1.3.

Analytical Errors and Validation

of Analytical procedures

1
 Errors in pharmaceutical analysis

 The terminology ‘error’ invariably refers to the difference in the numerical

values between a measured value and the true value.

 It has become universally accepted in methods of comparison that the

percentage composition of a ‘standard sample’ provided and certified by

the NIST, BPCRS or EPCRS must be regarded and treated as absolutely
correct, pure and authentic while evaluating a new analytical method
 Consequently, the differences thus obtained between the standard values and

those by the new analytical methods are then treated as ‘errors’ in the latest
procedure

NIST- National Institute of Standards and Technology based in US.

BPCRS- British Pharmacopoea Chemical Reference Substance,

EPCRS -European Pharmacopoea Chemical Reference Substance

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 Analytical errors may be broadly categorized into two heads, namely :

(i) Determinate (systematic) Errors, and

(ii) Indeterminate (random) Errors.

i) Determinate (systematic) errors

 These are errors that possess a definite value together with a reasonable

assignable cause.
 However, in principle these avoidable errors may be measured and accounted

for conveniently.

3
 Determinate (systematic) errors:
(a)Personal Errors : due to ‘personal equation’ of an analyst and have no

bearing whatsoever either on the prescribed procedure or

methodology involved.

(b) Instrumental Errors : due to faulty and uncalibrated instruments,

such as : pH meters, single pan electric balances, UV
spectrophotometers,

(c) Reagent Errors : due to individual reagents

 For instance : impurities inherently present in reagents ; unwanted introduction of

‘foreign substances’ caused by the action of reagents on either porcelain or glass

apparatus.

4
 (d) Constant Errors :
 Example : Assuming a constant equivalence point error of 0.10 ml is
introduced in a series of titrations, hence for a specific titration needing only
10.0 ml of titrant shall represent a relative error of 1% and only 0.2% for a
corresponding 50 ml of titrant consumed

(e) Errors due to Methodology : Both improper (incorrect) sampling and

incompleteness of a reaction often lead to serious errors.

 A few typical examples invariably encountered in titrimetric and

gravimetric analysis are cited below :

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Indeterminate (random) errors:
 Cannot be pin-pointed to any specific well-defined reasons.

 Usually manifested due to the minute variations which take place

inadvertently in several successive measurements performed by the

same analyst, using utmost care, under almost identical experimental
parameters.
 Mostly random in nature and ultimately give rise to high as well as low

results with equal probability.

 Neither be corrected nor eliminated, and therefore, form the ‘ultimate

limitation’ on the specific measurements.

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Minimizing systematic errors

 Systematic errors may be reduced substantially and significantly by

adopting one of the following procedures rigidly.

(i) Calibration of Instruments,

Apparatus and Applying Necessary Corrections

Most of the instruments, commonly used in an analytical laboratory,

 Such as : UV-Spectrophotometer, IR-Spectrophotometer, single—pan electric balance, pH-meter and the

like must be calibrated duly, before use so as to eliminate any possible errors.

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 In the same manner all apparatus, namely : pipettes, burettes, volumetric

flasks, thermometers, weights etc., must be calibrated duly, and the

necessary corrections incorporated to the original measurements. 

 In some specific instances where an error just cannot be avoided it may be

convenient to enforce an appropriate correction for the effect that it

ultimately causes ;

 For instance : the inherent impurity present in a weighed precipitate can be

estimated first and then deducted duly from its weight.

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 (ii) Blank Determination :

 In order to ascertain the effect of the impurities present in the reagents

employed and reaction vessels used ; a blank determination is an absolute

necessity.

 It may be accomplished by performing a separate parallel estimation,

without using the sample at all, and under identical experimental

parameters as employed in the actual analysis of the given sample.

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 iii) Using standard substance

External standard

Internal standard

Standard addition

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 External standard

 It essentially comprises of performing an altogether separate estimation

under almost identical experimental parameters with a quantity of a std

substance that consists of similar weight of the component as is present in

the unknown sample.

 Thus, the conc of the component present in the unknown sample:

Rsample
Csample  Cstd
Rstd

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 Internal standard

 Employed if there is variation in experimental parameters.

 The effect of such variations can be eliminated by use of internal std.

 An equal amount of an internal std, a component that is not present in the

sample, is added to both the sample and std solutions.

 Quantification is achieved by using ratios of response of the component to

the internal std (Rr):

Rr _ sample
Csample  C std
13 Rr _ std
 Standard Addition
 This is especially useful when there is a problem with interference from the

sample matrix, since it cancels out these effects.

 Here, a small known quantity of the component under estimation is added to the

sample, which is subsequently subjected to analysis for the total amount of

component present.

 The actual difference in the quantity of components present in samples with or without the

added component ultimately gives the recovery of the quantum added component.

 The analyte conc in the original sample:

RUnspiked
Cunknown  C spiked
Rspiked  Runspiked
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 Validation of Analytical procedures

 Validation is the conformation of a newly developed analytical methods that is

going to be used in QC to produce analytical results as designed and expected.

 The object of validation of an analytical procedure is to demonstrate that it is

suitable for its intended purpose determined by means of well-documented

experimental studies.

 Accuracy and reliability of the analytical results is crucial for ensuring quality,

safety and efficacy of pharmaceuticals.

 For this reason, regulatory requirements have been published for many years.

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 General recommendation in method validation

 The principle of the test procedure should be described briefly.

 Procedure should be sufficiently detailed to make repletion by experts or

other analysts & the following description should be provided:

 Parameters evaluated or tested

 Reagents preparation techniques

 Calculation technique of assessed parameters

 Equipment and parameters

 Reference standard used

 Precaution to be taken

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  The common parameters that should be verified in method validation:

Linearity

 The ICH defines the linearity of an analytical procedure as the ability (within a given
range) to obtain test results of variable data which are directly proportional to the
concentration (amount of analyte) in the sample.

 The equation of a straight line takes the form: y = ax + b,

 Where b intercept of y-axis, a slope of the line.

 At least five concentration levels should be used.

 Under normal circumstances, linearity is achieved when the coefficient of

determination (r2) is ≥0.997

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 Accuracy

 The ICH defines the accuracy of an analytical procedure as the closeness of

agreement between the values that are accepted either as conventional true

values or an accepted reference value and the value found.

 Accuracy is usually reported as percent recovery by assay, using the proposed

analytical procedure, of known amount of analyte added to the sample

 Typical accuracy of the recovery of the drug substance in the mixture is

expected to be about 98 to 102%.

 Values of accuracy of the recovery data beyond this range need to be

investigated.

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Precision

 Precision is the degree of agreement among a series of measurements of

the same quantity; it is a measure of the repeatability/reproducibility of

results rather than their correctness

 It does not imply anything with respect to their relation to the ‘true

value’

 Precision is usually investigated at three levels: repeatability,

intermediate precision, and reproducibility

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Repeatability
 is a measure of the precision under the same operating conditions over a
short interval of time.

 It is sometimes referred to as intra-assay precision

Intermediate Precision.
 Intermediate precision is defined as the variation within the same
laboratory.

 The extent to which intermediate precision needs to be established

depends on the circumstances under which the procedure is intended to be
used.

 Typical parameters that are investigated include day-to-day variation,

analyst variation, and equipment variation

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 Reproducibility.
 Reproducibility measures the precision between laboratories

 This parameter should be considered in the standardization of an

analytical procedure (e.g., inclusion of procedures in pharmacopoeias
and method transfer between different laboratories).

 To validate this characteristic, similar studies need to be

performed at other laboratories using the same homogeneous
sample lot and the same experimental design.

 The most common approach is the direct method transfer from the
originating laboratory to the receiving laboratory.

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1
 Accuracy without
Accuracy & Precision Precision

Precision without No Precision &

22 Accuracy No Accuracy
 Range

 The range of a method is related to its sensitivity.

 The interval b/n the upper & lower conc (amounts) of the analyte in the

sample (including these concs) for which it has been shown that the

analytical procedure has a suitable level of precision, accuracy & linearity.

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 Selectivity
 The selectivity of a method is a measure of how capable it is of measuring

the analyte alone in the presence of other cpds contained in the sample.
 Detection methods can be ranked according to their selectivity.

 A simple comparison is b/n fluorescence & UV spectrophotometery;

 There are many more cpds w/c exhibit UV absorption than fluorescence, thus

fluorescence spectrophotometry is more selective methods.

 B/c selective methods are based on more complex principles than non

selective methods, they may be less robust,

 e.g. fluorescence spectrophotometry is more affected by changes in the analytical

method than UV.

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 Robustness

 The measure of its capacity to remain unaffected by small, but deliberate variations

in method parameters and provides an indication of its reliability during normal usage.

Limit of detection

 ICH defines LOD as the lowest amount of analyte in a sample which can be detected

but not necessarily quantitated as an exact value.

Limit of quantitation

 ICH defines LOQ of an individual analytical procedure as the lowest amount of

analyte in a sample which can be quantitatively determined with suitable precision and

accuracy.

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 1.4. Basic calculations in
pharmaceutical analysis

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 Molarity
 The molar concentration (Cx) of the solution of the chemical species X is the number of

moles of that species that is contained in one liter of the solution (not one liter of the
solvent).
 The unit of molar concentration is molarity, M, which has the dimensions of mol L -1.

Cx= no mole solute = no m mole solute

no L solution no ml solution
 One liter of one molar solution will consist of one mole of solute plus enough solvent to

make a final volume of one liter.

Example:
 Calculate the molar concentration of ethanol in an aqueous solution that contains 2.30g of

C2H5OH (46.07 g/mol) in 3.50L of solution.

 Describe the preparation of 2.00L of 0.108M Bacl2 from BaCl2.2 H2O (FW= 244.3g /mol )

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Normality
 Normality (N) is defined as the number of equivalent weight of solute
dissolved in one liter of solution.
N = no of equivalent weight no of equivalent weight = weight of solute

Volume of so/n in Liter equivalent weight

 An equivalent weight is defined as the ratio of a chemical species’ formula

weight (FW) to the number of its equivalents

 The number of equivalents, n, is based on a reaction unit, which is that

part of a chemical species involved in a reaction

 Normality makes use of the chemical equivalent, which is the amount of

one chemical species reacting stoichiometrically with another chemical

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species.
 1.4. Basic calculations……

 Note that this definition makes an equivalent, and thus normality, a

function of the chemical reaction in which the species participates.

 Although a solution of H2SO4 has a fixed molarity, its normality

depends on how it reacts.

 In an acid–base reaction, the reaction unit is the number of H+ ions

donated by an acid or accepted by a base. For the reaction between

sulfuric acid and ammonia

n = 2 for H2SO4 and n = 1 for NH3

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 1.4. Basic calculations……

 In a precipitation reaction, for example, the reaction unit is the charge of

the cation or anion involved in the reaction; thus for the reaction

n = 2 for Pb2+ and n = 1 for I–.

 For a complexation reaction, the reaction unit is the number of electron

pairs that can be accepted by the metal or donated by the ligand. In the

reaction between Ag+ and NH3

n for Ag+ is 2 and that for NH3 is 1

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 1.4. Basic calculations……

 Finally, in an oxidation–reduction reaction the reaction unit is the

number of electrons released by the reducing agent or accepted by the

oxidizing agent; thus, for the reaction

n = 1 for Fe3+ and n = 2 for Sn2+

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 Molarity vs Normality
Molarity: M = moles of solute contained in one liter of
solution.
Normality: N = moles of reactive units per liter (equivalents
per liter).
Normality is always a multiple of molarity.
E.g.
In an acid-base reaction, normality is a measure of the protons
(H+) or hydroxides (OH-) that react with one another.
Consider a 1 M solution of sulfuric acid, H2SO4.
Normality is a measure of the moles of protons in the solution.
Since 2 protons are available to react on each molecule of
H2SO4, the normality is 2 N.
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 Dilutions
 Because solutions in science are often much more
concentrated than are desired or can be managed for a given
protocol, it is frequently necessary to dilute these solutions to a
desired level.

The number of moles of solute in the concentrated solution is

equal to the number of moles of solute in the dilute solution.

molescon = molesdil

CconVcon = CdilVdil
Dilution factor (DF): ratio of final volume/aliquot volume
(final volume = aliquot + diluent)

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 Example: how would you prepare 0.5 M of

HCl acid in 250ml from its stock solution

having 11.9 M?
Solution

CconVcon = CdilVdil

11.9M*Vcon = 0.5M*250ml

Vcon = 10.5ml

By transferring 10.5ml from stock sol to 250ml flask &

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adding a solvent to the mark.
 Weight, Volume, and Weight-to-Volume Ratios

 Weight percent (% w/w), volume percent (% v/v) and weight-to-volume percent

(% w/v) express concentration as units of solute per 100 units of sample

 Percent composition of a solution can be expressed as:

 Weight percent (w/w) = mass of solute X 100%

mass of soln

 Volume percent (v/v) = volume of solute X 100%

volume of solution

 Weight /volume percent (w/v) = mass of solute g X 100%

 volume soln ml

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 1.4. Basic calculations……

 Weight percent is frequently employed to express the concentration of commercial

aqueous reagents. E.g. 37% hydrochloric solution – this means the reagent contains 37g

of HCl per 100g of solution.

 Volume percent is commonly used to specify the concentration of a solution prepared

by diluting a pure liquid with another liquid.

E.g. 5% aqueous solution of methanol – usually means a solution prepared by diluting

5.0ml of pure methanol to give 100ml of solution with enough water.

 Weight /volume percent is after employed to indicate the composition of dilute

aqueous solutions of solid reagents.

E.g., 5% aqueous silver nitrate often refers to a solution prepared by dissolving 5g of

3
silver nitrate in sufficient water to give 100ml of solution
6
 Parts per million & parts per billion

 For very dilute solutions, parts per million (PPM) is convenient way to express

concentration:

Cppm = Mass of solute X 106 ppm

Mass of solution

 The units of mass in the numerator & denominator must agree.

 For even more dilute solutions we use parts per billion. 

Cppb = Mass of solute X109 ppb

Mass of solution

 1ppm is equivalent to 1 mg of solute per liter (mg/l) or 1 mg of solute per kg (mg/kg).

 E.g. 1µg/mL = 1ppm

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 1.5. Physical and chemical
properties of drug molecules

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 Chemical analysis is an important part of the quality
assessment of drugs.

A deeper understanding of the analytical method requires

knowledge about both the analytical technique and the
chemical properties of the analytes.

Therefore a basic knowledge of a number of

physicochemical properties of molecules is needed to be
able to understand and further develop analytical chemical
methods.

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 Calculation of pH

 The pH of a solution is defined as – log [H+], where [H+] is the conc of

hydrogen ion in so/n

 In pure water the conc. of hydrogen ion governed by the equilibrium :

 Ka is the dissociation constant for the equilibrium, is known as Kw in the case of

the dissociation of water

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  Since the conc of water does not change appreciably as a result of

ionization, its conc can be regarded as not having an effect on the

equiblirium and it can be omitted from the equation and this mean
that in pure water:

 If an acid is introduced into an aqueous so/n the [H+] increases

4
1
  Strong acid is completely ionized in water and [H+] is equal to

its Molarity
 e.g. 0.1M HCl contains 0.1M H+ and has a pH of log [0.1] = 1

 For a so/n of a strong base such as 0.1NaOH, [OH-]=1M and

 [H+] [OH-] = 1x 10-14, therefore [H+] = 1x 10-13 and

 pH = 13

 Weak acids are not completely ionized in aqueous so/n and are

in equilibrium with the undissociated acid, as is the case for

water, w/c is a very weak acid.

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  The dissociation constant Ka is given by the expression below:

 For instance in a 0.1M so/n of acetic acid (Ka=1.75 x 10-5) the equilibrium can be

written as follows:

 The pH can be calculated as follows:

 Since the dissociation of the acetic acid does not greatly change the conc. of the

unionized acid, the above expression can be approximated to:

 In comparison the pH of 0.1M HCl is 1

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4
4
 pKa

 The pKa value of a cpd is defined as:

 If pKa is used as a measure of acidic or basic strength,

 For an acid the smaller the pKa value the strongest the acid.

 For a base the largest the pKa value the stronger the base.

45
  For an acid the forward reaction used

 In the case of a base it is the protonated form of the base that act

as a proton donor

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 Buffer solution
 A solution containing a weak acid/ base and its conjugate

base/acid that is resistant to a change in pH when a strong acid

or strong base is added.

 Adding as little as 0.1 mL of conc HCl to a liter of H2O shifts the

pH from 7.0 to 3.0 (ΔpH = 4).

 The same addition of HCl to a liter solution that is 0.1 M in both a

weak acid and its conjugate base, however, results in only a

negligible change in pH (ΔpH = 0.09).
47
  A mixture of acetic acid and sodium acetate is one example of an

acid/base buffer
 The equilibrium position of the buffer is governed by the reaction

 The relationship between the pH of an acid–base buffer and the

relative amounts of CH3COOH and CH3COO– is derived by taking

the negative log of both sides of the above equation and solving
for the pH

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  Buffering occurs because of the logarithmic relationship

between pH and the ratio of the conjugate base and weak

acid concentrations.

 For example, if the equilibrium concentrations of CH3COOH

and CH3COO– are equal, the pH of the buffer is 4.76.

 If sufficient strong acid is added such that 10% of the

acetate ion is converted to acetic acid, the concentration

ratio [CH3COO–]/[CH3COOH] changes to 0.818, and the pH
decreases to 4.67.
49
  A more useful relationship relates the buffer’s pH to the initial

concentrations of weak acid and base.

 A general buffer equation can be derived by considering the following

reactions for a weak acid, HA, and the salt of its conjugate base, NaA.

 After several rearrangement, it provide us a general formula known as

henderson-Hasselbalch equation

 Where CNaA conc of salt, CHA conc of the acid

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  Hasselbalch equation provides a simple way to calculate

the pH of a buffer and to determine the change in pH

upon adding a strong acid or strong base.

 For a base, Henderson-Hasselbalch equation is written

as

51
 • Using Hasselbach equation it is possible to determine degree of

ionization of a drug at a given pH.

• E.g. degree of ionization of acetic acid at pH of 4.76

acetic acid ionized 50% at pH of

4.76

5
2
  E.g1. Calculate the pH of a buffer that is 0.020 M in NH3 and 0.030M in NH4Cl.

What is the pH after adding 1.00 mL of 0.10 M NaOH to 0.10 L of this buffer? (Ka
= 5.7x10-10) Solution

 Initial pH of the buffer is:

 Adding NaOH converts a portion of the NH4+ to NH3 due to the ff reaction:

 The new concentrations of NH4+ & NH3 are:

 Final pH of the buffer is:

53
 E.g2 How many moles of sodium acetate and acetic acid
are required to prepare 1 liter of a buffer, pH 5.0, which is 0.
1 M in total available acetate (dissociated and undissociated).
Acetic acid has a pK of 4.74.
Solution: From Buffer Equation ,
           5.0 = 4.74 + log [salt]/ [acid]
            or log [salt]/[acid] = 0.26.
   [salt]/[acid] = 1.82 , [salt] + [acid] = 0.1
Acid = 0.1/2.82 = 0.035M
Salt = 0.1 x 1.82/2.82 mole = 0.065 mole.  

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Have you ever taken an over-the-counter (OTC) drug called
ibuprofen?
with the carboxylic acid moiety (COOH) the proton donor, which
has a pKa of around 4.4.
What happens once it’s swallowed?
Solution

First it goes to the stomach. We’ll assume the pH of the stomach is

around 3.

55
 [S ] [S ]
3  4.4  log ,,,  10 1.4  0.03981
[ A] [ A]
0.03981
% _ Salt  x100  4%
1.03981
 Most of the drug is in acid form and therefore could cross the stomach lining to enter the
bloodstream; i.e., ibuprofen can be absorbed from the stomach.
 Next the large intestine, assuming pH = 8
[S ] [S ]
8  4.4  log ,,,  103.6  3981
[ A] [ A]
3981
% _ Salt  x100  99.97%
3982
 Most of the drug is in salt form and so shouldn’t readily cross the intestinal lining and
enter the bloodstream.
 This is not true in real life, however. About 80% of ibuprofen is absorbed. Why so much? Partly
because there is a very large surface area in the gut, maximizing the amount of
unionized drug coming into contact with the membrane.
 Partly because some absorption takes place in the duodenum, where the pH is lower (closer to
6) and more drug is unionized. There may be some that crosses the membrane via pores,
since it is not a very large molecule (MW≈187). The most likely reason that the drug
is pretty well absorbed, though is by active transport across the gut wall.
56
 Stability of drugs
Many drugs are quite stable but functional groups such as
esters and lactam rings w/c occur in some drugs are
susceptible to hydrolysis and functional groups such as
catechols and phenols are quite readily oxidized.

The most common type of degradation w/c occur and

formulated drugs obey zero or first order kinetics

57
 Zero order degradation
 In zero order kinetics the rate of degradation is independent of

the conc of the reactant.

 This type of degradation is typically of hydrolysis of drugs in

suspension or tablets where the drugs is initially in the solid

state and gradually dissolve at the same rate as the drug in so/n
is degraded

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 First order degradation
 This type of degradation would be typical of hydrolysis of a drug in so/n

 In first order kinetics the rate constant k has units h-1 or s-1 and the
rate of the reaction for a drug is governed by the expression

 From this expression by integration and rearrangement the following

expression arises:

 The half life of the drug (the time taken for 50% of a sample drug to

degrade, i.e. where x is a/2) is thus given by the following expression

59
 t0.9 = 7.85 h

60
 Quiz
 Calculate the percentage of ionization of diphenhydramine
at pH of 7 (show the necessary steps).

61