FIELD OF INVENTION
The present invention relates to a new HPLC method for the analysis of the drug
substance bosentan and related substances and use of said substances as reference
standards and markers.
BACKGROUND ART
In order to secure marketing approval for a pharmaceutical product, a manufacturer must
submit detailed evidence to the appropriate regulatory authorities to prove that the
product is suitable for release on to the market. It is, therefore, necessary to satisfy
regulatory authorities that the product is acceptable for administration to humans and that
the particular pharmaceutical composition, which is to be marketed, is free from
impurities at the time of release and that it has acceptable storage stability.
Submissions to regulatory authorities must include analytical data which demonstrate that
impurities are absent from the active pharmaceutical ingredient (API) at the time of
manufacture, or are present at acceptable levels, and that storage stability of the
pharmaceutical composition is acceptable.
The likely impurities in APIs and pharmaceutical compositions include residual
quantities of synthetic precursors (intermediates), by-products which arise during
synthesis of the API, residual solvents, isomers of the API (e.g. geometrical isomers,
diastereomers or enantiomers), contaminants, which are present in materials used in the
synthesis of API or in the preparation of the pharmaceutical composition and unidentified
adventitious substances. Other impurities which may appear during storage include
degradants of the API, such as those formed by hydrolysis or oxidation.
The Health Authorities have very stringent standards and manufacturers must
demonstrate that their product is relatively free from impurities or within acceptable
limits and these standards are reproducible for each batch of pharmaceutical product that
is produced.

The tests required to demonstrate the API or pharmaceutical compositions are safe and
effective include purity assays, related substances testing, content uniformity testing and
dissolution testing. The assay test determines the purity of the test product when
compared to a standard of known purity, while the related substances test is used to
quantify all the impurities present in the product. The content uniformity test ensures that
batches of product like a tablet contain a uniform amount of API and the dissolution test
ensures that each batch of product has a consistent dissolution and release of the API.
The technique of choice for the analysis of API or pharmaceutical compositions (e.g.
tablets and capsules) is usually High Performance Liquid Chromatography (HPLC)
coupled with a detector. Detectors include UV-visible detectors or mass-spectrometry
(MS) detectors. The API and the impurities present, if any, are separated on the HPLC
stationary phase and they can be detected and quantified by said detectors.
HPLC is a chromatographic separation technique in which high-pressure pumps force the
substance or mixture being analyzed together with a liquid solvent-mobile phase, also
referred to as the eluant - through a separating column containing the stationary phase.
HPLC analysis may be performed in isocratic or gradient mode. In isocratic mode, the
mobile phase composition is constant throughout. A gradient HPLC separation is carried
out by a gradual change over a period of time in the percentage of the two or more
solvents making up the mobile phase. The change in solvent is controlled by a mixer
which mixes the solvents to produce the mobile phase prior to its passing through the
column.
If a substance interacts strongly with the stationary phase, it remains in the column for a
relatively long time, whereas a substance that does not interact with the stationary phase
as strongly elutes out of the column sooner. Depending up on the strength of interactions,
the various constituents of the analyte appear at the end of the separating column at
different times, known as retention times, where they can be detected and quantified by
means of a suitable detector, such as a UV detector.

Bosentan belongs to a class of highly substituted pyrimidines and is used for the
treatment of pulmonary arterial hypertension by blocking the action of endothelin.
Bosentan, having a chemical structure as shown in formula I has a molecular weight of
551.615 and its molecular formula is C27H29N5O6S. Bosentan is a white to yellowish
white powder and is freely soluble in acetonitrile.

The prior art discloses three process impurities Ro 47-0005, Ro 47-4056 and Ro 47-9931
obtained during the synthesis of bosentan (EMEA 2005) However, the structures of these
impurities were not described. Further, the report also described the formation of three
metabolites: Ro 48-5033 (hydroxylation product of tertiary butyl group), Ro 47-8634
(free phenol metabolite) and Ro 64-1056 (a secondary metabolite, which is free phenol
and hydroxylated tertiary butyl group).
Several HPLC methods to detect these impurities are reported in the literature for
example: 1) "Evolution of bioanalytical methods for the cardiovascular drug bosentan",
Chromatographia, 55 (1), 2002 and 2) "Determination of endothelin receptor antagonist
in human plasma by narrow-bore liquid chromatography and ion spray tandem mass

spectrometry", J. Chromatography A, 712, p 75-83 (1995). These publications describe
isocratic HPLC methods using mixtures of ammonium acetate and acetonitrile and
reverse phase chromatography (RP-18 or RP-8).
None of the current HPLC methods are suitable for the detection and quantification of all
synthetic intermediates and other related substances that are present in a bosentan sample,
particularly a sample synthesised by alternative novel routes, such as the route disclosed
in Indian Patent Appl. No. 1245/MUM/2007. Current methods are also deficient in
estimating the total impurities in bosentan and its salts.
Therefore, the HPLC methods reported in the prior art are not convenient or suitable for
analyzing bosentan and its salts as an API, particularly with respect to related substances
present in sample synthesised by the route disclosed in Indian Patent Application No.
1245/MUM/2007.
Consequently, although several HPLC methods have been reported in the literature for
the analysis of bosentan and/or its salts and its impurities, there is still a need for an
alternative method which avoids the problems associated with the known methods as
discussed above.
Studies by the present inventors have lead to the development and validation of a new,
efficient, reproducible and simple HPLC method for the analysis of Bosentan,
particularly with respect to the related substances formed during the synthesis.
OBJECT OF INVENTION
It is, therefore, an object of the present invention to provide a new, alternative method for
analyzing Bosentan, its impurities and related substances, whilst avoiding the typical
problems associated with the prior art methods.
A particular object of the invention is to provide a new, accurate and sensitive HPLC
method for the detection and quantitation of intermediates and related substances that are

formed and may remain in the batches of Bosentan and/or its salts synthesised by the
route disclosed in Indian Patent Appl. No. 1245/MUM/2007.
A further object is the provision of reference markers and reference standards for use in
the detection of impurities designated A-E which are formed in processes for the
preparation of bosentan, in particular by the route disclosed in Indian Patent Appl. No
1245/MUM/2007.
SUMMARY OF THE INVENTION
The current invention provides a HPLC method for analyzing bosentan wherein the
mobile phase comprises two or more liquids and the relative concentration of the liquids
is varied to a pre-determined gradient.
The inventors have also appreciated that five impurities, designated compounds A-E, can
be utilised as reference markers or standards for the analysis of bosentan or of
pharmaceutical dosage forms comprising bosentan. The impurities A-E have not been
previously disclosed in the prior art.
Accordingly a first aspect of the present invention provides a Compound A having the
chemical name N-[6-(2-(2-hydroxyethoxy)emoxy)-5-hydroxy-2-pyrirnidin-2-yl-
pyrimidin-4-yl]-4-tert-butyl benzene sulphonamide and structure:


A second aspect provides a compound B having the chemical name N-[6-(ethene-1-oxy)-
5-hydroxy-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-butyI benzene sulphonamide and
structure:

A third aspect provides a compound C having the chemical name N-[6-2-ethoxy-(2-
hydroxyethoxy)-5-(2-methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-butyl-
benzene sulfonamide and structure:


A fourth aspect provides a compound D having the chemical name N-[6-hydroxy-5-(2-
methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-butyl-benzene sulfonamide
and structure:

A fifth aspect provides a compound E having the chemical name N-[6-(ethene-l-oxy)-5-
(2-methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-butyl benzene
sulphonamide
and structure:

The compounds A-E for use as reference markers are by-products formed during the
synthesis of bosentan. In a particularly preferred embodiment the compounds A-E
according to the invention are in isolated form. Most preferably the isolated form is in

substantially pure form preferably having a purity of greater than about 90%, preferably
greater than 95% most preferably greater then 99%.
A sixth aspect according to the invention provides a method of testing the purity of a
sample of bosentan or a pharmaceutical dosage form comprising bosentan, which method
comprises assaying the sample for the presence of one or more of the compounds A-E
according to the invention. In the method of the invention said compounds are acting as
reference markers.
According to a seventh aspect of the present invention, there is provided a method for the
characterization of the compounds A-E using a HPLC method for the analysis of said
process impurities A-E in bosentan. Preferably, the HPLC method is a LC-MS
compatible method.
Accordingly, there is provided the use of compounds A-E according to the invention as
reference marker(s) or alternatively as reference standard(s) in testing the purity of a
sample of bosentan or a pharmaceutical dosage form comprising bosentan.
A further aspect provides a HPLC method for testing the purity of a sample of bosentan
said method comprising determining the presence of any one or more of compounds A -
E in a sample by utilizing a reference marker or in alternative embodiments a reference
standard according to the invention.
HPLC method for testing the purity of a sample of bosentan said method comprising
determining the presence of any one or more of compounds A - E in a sample by
utilizing a reference marker
A further aspect provides a chromatographic method for testing the purity of a sample of
bosentan by determining the presence of any one of impurities A - E in a sample
comprising bosentan said method comprising:

i. dissolving a sample of bosentan or a dosage form comprising
bosentan in a solvent to produce a sample solution;
ii. dissolving a sample of a compound according to any of claims 1 -8
in a solvent to produce a reference marker solution;
iii. subjecting the sample solution and the reference solution to a
chromatographic technique and
iv. determining the presence of one or more of compound A-£ in the
sample by reference to the position of the known compound(s)
present in the reference solution.
In one embodiment the chromatographic method is HPLC, preferably the method is a
gradient HPLC method.
Preferably, the stationary phase used in the current invention is reverse phase. Suitable
stationary phases include octadecylsilyl silica gel or octylsilyl silica gel.
In a preferred embodiment of the invention there is provided a gradient HPLC method
wherein the mobile phase comprises a combination of aqueous solution of buffer (A) and
organic solvent (B) preferably the buffer (A) is an aqueous solution of a phosphate salt,
an acetate salt, a formate salt or trifluoroacetic acid or mixtures thereof. More preferably
the buffer (A) is an aqueous solution of an acetate salt, most preferably ammonium
acetate or alternatively ammonium formate, which in particularly preferred embodiments
are present at a concentration of between about 0.01M to 1.0M.
Further preferred embodiments according to the invention provide a mobile phase
wherein the organic solvent (B) is selected from the group comprising methanol
acetonitrile, propanol or isopropanol most preferably acetonitrile or alternatively
methanol or mixtures thereof.
A particularly preferred mobile phase comprises a combination of ammonium acetate (A)
and acetonitrile (B).

There is further provided a gradient HPLC method according to the invention wherein the
mobile phase comprises a gradient programming as follows:

A particularly preferred gradient HPLC method is also provided wherein the mobile
phase comprises ammonium acetate as the buffer (A). In another particularly preferred
embodiment the mobile phase comprises acetonitrile as the organic solvent (B). A further
preferred embodiment comprises a HPLC method wherein the pH of the buffer is about 2
to 6. In other embodiments the chromatography is carried out at a temperature of between
about 15-40°C.
The HPLC method according to the current invention efficiently detects and quantifies in
a single run all impurities including those selected from the following compounds:
Compound A: N-[6-(2-(2-hydroxyethoxy)ethoxy)-5-hydroxy-2-pyrimidin-2-yl-
pyrimidin-4-y]]-4-tert-butyl benzene sulphonamide.
Compound B: N-[6-(ethene-l-oxy)-5-hydroxy-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-
butyl benzene sulphonamide.
Compound C: N-[6-(2-(2-hydroxyethoxy)ethoxy)-5-(2-methoxyphenoxy)-2-pyrimidin-2-
yl-pyrimidin-4-yl]-4-tert-butyl benzene sulphonamide.
Compound D: N-[6-hydroxy-5-(2-methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-
tert-butyl benzene sulphonamide.

Compound E: N-[6-(ethene-l -oxy)-5-(2-methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-
4-yl]-4-tert-butyl benzene sulphonamide.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: analytical HPLC chromatogram of sample A
Figure 2: analytical HPLC chromatogram of sample B.
Figure 3: Structures of impurities A-E
Figure 4: MS/MS Fragmentation of Impurity A
Figure 5: MS/MS Fragmentation of Impurity B
Figure 6: MS/MS Fragmentation of Impurity D
Figure 7: MS/MS Fragmentation of Impurity E
DETAILED DESCRIPTION
The current invention can be used to analyse bosentan and/or its salts as an API or
bosentan and/or its salts when formulated in a pharmaceutical composition.
The pharmaceutical compositions that can be analysed by the current invention include
solid and liquid compositions and optionally comprise one or more pharmaceutically
acceptable carriers or excipients. Solid form compositions include powders, tablets, pills,
capsules, cachets, suppositories, and dispersible granules. Liquid compositions include
solutions or suspensions which can be administered by oral, injectable or infusion routes.
The term "Bosentan" as used herein throughout the description and claims refers to
bosentan and/or any salt, solvate, isomer or enantiomer thereof. The current invention is
particularly useful for the analysis of bosentan free base.
The term "impurities" or "related substances" as used herein throughout the specification
can mean either impurities formed in the manufacture of the API or the pharmaceutical
composition and/or formed by degradation of the API or in the pharmaceutical
composition on storage.

As discussed above, the HPLC methods reported in the prior art are not suitable for
analysing bosentan, particularly with respect to the related substances formed in the
synthesis of Bosentan and/or its salts prepared by the process disclosed in Indian Patent
Application No. 1245/MUM/2007. A reason for the difficulties encountered in the prior
art could be due to the large polarity differences between the related substances and
bosentan.
However, a particularly preferred embodiment of the current invention solves this
problem and efficiently detects and quantifies, in a single run, all impurities and
intermediates formed in this particular synthetic process. The present invention is
advantageous as the gradient method allows the elution of all polar to non-polar
impurities.
The present invention is particularly suitable for determining and quantifying the
presence of one or more of compounds or impurities A-E in a sample. The term
"impurity" and "compound" insofar as they relate to compounds A-E are used
interchangeably herein unless described otherwise.
The current invention is also advantageous as the method is selective, sensitive, linear,
precise, accurate and robust for the analysis of related substances in bosentan and/or its
salts. In addition, the current invention is highly sensitive and allows detection and
quantification of related substances in bosentan and/or its salts at levels much lower than
acceptance limits specified by health authorities and in ICH Guidelines.
In addition, the method of the current invention can be used to easily detect and quantify
all degradation impurities formed on storage of samples of bosentan. This was established
by carrying out forced degradation studies as per ICH Q1A Guidelines and validated as
per ICH Q2A Guidelines covering the parameters Specificity, Linearity and Range,
Precision (Repeatability, Reproducibility and Intermediate Precision), Accuracy, Limit of
Detection (LOD), Limit of Quantitation (LOQ), Robustness and System Suitability.

The present inventors have developed a novel gradient HPLC method to characterise five
process impurities A-E by LC-MS and LC-MS/MS. Said method is robust enough to be
used in the analysis of the presence of other known related substances such as precursors
in bosentan synthesis particularly bosentan synthesised by the route described in Indian
Patent Appl No. 1245/MUM/2007. Due to large polarity differences between the
impurities, precursors and Bosentan, a gradient programming was considered to be most
suitable by the inventors.
The inventors of the present invention have further used LC-MS and LC-MS/MS
techniques to characterise the structures of new process impurities A-E,
In the working of the invention the inventors of the present invention have found that
stationary phases comprising octadecylsilyl silica gel (RP-18) or octasilyl silica gel (RP-
8) to be most advantageous. A particularly preferred stationary phase comprises a Waters
XTerra RP18 (250 mm x 4.6 mm), 5µ; column.
The method of the current invention preferably comprises a gradient programming so that
the relative concentration of the liquids A and B are typically varied to a gradient
between 100 % A: 0 % B to 0 % A: 100 % B over a period of 10 to 180 minutes.
Preferably, the gradient is between 100% A: 0 % B to 0 % A: 100 % B over a period of
25 to 120 minutes. More preferably, 100 % A: 0 % B to 0 % A: 100 % B over a period of
25 to 60 minutes most preferably the gradient is between about 90 % A: 10 % B to 10%
A: 90%o B over about 40 minutes. The advantage of such a gradient method, allows the
elution of all polar to non-polar impurities.
The mobile phase used is preferably selected from combinations of one or more buffer(s)
(A) and one or more organic solvent(s) (B).
The buffer(s) is/are preferably selected from the group comprising an aqueous solution of
a phosphate salt, an acetate salt, a formate salt and trifluoroacetic acid or mixtures
thereof.

The buffer can be present at a concentration of 0.001 to 0.1 M, preferably at a
concentration of 0.001 to 0.05 M, more preferably at a concentration of 0.005 to 0.05 M.
A particularly preferred mobile phase comprises a combination of ammonium acetate (A)
and acetonitrile (B).
In a particularly preferred embodiment according to the invention there is further
provided a gradient HPLC method wherein the mobile phase comprises a gradient
programming as follows:

A particularly preferred gradient HPLC method is also provided wherein the mobile
phase comprises ammonium acetate as the buffer (A). In another particularly preferred
embodiment the mobile phase comprises acetonitrile as the organic solvent (B). The
inventors have found that the gradient programming is particularly effective when the
mobile phase comprises ammonium acetate (A) and acetonitrile (B).
The buffer (A) may contain one or more additional solvent(s) which are organic solvents
selected from methanol, acetonitrile, propanol or isopropanol or a mixture thereof. The
additional solvent in the buffer (A) may or may not be the same solvent as the organic
solvent (B). The additional solvent in the buffer (A) is preferably acetonitrile.
The pH of the buffer is selected to be between about 2 to 7.
Typically, the method of the current invention is carried out at a column temperature
between approximately 15 to 40°C.

A further aspect of the invention provides an internal reference solution. The reference
solution will comprise one or more of compounds A-E dissolved in an appropriate
solvent. Said reference solution may be used in determining the presence of any of
compounds A-E as impurities in a sample being analysed using chromatographic
techniques according to the invention. The method of said analysis will be apparent to the
skilled person.
A further aspect according to the invention provides a reference standard solution
wherein a known amount of one or more of compounds A-E is dissolved in an
appropriate solvent. Said reference solution may be used in determining the presence and
amount of any of compounds A-E as impurities in a sample being analysed using
chromatographic techniques according to the invention. The method of said analysis will
be apparent to the skilled person.
The inventors have tested the methods of the current invention extensively to show that
they are reproducible, accurate, precise and linear with respect to concentration and
robustness.
While the present invention has been described in terms of its specific embodiments,
certain modifications and equivalents will be apparent to those skilled in the art and are
intended to be included within the scope of the present invention.
The methods of the invention disclosed herein can also be used for the analysis of
compounds with similar chemical structures and/or similar chemical or physical
properties to bosentan, and its salts and/or isomers or enantiomers.
The present invention is illustrated but in no way limited by the following example.
EXAMPLE
HPLC Method and Analysis:
The five process impurities A to E in Bosentan observed during HPLC analysis were
found to be above 0.1 % by area normalization and are required to be identified as per

ICH Q3A guidelines. The method used for the said analysis is a gradient HPLC method
according to the invention. The experimental conditions used are as follows:
Experimental Conditions:
Column: Waters XTerra RP18 (250 mm x 4.6 mm), 5µ;
Flow rate: 1 ml/min;
Detection: 225 nm;
Sample concentration: 1000 ppm;
Diluent: Acetonitrile;
Mobile phase: 0.03 M Aqueous Ammonium acetate (A)-Acetonitrile (B) gradient;
The gradient program is described below:

Mass: API 2000 Triple Quadrapole
Ionization mode: Positive and Negative modes
Samples A and B of bosentan were analyzed for process impurities by LC-MS using the
above HPLC method. Figures 1 and 2 show the analytical HPLC chromatograms of said
samples respectively.
The retention times (RT), relative retention times (RRT) and % area of each impurity by
a area normalization method, molecular ions determined from the relevant mass spectra
and fragments from the secondary mass spectra (MS/MS) for each impurity are
summarised in Tables 1 and 2.

Characterisation:
HPLC analysis of the samples showed impurities A to E could be detected during mass
analysis. Based on the molecular ions obtained in the mass spectra of the impurities and
process conditions, the structures of impurities A to E were identified as depicted in
Figure 3.
Further, on the basis of fragmentation pattern reported in literature [J. Am. Soc. Mass.
Spectrum., vol 10 (12), p 1305-1314 (1999)] for Bosentan, the structures of the impurities
A, B, D and E were confirmed by the interpretation of the fragment ions observed in
MS/MS spectra. The fragmentation pattern of the impurities A, B, D and E are shown in
figures 4 to 7 respectively. In the case of impurity C the molecular ion peak observed at
m/z 594.3 (M-H)+ by MS fragmented so much that during MS/MS studies none of the
peaks were informative as to the nature of impurity C. Thus, due to the absence of
diagnostic fragments in the MS/MS spectra of impurity C, the structural confirmation
could not be performed.
The chemical names of the impurities A to E are:
Impurity A: N-[6-(2-(2-hydroxyethoxy)ethoxy)-5-hydroxy-2-pyrimidin-2-yl-pyrimidin-
4-yl]-4-tert-butyl benzene sulphonamide.
Impurity B : N-[6-(ethene-l-oxy)-5-hydroxy-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-/er/-
butyl benzene sulphonamide.

Impurity C : N-[6-(2-(2-hydroxyethoxy)ethoxy)-5-(2-methoxyphenoxy)-2-pyrimidin-2-
yl-pyrimidin-4-yl]-4-tert-butyl benzene sulphonamide.
Impurity D : N-[6-hydroxy-5-(2-methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-
tert-butyl benzene sulphonamide.
Impurity E : N-[6-(ethene-l-oxy)-5-(2-methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-4-
yl]-4-tert-butyl benzene sulphonamide.

We claim:
1. Compound A having the chemical name N-[6-(2-(2-hydroxyethoxy)ethoxy)-5-
hydroxy-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-butyl benzene sulphonamide and
structure:

2. Compound B having the chemical name N-[6-(ethene-l-oxy)-5-hydroxy-2-
pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-butyl benzene sulphonamide and structure:

3. Compound C having the chemical name N-[6-(2-(2-hydroxyethoxy)ethoxy)-5-(2-
methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-butyl benzene
sulphonamide and structure:


4. Compound D having the chemical name N-[6-hydroxy-5-(2-methoxyphenoxy)-2-
pyrimidin-2-yl-pyrimidin-4-yl]-4-tert-butyl benzene sulphonamide and structure:

5. Compound E having the chemical name N-[6-(ethene-l-oxy)-5-(2-
methoxyphenoxy)-2-pyrimidin-2-yl-pyrimidin-4-yI]-4-tert-butyI benzene
sulphonamide and structure:


6. A compound according to any of claims 1 to 5 in isolated form.
7. A compound according to claim 6 which is in substantially pure form.
8. A compound according to any of claims 6 or 7 which has a purity of greater than
about 90%.
9. Use of a compound according to any of claims 1-8 as a reference marker in
testing the purity of a sample of bosentan or a pharmaceutical dosage form
comprising bosentan.
10. Use of a compound according to any of claims 1-8 as a reference standard in
testing the purity of a sample of bosentan or a pharmaceutical dosage form
comprising bosentan.
11. A HPLC method for testing the purity of a sample of bosentan said method
comprising determining the presence of any one or more of compounds A - E in a
sample by utilizing a reference marker according to claim 9 or a reference
standard according to claim 10.
12. A chromatographic method comprising a mobile phase and a stationary phase for
testing the purity of a sample of bosentan by determining the presence of any one
of impurities A to E in a sample comprising bosentan said method comprising:

i. dissolving a sample of bosentan or a dosage form comprising
bosentan in a solvent to produce a sample solution;
ii. dissolving a sample of one or more compound(s) according to any
of claims 1 -8 in a solvent to produce a reference marker solution;
iii. subjecting the sample solution and the reference solution to a
chromatographic technique, and
iv. determining the presence of one or more of compound A-E in the
sample by reference to the or presence of the known compound(s)
present in the reference solution.
13 A method according to claim 12 wherein the chromatographic method is HPLC.
14. A method according to claim 13 wherein the method is a gradient HPLC method
15 A method as claimed in claim 13 or 14 wherein the stationary phase is reverse
phase.
16. A gradient HPLC method according to any of claims 12 to 15, wherein the
stationary phase used is octasilyl silica gel.
17 A gradient HPLC method according to any of claims 12 to 15, wherein the
stationary phase used is octadecylsilyl silica gel
18. A gradient HPLC method according to any of claims 12-17, wherein the mobile
phase comprises a combination of a buffer (A) and organic solvent (B).
19. A gradient HPLC method according to claim 18 wherein the buffer (A) is an
aqueous solution of either a phosphate salt, an acetate salt, a formate salt or
trifluoroacetic acid or mixtures thereof.

20. A gradient HPLC method according to claim 19 wherein the buffer (A) is an
aqueous solution of an acetate salt
21. A gradient HPLC method according to claim 20, wherein the buffer (A) is
aqueous solution of ammonium acetate.
22. A gradient HPLC method according to claim 21, wherein the concentration of
ammonium acetate is 0.01 M to 1.0M.
23. A gradient HPLC method according to any of claims 18-22, wherein the organic
solvent (B) is selected from the group comprising methanol, acetonitrile, propanol
or isopropanol or mixtures thereof.
24. A gradient HPLC method according to claim 23 wherein the organic solvent (B)
comprises acetonitrile.
25. A gradient HPLC method according to claim 23 wherein the organic solvent (B)
comprises methanol.
26. A gradient HPLC method according to any of claims 18-25 wherein the mobile
phase comprises a combination of ammonium acetate (A) and acetonitrile (B).
27. A gradient HPLC method according to claims 18-26, wherein the mobile phase
comprises a gradient programming as follows:


28. A gradient HPLC method according to claim 27 wherein the mobile phase
comprises ammonium acetate as the buffer (A).
29. A gradient HPLC method according to either of claims 27 or 28 wherein the
mobile phase comprises acetonitrile as the organic solvent (B).
30. A gradient HPLC method according to any of claims 18-29, wherein the pH of the
buffer is about 2 to 6.
31. A gradient HPLC method according to any of claims 16-30, wherein the
chromatography is carried out at a temperature of between about 15-40°C.

A new HPLC method for the analysis of the drug substance bosentan and related
substances and use of said substances as reference standards and markers.