A critical evaluation of the methods used for the quality control of XXXXX

XXXXX is an .., indicated for use in the treatment of mild to moderate essential
hypertension alone or with thiazide therapy, and severe hypertension resistant to other
treatment. It is also used as an adjunct in congestive heart failure, following myocardial
infarction, and for diabetic nephropathy in insulin dependent diabetes1.

Figure 1 Structure of XXXXX

Adequate quality control of active drugs such as XXXXX is an important part of the
pharmaceutical industry, and good control should be both qualitative and quantitative. Many
analytical techniques have been developed for the purpose of quality control, and validation
and control of these analytical methods is also required to fulfil the requirements of GLP
(Good Laboratory Practice) and VAM (Valid Analytical Measurements) regulations.

QUALITY CONTROL OF THE BULK DRUG XXXXX

When considering the quality control of the bulk drug XXXXX, we are mainly concerned with
the issues of identification and purity. It is important that the sample of XXXXX taken for
these tests be representative of the material as a whole.
Identification & Purity
The British Pharmacopoeia provides us with four methods to test for the identification of
XXXXX as a bulk material. Test B is that of preference, and if this technique is not viable,
then tests A, C and D should be performed.
A

Melting point

105 - 108C

Infra-red (IR) spectrophotometry

examine and
reference

Thin layer chromatography

as detailed in BP

Chemical test

as detailed in BP

Figure 2 Methods for the Identification of XXXXX 2

compare

to

Infra Red Spectrophotometry (IR)

IR is described as the test of preference as it is a highly specific qualitative technique. If the
spectrum of the sample matches that of the reference trace, then XXXXX has been positively
identified.
The process of infrared spectrophotometry involves passing electromagnetic radiation of
different wavelengths through the sample, which is in the form of a potassium bromide (KBr)
disk. The radiation is absorbed by the bonds of the molecule, causing them to stretch or bend.
The spectrum then shows absorbencies characteristic to the particular bonds in the molecule.
The far IR region (<1600cm-1) is the important part of the spectrum when identifying the test
compound, as this is the so-called fingerprint region. The reference trace for XXXXX, RS
038 is shown below:

Figure 3 XXXXX Reference Spectrum (RS 038) 2

Infra-red spectrophotometry produces a complex spectrum unique to the compound under
test, so it is highly accurate. The scan of the sample is very quick, so once the disk has been
prepared, an FTIR machine will acquire the spectra in about one second. The machine is linked
to a computer so that the spectrum can be checked against a library of standard spectra, so a
degree of automation is possible.
IR can also be used for identifying different polymorphs of the test substance. XXXXX exists
as two different crystalline polymorph forms3, one of which is stable, and the other unstable.
It is important that the correct polymorph is manufactured, as it will affect drug bioavailability,
chemical processing of the material, and patent lifetime.
Although IR is a very useful tool for identification purposes, it is rarely used as a quantitative
technique because the spectra obtained are so complex. Impurities have to be present in quite
large amounts to be detected, hence the use of IR as an identification tool only. The
preparation of samples (especially KBr disks) is quite difficult, and so requires time and a
degree of skill by the operator. Also, the technique lacks robustness, as handling of the samples
can affect the spectra obtained.
Melting point

The melting point of XXXXX should be between 105-108C, when carried out by the method
stated in the BP2. Measuring the melting point is a very basic qualitative identification
technique, and can also be used to indicate the presence of impurities in a sample, as they will
lower the melting point. Melting point can also be used to differentiate between the two
polymorphs of XXXXX, as the stable polymorph melts at 106C, and the unstable form at
88C3.
Melting point is a good beginning in terms of analysis in order to identify XXXXX. The
materials used are cheap, and it is a simple method to carry out. The technique is fairly robust,
and with newer technology, several samples can be run concurrently so increasing automation
and batch analysis of the process.
The disadvantages of using melting point as an analytical technique arise mainly when using
the BP method of determination. It is cumbersome, and it can take several minutes to achieve
results. However, when using both the BP method and automatable modern day machinery, it
is important to realise that results obtained can be affected by small variables. The presence of
large crystals in the sample, dust, or other laboratory debris in the testing tube can all either
increase or decrease the melting point, or cause a no result to be read by the machine.
The melting point test should be carried out in conjunction with TLC and a solution test to
positively identify XXXXX, as it is not suitable as a stand-alone method. It cannot identify the
compound absolutely, as many other substances will have a melting point within this range.
Thin Layer Chromatography (TLC)
TLC is a commonly used industrial method that provides both qualitative and quantitative
information with regards to the identification and purity of bulk drug samples. It is a simple,
cheap and robust method, and has the advantage over HPLC and GLC that all components of
the sample will be eluted and so detected. A wide variety of mobile phases, stationary phases
and visualising agents can be used, making the technique easily adaptable. Batch
chromatography is also possible, so that many samples can be analysed in tandem.
TLC involves the migration of the sample up a layer of stationary phase. The components of
the sample have different affinities for the mobile and stationary phases, so when separation
has occurred on the plate, all components of that material should be visible, either to the naked
eye or with the use of suitable visualising agents. 5,5-dithiobis-2-nitrobenzoic acid is the
named agent specific for XXXXX, showing a yellow colour when sprayed onto the plate. The
chromatography should also give an indication as to the relative concentrations of all
components of the sample.
The disadvantages of TLC lie in the sample preparation and preparation of the plate. More
operator skill is required for optimal use when compared with HPLC.
Chemical Solution test
The BP states that when about 20mg XXXXX is dissolved in 2ml water and 0.05M iodine
added, the colour is discharged immediately.
Tests such as this where a colour change is involved are useful because the results can be seen
easily. They are also cheap, and do not require specialised apparatus. The disadvantages
though are that they are not quantitative, and although they detect the presence of XXXXX
3

they will not detect its concentration or the presence of impurities. As for melting point and
TLC, it is not a stand-alone method.
High-Performance Liquid Chromatography (HPLC)
As stated in the BP, the purity of XXXXX should be 98.0-101.5% as calculated with reference
to the dried substance. Impurities in the bulk drug will include starting materials and
degradation products, by-products, reaction intermediates, reagents, ligands and catalysts. Any
impurities found should be identified and quantified.
In HPLC, a liquid mobile phase is pumped under pressure through a stainless steel column of
mobile phase. The sample in solution is injected in to the system, and the components of that
sample elute from the column at different times, depending upon their differing affinities for
the mobile and stationary phases. Monitoring of the effluent is carried out by a detector, which
in the case of XXXXX HPLC is a spectrophotometer set at 220nm2.
HPLC is the industrial method of choice for determining the purity of bulk drugs and
identifying the bulk drug and impurities present. The technique is both qualitative and
quantitative. It is sensitive, accurate, reproducible, and automatable. Precise sample injection
via an injection port/loop means the results obtained are very precise.
As technology advances, there have been many developments in HPLC technique. There are
more columns with improved separation, a greater choice of detectors, and a greater degree of
software control. Columns are now being developed under the banner of chiral stationary
phases, which are useful in the separation of the different enantiomers of a substance.
The disadvantages of HPLC are that it is an expensive technique. The equipment required is
costly, and with todays world of branding, the components of the machine often have to be
made by the same company to be compatible with each other. HPLC also produces a lot of
organic solvent waste. There is great loss of money through loss of reagents, and in the
disposal of the chemicals as they are a hazard to the environment.
The disadvantage of HPLC when applied to the analysis of XXXXX is that a derivitisation
process has to be carried out prior to injection to render the molecule detectable by UV. The
absence of a strong chromophore in XXXXX makes this necessary.

QUALITY CONTROL OF XXXXX IN FORMULATIONS

XXXXX is manufactured by a number of pharmaceutical companies, as tablets containing
12.5mg, 25mg or 50mg of the active drug. Squibb also manufacture Capozide tablets, which
contain the actives XXXXX (25mg or 50mg) and hydrochlorothiazide (12.5mg or 25mg).
During the manufacture of the formulated product from the bulk drug, there are many
opportunities to introduce contamination and impurities to the tablet. These include cross
contamination from other active compounds being processed in the same tablet compactors
and presses; particulate matter from the environment, and any impurities present in the tablet
excipients. As a result, tablets are subjected to a variety of strict quality control procedures,
some of which are pharmacopoeial, and others, which are non-pharmacopoeial.

Pharmacopoeial Tests
The BP monograph for XXXXX states that the tablets comply with the requirements stated
under Tablets and with the following requirements. The various requirements include tests for
weight and content of the tablets, and also identification and purity.
General standards and test methods for XXXXX tablets (and all other drugs formulated as
tablets) are as follows, and are designed to control the stated properties. The actual methods
themselves form an appendix to the main pharmacopoeia.

Uniformity of weight, uniformity of content, and content of active ingredient tests

these are designed to monitor the content of the active ingredient in the tablet
Disintegration and dissolution tests
these standards check the ability of the drug to be released from the tablet

XXXXX in tablets is identified by IR spectrophotometry and HPLC assay. The IR spectrum

should be concordant with the reference spectrum for XXXXX. In the assay, the principal
peak obtained with the test solution should have the same retention time as the peak due to
XXXXX in the chromatogram of the standard for that test. The benefits and disadvantages of
these techniques have been discussed previously.
With regards to the content of XXXXX in the tablet formulation, the British Pharmacopoeia
says it should be 95.0- 105.5% of the stated amount, which is determined by HPLC. As
discussed earlier, HPLC is the method of choice for this type of analysis due to its high
accuracy and automatability. However, the disadvantage of using HPLC for the determination
of XXXXX in formulations is that the active drug has to be extracted from its formulation
prior to injection and analysis.
Due to the highly reactive nature of the thiol group in XXXXX it undergoes rapid oxidation,
producing XXXXX disulphide. The HPLC test is not valid unless the resolution between the
peaks for XXXXX and XXXXX disulphide is at least 2.0.
Many other methods have also been developed for the quality control and determination of
XXXXX in tablets. Such methods include determination by anion exchange HPLC using
indirect photometric detection4, and flow injection analysis (FIA)5.
High performance TLC (HPTLC) with fluorescence densitometry has been applied to the
analysis of many pharmacologically active thiols, including XXXXX6. XXXXX does not have
a strong chromophore, and as discussed earlier, derivitisation of the sample is required to
render the active detectable in both HPLC and TLC.
In this method, the thiol group was reacted with a thiol specific reagent which produced
fluorescent derivatives. These were then analysed by TLC. The limit of detection for the thiols
in this study was found to be in the low picogram range, making this a very sensitive
technique.
Non-Pharmacopoeial Tests

As part of in process and quality control programmes, tablets are subjected to a wide variety
of other non-pharmacopoeial tests. Theses include extra physical tests on the tablets
themselves, and also stability tests.
Physical Tests
These are designed to assess parameters such as individual tablet weight, thickness and
diameter as part of in-house processing. The mechanical strength of tablets is also routinely
measured by crushing tests (the compression force which when applied to a tablet
diametrically, just causes it to fracture), friability tests (measure the resistance of the tablets to
continued abrasion as a percentage weight loss), and tablet toughness tests.
Stability Tests
Information about stability gives an indication as to how tablets should be packaged and
stored, and what their shelf life should be. The stability of a formulated product is assessed by
the application of a suitable stress on the product over an extended period of time. The effect
of the particular stress is then assessed in terms of active drug content, levels of degradation
products, and dissolution of the tablet, for example.
The applied challenge should be relevant to the physicochemical properties of the drug, and
the conditions likely to be encountered during transport, storage, and use 3. Examples of
commonly used stresses are elevated temperature and relative humidity, high partial pressure
of oxygen, and microorganisms.
Accelerated Storage Testing
Accelerated storage tests are required to provide information about the extended stability of
the drug, and how the stability is affected by various storage conditions.
The four main processes by which drug degradation occur are hydrolysis, oxidation,
photolysis, and by the action of trace metal catalysts 7. These reactions occur more quickly at
elevated temperature, and so the decomposition of the active drug can be predicted in shorter
periods of time. For every 10C rise in temperature, the speed of decomposition is twice as
fast.

XXXXX (AND ITS METABOLITES) IN BIOLOGICAL SAMPLES

Approximately 60-75% of XXXXX is rapidly absorbed from the gastro-intestinal tract 8, and
its levels can be measured in the blood 15 minutes after ingestion9. Peak plasma concentrations
are achieved within about an hour (see figure 3), although the absorbance is reported to be
decreased in the presence of food.

Figure 3 Plasma concentration-time profile for XXXXX10

XXXXX is approximately 30% bound to plasma proteins. It can cross the placenta, and is
found in breast milk at approx. 1% maternal blood concentrations8.
XXXXX is largely excreted in the urine, and almost half is as the unchanged drug. The rest of
the excreted amount is as XXXXX disulphide and other complex metabolites such as its dimer
disulphide, and mixed disulphides with glutathione, cysteine, and serum albumin9. The
elimination half-life of XXXXX is two to three hours, but this is increased in renal failure.
XXXXX and its metabolites can be detected in all tissue types, including the liver, kidney, and
whole blood and plasma. However, due to the highly reactive nature of the thiol species in
XXXXX, its rapid oxidation results in the formation of XXXXX disulphide. This instability of
XXXXX in biological media necessitates the immediate derivitisation of the thiol with a
stabilising agent, usually N-ethylmaleimide (NEM). Stabilisation of the urine metabolites is
achieved with metal chelators, acid pH and 5C storage.

Thin Layer Radiochromatography (TLRC)

This reported technique allows the analysis of XXXXX in whole blood, and has a limit if
detection of about 10ng XXXXX / ml blood. N-ethylmaleimide (NEM) is used as the
stabilising agent, and the prepared samples are analysed for total radioactivity11.
The disadvantages of this technique are most obviously the use of radioactive substances by
the analyst. As with all the analyses involving XXXXX, the sample also requires extensive
preparation beforehand to prevent the oxidation of XXXXX.
Gas Chromatography
Gas chromatography-mass spectrophotometry (GC-MS) in the selected-ion mode can
detect NEM-XXXXX in whole blood, urine, amniotic fluid, milk, and tissues such as the
liver, kidney, lung and placenta with only small adjustments to the procedure for each
biological medium12.
Sample preparation for this technique is a lengthy process involving various extraction steps,
and the limit of detection is 5.5ng/ml of blood.

Gas chromatography-flame photometric detection (GC-FPD) is used for the

determination of XXXXX in blood and urine, and the detector is sulphur selective.
However, the sensitivity of this technique is in the microgram range, whereas it is needed
in the nanogram range. As a result it is not satisfactory for human blood studies.

High Performance Liquid Chromatography

7

The HPLC analysis of XXXXX can be carried out on whole blood and urine samples. It is
normally determined by reverse phase HPLC, i.e. by using a stationary phase that is less polar
than the mobile phase. Reverse phase analysis is common for analysing biological systems.
HPLC analysis of samples is highly qualitative and quantitative, and methods have been
reported that are as sensitive and precise down to 5ng/ml of whole blood, and 0.1mcg/ml of
urine13.
The main disadvantage of using HPLC for analysis of biological samples such as whole blood
and urine is that they cannot be directly injected into the system before they have been cleaned.
This is usually achieved by solid phase extraction (SPE), which separates the desired analyte
from the unwanted components of the sample. The process of SPE is automatable, so bulk
analyses can be simultaneously undertaken.
Other analyses
The above described techniques are more commonly used as they are variations of the most
popular analyses for determination of XXXXX. However, other techniques reported include
spectrofluorimetry, radioimmunoassay, and colourimetry, and can detect XXXXX in a wide
range of biological media. As technology advances, then maybe these analyses will be used
more in the future.

CONCLUSION
There are many analytical techniques available for the quality control of XXXXX; in the bulk
drug and its formulations. None of the tests are absolute when carried out individually, but
coupled with other techniques they ensure the continuing high quality control of XXXXX in
all its manifestations.
As human thinking and time advance, developments in analytical processes will continue, and
some widely used techniques will become obsolete as they are superseded by newer
technologies. Hyphenated techniques may become more widespread, thus reducing the human
need in preparation and analysis. Automation is the key to industrial analysis, as the search to
reduce costs and overheads is pursued.
The determination of XXXXX and its metabolites in biological media is of great importance.
Results obtained from these sorts of analyses help to build a profile of the pharmacokinetics
and pharmacodynamics of the drug.
Determination of these important parameters is essential in the search for patents for drugs, as
they help profile bioavailabilty, mode of metabolism, and the toxicity of the active drug.

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British Medical Association, British National Formulary, Edition 37

(March 1999, British Medical Association & Royal Pharmaceutical Society of Great
Britain)

2.

British Pharmacopoeia, 2001 Edition

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Edited by Lund W. The Pharmaceutical Codex, Twelfth Edition

(1994, Pharmaceutical Press)

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Tahseen M. et al. Journal of Pharmaceutical and Biomedical Analysis, 25, 39-52

(2001)

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Zhang Z.D. et al. Journal of Pharmaceutical and Biomedical Analysis, 14, 939-945
(1996)

6.

Lin Ling B. et al. Journal of Pharmaceutical and Biomedical Analysis, 7, 1663-1670

(1989)

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Edited by Aulton M.E. Pharmaceutics: The science of dosage form design, Second
Edition (2002, Churchill Livingstone)

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Martindale W. Martindale Edition 32 (1999, Pharmaceutical Press)

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Edited by Florey K. Analytical Profiles of Drug Substances, Volume 11 (1982,

Academic Press)

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Franklin M.E. et al. Journal of Chromatography B: Biomedical Sciences &

Applications, 705(1), 47-54 (1998)

11.

Migdalof B.H. et al. Journal of Liquid Chromatography, 3(6), 857 (1980)

12.

Edited by Florey K. Analytical Profiles of Drug Substances, Volume 11 (1982,

Academic Press)

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Kawahara Y. et al. Chem. Pharm. Bulletin, 29(1), 150 (1981)