‫بسم هللا الرحمن الرحيم‬

Fundamental of pharmaceutical analysis

Dr. Suha Elderderi Mohmed

B. Pharm. (Faculty of Pharmacy, University of Gezira , 2011)

M. Pharm. in Pharmaceutical Analysis (Faculty of Pharmacy, University of Gezira , 2016)
PhD student in pharmaceutical analysis (university of Tours, France)
 Why we studying this course ??

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What is pharmaceutical analysis ?

• A pharmaceutical analysis is intended to either identify or quantify

one or more substances in a given sample of pharmaceutical interest.
• A substance to be identified or quantified is called the analyte.

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 Questions pharmaceutical analysis methods are used to answer ?

 Is the identity of the drug in the Do the identity and purity of a pure
formulated product correct? drug substance to be used in the
What is the percentage of the stated preparation of a formulation meet
content of a drug present in a formulation? specification?
Does this formulation contain solely the Do the identity and purity of excipients
active ingredient or are additional to be used in the preparation of a
impurities present? formulation meet specification?
What is the stability of a drug in the
formulation and hence the shelf-life of the What are the concentrations of
product? specified impurities in the pure drug
At what rate is the drug released from its substance?
formulation so that it can be absorbed by What is the concentration of the drug
the body? in a sample of tissue or biological
fluid?
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 Qualitative and quantitative analysis
• Qualitative analysis is intended to Quantitative analysis is intended to
confirm the identity of the analytes. measure the exact concentration or the
exact amount of the analyte in a given
sample.

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for example
paracetamol tablets containing 500 mg paracetamol per tablet have to be
controlled prior to release from production by pharmaceutical analysis.

Qualitative analysis Quantitative analysis

Identification of paracetamol in the tablets determination of paracetamol in the tablet

is performed to make sure that the tablets is performed to measure the content of
contain the correct active pharmaceutical paracetamol and to check that this result
ingredient, is exactly or close to 500 mg per tablet.
Basic concepts of the control of analytical quality

Control of the quality of

= Validation
methods of analysis
 Validation of analytical procedure
 The Analytical procedure provides an exact description of how the analysis is carried out in
details for each step.

The full method should describe:

1. the quality and source of the reference standard for the compound being analysed
2. the procedures used for preparing solutions of the reference standard
3. the quality of any reagents or solvents used in the assay and their method of
preparation
4. the procedures and settings used for the operation of any equipment required in
the assay
5. the methodology used for calibration of the assay and methodology used for the
processing of the sample prior to analysis.
 The most fundamental requirements of analysis is to be: accurate and precise .
 The level of accuracy and precision describe the quality of a measurement.

Valid method = accurate and precise

 Accuracy:

 expresses how well the analytical result matches the “true

value”.
 Can be expressed by :
 mean percentage
Equal to mean of analytical results/ real or true value*100
(close to 100% is the better is the accuracy)
 Relative error %
Error = the difference between the true value (Xt) and the
analytical result (X)
Error % = error/true value * 100

(The lower is the error/ difference/ or error % is the better is the

accuracy)
 To establish the accuracy of an analytical method, the true value has to be known

 But how can the “true” value be obtained for unknown samples?

There are several ways to do this:

 A final possibility is to add
 Another is to analyze
known amounts of analyte
 One is to let an the same sample with
(as a chemical reference
experienced analyst different methods and
substance) to the sample
do the analysis on to compare the results.
(standard addition). The
the same samples Good agreement
analytical results before and
with a well tested between several
after standard addition can
procedure. This may methods gives a good
be compared, and this
give an indication of indication, but never a
difference can be compared
the “true” value. proof that the results
to the exact amount added to
are accurate.
the sample.
Precision :

 The precision of a method indicates how close

the different measurements are.
 Precision is expressed by
 The standard deviation or relative standard
deviation.
 SD =
 High precision means that the standard deviation
is small, which of course is desirable in
pharmaceutical analysis.
 RSD= SD/ mean *100

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 Is there an absolute guideline for how the good precision for API in formulation should be?

No. In general a precision of <

 The achievable precision depends on the sample being analysed
For example:
RSDs greater than 1.0% may be achieved in the
analysis of trace impurities in bulk drugs
or drugs in biological fluids

 Precision generally obtained by repeating the assay procedure a minimum of 5 times

from 5 separate aliquots of samples , giving total of 25 measurements
 High precision means that a relatively low
number of measurements can be performed
on the same sample without affecting the
accuracy of the results significantly
 Validation of analytical procedure

 levels of precision:

Intermediate
Repeatability Reproducibility
precision

 expresses the precision  expresses within laboratory  expresses the

obtained under the same variation of precision precision
conditions over a short  (analysis carried out by between
interval time. different analysts, on different laboratories
 (repeated by the same days or with different
person using a single equipment)
instrument)  {standardized method}
 example
 The mean% and RSD for four analysts results of stated content of a drug are:

Analyst 1: 99%+/- 5.8%

Analyst 2: 110.9% +/- 7.7%

Analyst 3: 99.6% +/-0.9%

Analyst 4: 91.9 %+/-0.9%

 Comment on accuracy and precision of each results?

 Types of Errors in analysis
Gross errors Systematic errors Random errors

 easily recognized  are errors that  are errors that

 involve a major breakdown can be cannot be
in the analytical process. detected and detected and
(samples being spilt, which which therefore
instruments breaking down therefore can cannot be
or being used in the wrong be corrected. corrected.
way).
 If a gross error occurs the
results are rejected and the
analysis is repeated from
the beginning.

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 Random and systematic errors can be distinguished in the following example

 A batch of paracetamol tablets are stated to contain 500 mg of paracetamol per

tablet; for the purpose of this example it is presumed that 100% of the stated
content is the correct answer.

 Four students carry out a spectrophotometric analysis of an extract from the tablets
and obtain the following percentages of stated content for the repeat analysis of
paracetamol in the tablets:
 Student 1: 99.5%, 99.9%, 100.2%, 99.4%, 100.5%
 Student 2: 95.6%, 96.1%, 95.2%, 95.1%, 96.1%
 Student 3: 93.5%, 98.3%, 92.5%, 102.5%, 97.6%
 Student 4: 94.4%, 100.2%, 104.5%, 97.4%, 102.1%
 The means of these results can be simply calculated according to the formula:
𝑋𝑖
𝑥=∑
𝑖 𝑛
 Student 1 :
 results are all clustered All the steps in
close to 100% of the stated precise and
the assay have
content accurate
been controlled
 a mean for the five measurements
very carefully
measurements very close
to the correct answer. The analysis has been
mainly well controlled
except for probably
 Student 2: precise assay one step, which has
 results are closely clustered, but not caused the inaccuracy
 a mean is lower than the accurate and thus the assay is
correct answer. measurements precisely inaccurate
the student 2 has
produced an analysis
containing a
systematic error.
 Student 3:
 results are widely The analysis was thus poorly
scattered and hence imprecise and controlled and it would
imprecise, inaccurate require more work than that
 a mean is lower than measurements required in the case of
the correct answer. student 2 to
. eliminate the errors
the analysis contains random
 Student 4: errors (scattered results)
 results are widely imprecise and
scattered accurate although the answer obtained is
 yet a mean is close measurements accurate, it would not be wise to
trust it to always be so.
to the correct
answer.
What are The factors leading to imprecision and inaccuracy in assay results
are???

Incorrect weighing and transfer of analytes and standards

Inefficient extraction of the analyte from a matrix, e.g. tablets
Incorrect use of pipettes, burettes or volumetric flasks for volume measurement
Measurement carried out using improperly calibrated instrumentation
Failure to use an analytical blank
Selection of assay conditions that cause degradation of the analyte
Failure to allow for or to remove interference by excipients in the measurement
of an analyte

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 Standard Operating Procedure (SOP):
 This particular section of the
operating procedure
describes the assay itself,
 there would also be other sections
in the procedure dealing with
 safety issues,
 the preparation and storage of the
solutions used for extraction and
dilution,
 the glassware required and
 a specification of the
instrumentation to be used.
Each of the sections within an assay
would have other SOPs associated with
them, governing, for instance, the correct
use and care of balances
 Calibration

 The calibration of a method involves comparison of the value or values of particular

parameter measured by the System under strictly defined condition with pre-set standard
values

Analytical blank
This consists of all the reagents or solvents used in an analysis without any of the analyte
being present.
A true analytical blank should reflect all the operations to which the analyte in a real sample
is subjected.
 limit of detection

This is the smallest amount of an analyte which can be detected by a particular method.

Limit of quantification

The limit of quantification is defined as the smallest amount of analyte which can be quantified
reliably
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 Linearity
 Most analytical methods are based on processes where the method produces a
response that is linear and which increases or decreases linearly with analyte
concentration. The equation of a straight line takes the form:
𝑦 =𝑎+𝑏𝑥
 where a is the intercept of the straight line with the y
axis and b is the slope of the line

 The statistical measure of the quality of fit of the line through the data is the
correlation coefficient r. A correlation coefficient of > 0.99 is regarded as
indicating linearity.
 Robustness

 Robustness is evaluated in order to determine how resistant the precision and accuracy of
an assay is to small variations in the method.

 The types of parameters which are assessed in order to determine the robustness of a
method include:
• the stability of analytical solutions;
• the length of the extraction time;
• the effect of variations in the pH of a HPLC mobile phase;
• the effect of small variations in mobile phase composition;
• the effect of changing chromatographic columns;
• the effect of temperature and flow rate during chromatography
 Selectivity

 The selectivity of a method is a measure of how capable it is of measuring the analyte

alone in the presence of other compounds contained in the sample.

The most selective analytical methods involve a chromatographic separation.

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 Sensitivity

 The sensitivity of method indicates how responsive it is to a small change in the

concentration of an analyte.
References
• David G Watson (2012). Pharmaceutical analysis

 Steen_Hansen, _Stig_Pedersen Bjergaard,

Knut_Rasmu. Introduction to pharmaceutical chemical
analysis, 2012

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 Thank you for attention

10/11/2022 by: Suha Elderderi