Derivatizing reagent: Prepare accurately 100 mL of

Standard procaine hydrochloride solution: In duplicate, prepare accurately 100 mL of

Ammonium sulfamate solution: Prepare 100 mL of approx. 0.5% ammonium

Sodium nitrite solution: Prepare 100 mL of 0.1% sodium nitrite.

PHRM 2041 DRUG ANALYSIS

Preparation of procaine derivatives

In duplicate, pipette 0 (blank), 1, 2, 3, 4, and 5 mL of stock solution (~0.005% procaine

Add 1 mL of 2 M H2SO4 followed by 5 mL of sodium nitrite solution to each of the 14

Determination of analytical wavelength

PHRM 2041 DRUG ANALYSIS

Expt 1 Worksheet - Syn #____ Names_________________Date_____

Mass of procaine HCl in 100 mL:

PHRM 2041 DRUG ANALYSIS

4. The plot of absorbance vs concentration is a straight line (y = mx + c). Which terms in

molar = _____ L mol-1 cm-1.

PHRM 2041 DRUG ANALYSIS

Lambda max = ~290 nm

1% = ~700 dL g-1 cm-1

The advantages of chemical derivatisation are:

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

Experiment 2: SPECTROFLUOROMETRIC ANALYSIS

PHRM 2041 DRUG ANALYSIS

Preparation of the standard solutions

Preparation of the unknown quinine samples

PHRM 2041 DRUG ANALYSIS

Determination of Emission Wavelength

Open the Cary Eclipse folder on the desktop

Analysis of the standard solutions and the unknown quinine sample

Open the Cary Eclipse folder on the desktop

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

Expt 2 Worksheet - Syn #____ Names_________________Date_____

R2 value for six standards of lowest concentration:

PHRM 2041 DRUG ANALYSIS

PHRM 2041 DRUG ANALYSIS

8. Examine the cuvettes used for

Expt 1 Worksheet - Syn # Names_________Date_

Expt 2 Worksheet - Syn # Names_________Date_

Expt 3 Worksheet - Syn # Names_________Date_

Expt 4 Worksheet - Syn #_Names_________Date_

Expt 5 Worksheet - Syn # Names_________Date_