The renin-angiotensin system (RAS) is a physiological system that regulates blood pressure (BP). The enzyme renin or angiotensinogenase is secreted into the blood from juxtaglomerular kidney cells. The renin-secreting cells are sensitive to changes in blood flow and BP. An important stimulus for increased renin secretion is a decreased blood flow to the kidneys, which may be caused by loss of sodium and water (as a result of diarrhea, or persistent vomiting) or by narrowing of a renal artery. The renin enzyme circulates in the blood stream and catalyzes the conversion (hydrolysis) of angiotensinogen, secreted from the liver, into the peptide angiotensin I. An enzyme in the serum called angiotensin-converting enzyme (ACE) then converts angiotensin I into angiotensin II. Angiotensin II is the key effector peptide of RAS, which is central to the development of hypertension (Figure 1).1 Angiotensin II acts via receptors in the adrenal glands to stimulate the secretion of aldosterone, which stimulates salt and water reabsorption by the kidneys, and the constriction of small arteries (arterioles), which causes the blood pressure to rise. Patients with high levels of plasma renin activity have increased angiotensin II activity, the effects of which may lead to high BP.2 Candesartan Cilexetil is one of the most often-used first-line drugs regarding the management of arterial hypertension (Figure 2).
Figure 1. The renin-angiotensin system. Angiotensin II receptor antagonists, such as Candesartan, act by blocking binding of angiotensin II to the receptor subtype 1 (AT1). ACE inhibitors prevent conversion of angiotensin I to angiotensin II.1 (AT 2 = angiotensin II receptor subtype 2; BK = bradykinin receptor)
1.2 Candesartan Cilexetil
Candesartan Cilexetil is an esterified prodrug of Candesartan (receptor antagonist), which is converted to Candesartan by hydrolysis during absorption in the gastrointestinal tract (Figure 2). It is used in the treatment of hypertension. The prodrug is a non-peptide and classified as a selective angiotensin II receptor blocker (Figure 1). When angiotensin II is formed in the blood by the activity of ACE, it attaches to the angiotensin receptors. These receptors are located in tissues all over the body but primarily in smooth muscle cells surrounding blood vessels. Due to the binding of angiotensin to their receptors, vasoconstriction is caused (contraction of the muscles and narrowing of the blood vessels), resulting in an increase in BP. Candesartan enables the blood vessels to relax and to expand. Consequently, the BP will drop.3,4,5
Candesartan Cilexetil is most known by the following systematic chemical name: (±)-l-Hydroxyethyl 2-ethoxy-l-[p-(o-1H-tetrazol-5-ylphenyl) benzyl]-7-benzimidazole-carboxylate, cyclohexyl carbonate ester. C33H34N6O6 is referred to as its empirical formula. Its molecular weight is determined to be 610.66 g/mol.4,5,6
Figure 2. Chemical structure of Candesartan (left) and Candesartan Cilexetil (right)7
This receptor antagonist consists of a white powder. Its solubility across the physiologically pH range is very low: practically insoluble in water, sparingly soluble in methanol, and soluble in acetonitrile.4,8 In contrary, the ester prodrug is a highly lipophilic substance hence it shows a high solubility in tri- and diglyceride oils. Candesartan Cilexetil has a pKa value of 6.0.8 At 163ºC the compound starts to melt as well as to decompose.5
In the beginning, two polymorphic forms and the amorphous form of Candesartan Cilexetil were identified. These two polymorphisms were identified as Form-I (melting point at 120ºC) and Form-II (melting point at 163ºC). They were both isolated by recrystallization. To isolate Form-I, a mixture of 3:1 acetone: water is used while for the recrystallization of Form-II only acetone is needed. Nowadays, scientists discovered additional polymorphic forms of Candesartan Cilexetil. Currently, there are 31 polymorphic forms of Candesartan Cilexetil reported. Most of them have been characterized by X-ray powder diffraction or infrared absorption spectroscopy.5
1.3 Impurities
In Candesartan Cilexetil, various potential impurities are found. Impurities can be described as substances which do not provide any beneficial therapeutic effect, but have the ability to cause adverse effects.9 They can be subdivided into five different types: 1. organic/related substances; 2. residual solvents; 3. inorganic impurities; 4. potential genotoxic impurities and 5. special reagents. In this present project, the focus was on the fourth type of impurity.10
Potential genotoxic impurities (PGI) are impurities that are proven to be harmful to the human body even at extremely low concentrations. They are able to bring severe damage to the genetic information within a cell, which may lead to mutations and, in turn, may lead to cancer. Therefore, it is very important to identify these impurities and define their limits at an early stage.9 The concentration of all PGI’s in this drug substance is limited to 47 ppm. The concentration limit is based on the next equation10:
(Threshold of Toxicological Concern [µg/day] )/(dose [g/day ])= (1.5µg/day)/(0.032g/day)=47 ppm.
Currently, seven PGI’s have been found in Candesartan Cilexetil: Cilexetil, Azide, PGI-1, PGI-2, PGI-3, PGI-4 and PGI-5. Different chromatographic methods are used to separate and identify PGI’s from the active pharmaceutical ingredients (API).10
1.4 Determination/Separation of Candesartan Cilexetil and its potential genotoxic impurities
1.4.1 Chromatography
Chromatography is one of the most versatile and powerful techniques in analytical chemistry to separate and dose components in a complex mixture of chemical substances. Nowadays the technique includes a lot of different methods which makes it hard to give one single and comprehensive definition of chromatography. According to the International Union of Pure And Applied Chemistry (IUPAC) chromatography is defined as: ‘A physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.’ 11,12
There are many chromatographic systems that can be classified in different manners e.g. based on physicochemical processes, based on the physical nature of the stationary and mobile phase. The most common examples of chromatographic systems are: gas chromatography, liquid chromatography which includes normal phase chromatography (NPC) and reversed phase chromatography (RPC), hydrophilic interaction chromatography (HILIC), size exclusion chromatography (SEC), affinity chromatography (AC) and ion exchange chromatography (IEC).
In the present literature review, only the concept of liquid chromatography will be discussed in depth.
1.4.1.1 High Performance Liquid Chromatography
HPLC is the most common separation technique used all over the world. A typical HPLC system consists of the following parts: an injection system, a pump, a column system which, in turn, consists of an analytical column (optional a guard column), peek/stainless steel tubing with fittings, and a detector.13
The technique involves the injection of a small volume (usually in µl) of the sample into the system by means of a syringe. The sample will be injected in the mobile phase, which moves through the column by gravity. In the case of HPLC, the mobile phase is a liquid. The substances of the drug mixture will be forced through a column with the mobile phase under high pressure up to 400 atmospheres delivered by a pump.13 The column is a tube packed with porous, spherical particles with an internal diameter that varies between 3 – 5 mm. These particles consist mostly of porous silica (SiO2) that makes up the stationary phase. The components of the sample separate due to a balance between both (mobile and stationary) phases (Figure 3). The affinity of the molecules for the column is determined by various chemical and/or physical interactions as explained in section 1.4.1.3. The separated substances pass through a detector. The mechanism of obtaining peaks includes a flow cell that detects each compound against a background of mobile phase at a set wavelength. The detector sends an electrical signal to a computer data station where results are processed.
The outcome is a chromatogram. The peaks of a chromatogram represent the different compounds. The output can also be identified by an external measurement technique such as a mass spectrometer which calculates the amount of substances by measuring their mass. 14,15,16
Figure 3. Principle of HPLC15
1.4.1.2 Mobile/Stationary phase
Two important variants that are commonly used in HPLC include: normal phase chromatography and reversed phase chromatography. The major difference lies in the polarity of the solvent used and in the composition of the stationary phase. The efficiency of migration and separation is dependent on the polarity of the mobile phase and the type of adsorbent material used as the stationary phase.14
1.4.1.3 Reversed phase chromatography
RPC is one of the most applied methods worldwide.14 In this system the mobile phase is significantly more polar than the stationary phase. Reverse phase operates on the basis of hydrophobic interactions.17
The stationary phase is build out of silica-based particles modified by the binding of n-alkyl chains to its surface. The longer the alkyl chain, the greater the capacity of the column to retain hydrophobic molecules. Typically columns with chain length of 8, 12 or 18 carbons are used, referred to as C8, C12 or C18 columns.17
The mobile phase is a polar solvent that consists of a small amount of organic solvent (e.g. acetonitrile or methanol) dissolved in water. When decreasing the concentration of organic solvent, the polarity will increase which, in turn, increases the interaction with the stationary phase. Consequently, the ‘elution power’ will decrease and the components will elute at a later time.18
The polar molecules, moving with the polar mobile phase, will have a low affinity to the non-polar stationary phase, and will pass rapidly through the column (= short retention time). Non-polar molecules in the sample will have a higher affinity to the non-polar stationary phase and tend to stick to the column. They will pass the column more slowly (= high retention time). Consequently, hydrophilic molecules elute from the column first, while hydrophobic molecules will be retained.14
1.4.1.4 Normal phase chromatography
Normal phase HPLC is similar to thin layer chromatography or column chromatography.14
The solvent used in this system is a non-polar solvent for example hexane or diethyl ether. The stationary phase consists of very tiny silica particles. Polar components in the mixture will form attractions with the polar silica. Non-polar components will not adhere easily to the stationary phase and will therefore pass through the column more quickly.14
1.4.2 Mass spectrometry
The technique used in the present project is ‘Liquid Chromatography – Mass Spectrometry’ (LC-MS), in which HPLC and mass spectrometry are combined.
Mass spectrometry enables the identification of molecules based on their mass-to-charge (m/z) ratio. Mass spectrometry is based on these main steps19,20:
Molecules in the liquid sample get bombed by electrons (e-) with a very high energy level. This causes elimination of an e- out of the samplemolecule. An ion radical with a positive charge is formed.
This positively charge ion radical is very unstable due to an excess of vibrational energy. This results in fragmentation of the ion radical into smaller, more stable radicals and positive ions. Often these fragments are unique to the compound.
The ions will separate according to (m/z) ratio.
The seperated ions reach the detector one by one.
A mass spectrum is formed in which the absolute or relative intensity of the produced ions is plotted against their mass/charge ratio. This mass spectrum provides information about the molecular mass of the substance and can also provide structural information about the molecules.
1.4.2.1 Electrospray Ionization
The conversion of sample solution into gaseous phase is performed by Electrospray Ionization (ESI) which uses electrical energy to support the transfer of ions (Figure 4).
To get the liquid stream in the mass spectrometer into charged droplets, the liquid is compressed in a Taylor cone and sprayed out of a quartz silica capillary tube or a stainless steel to which a high voltage is applied. A cloud of tiny, charged droplets will be released. Electrospray ions can be classified in two categories: positive electrospray ions (addition of a positive ion to a molecule e.g. H+, Na+,…) and negative electrospray ions (removal of a proton from a molecule). The charge of the ions depends on the potential gradient which is applied to the electrospray tip. 19,21,22
The size of the droplets will decrease due to evaporation of the solvent. A desolvation gas accelerates the evaporation process.. A nebulizing gas ensures that the spray of ions is transported in the right direction. Both gasses consist of nitrogen. This evaporation process continues until the charge density on the surface of the droplet comes to a point where the repulsive force between charges overcomes the liquid surface tension that keeps the drop together. At this point, the Rayleigh limit is reached and small gaseous droplets will be expelled from the main droplet by a process called Coulomb fission. 19,21,22
The charged ions will be sampled in a cone and transported throughout an electric field to the mass-analyzer where they will be fragmented sequentially and analyzed. To get more ions into the mass spectrometer Z-spray is used. 19,21,22
Figure 4. ESI process23
1.4.1.2 Quadrupole detector
In this experiment a Quadrupole mass analyzer (QDa) carried out the analysis of ions. This is a single quadrupole mass detector. Main components of the quadrupole analyzer are the four parallel, conductive, cylindrical rods. The rods are located opposite to each other in a bundle, and are connected by electrical forces. There are two pairs of electrodes, each with an equal but opposite potential gradient. The ion beam moves through the quadrupole to the detector (Figure 5). Since the applied voltage has an effect on the trajectory of the ions between the four rods, only ions with a certain (m/z)-ratio will reach the detector. Other ions follow an unstable route and will collide with the rods in which case they will not get detected.20,22
Figure 5. Quadrupole analyzer24
1.5 Validation
To obtain reliable, consistent, qualitative and accurate data it is highly recommended to validate the method used in an analytical practice. In order to accomplish validation, a couple of validation parameters are tested. In some cases it is required to perform revalidation e.g. when there are changes in the synthesis of the drug substance, in the composition of the final product or in the analytical process.25
1.5.1 Precision
Precision is the closeness of agreement between the results of multiple measurements of the same homogenous sample. This parameter is usually expressed as the standard deviation or variation coefficient. Precision may be considered at three levels: intermediate precision, repeatability and reproducibility.25,26
The aim of intermediate precision is to prove that the method is able to provide the same results within the same laboratory. So this factor determines the variation within the laboratory. These variations are investigated by performing the method on different days, by different analysts or by using different equipment. 25,26 In this dissertation, intermediate precision is not discussed as it was not performed due to limited time of the lab analysts.
Repeatability expresses the ability of reproducing the same precise results over a short interval of time under the same operating conditions. Intra-assay is another term to indicate repeatability.25,26
To check the precision between laboratories, reproducibility is determined. Reproducibility is an important parameter to discuss when the analytical method is to be used in different laboratories. Differences in room temperature or humidity, columns from different suppliers are few of the many factors that can influence reproducibility.25,26
1.5.2 Linearity
The International Conference on Harmonization (ICH) defines linearity as: ‘The ability (within a given range) to obtain test results that are directly proportional to the concentration (amount) of analyte in the sample.’25,26 Linearity is tested by measuring a series of five or six injections of standards whose concentrations cover 80%-120% of the expected concentration range.25,26
1.5.3 Specificity
Specificity is achieved when the method is capable of assessing unambiguously the analyte of interest in a mixture where other components might be present together with the analyte.25,26
1.5.4 Accuracy
An analytical method is said to be accurate when the average value obtained by a series of measurements is close to the accepted true value.25,26
1.5.5 Limit of Detection
The lowest amount of analyte in a sample which can be detected is referred to as the limit of detection (LOD). This concentration does not necessarily need to be quantified as a precise value.25,26,27
1.5.6 Limit of Quantitation
The limit of quantitation (LOQ) describes the smallest concentration of analyte in a sample that can be determined with a reliable level of precision and accuracy. At this point all acceptance criteria are met and the quantitative value lies 20% of the target concentration.27
1.5.7 Robustness
To call an analytical method robust, the method has to remain unaffected by small, calculative differences in method parameters. This factor tells us more about the reliability of the method.25,26
1.6 Study aims
The aim of this study was i) to develop an HPLC technique that is able to separate Candesartan Cilexetil from the potential genotoxic impurity 5 (PGI-5) and ii) to test and further validate the HPLC method on an LC-MS.
Based upon the process validation for the active pharmaceutical ingredient (API) provided by the supplier, only traces of PGI-5 were found. All other PGI’s were not detected. Based on these results, PGI-5 is the only impurity included in the specification. Because the limit for the presence of PGI-5 in Candesartan Cilexetil is so low, it is not possible to detect PGI-5 accurately with an HPLC technique. Hence a Liquid Chromatography – Mass Spectrometry (LC-MS) method is needed to perform the separation.10
The chemical name of PGI-5 is ethyl 2-(2’-cyano-biphenyl-4-yl-amino-)-3-nitrobenzoate (Figure 6). Its molecular formula is C23H19N3O4 and its monoisotopic mass is equal to 401.14 Da.10
Figure 6. Chemical structure of PGI-510
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5. Adriana, F., Indrayanto, G. and Lestari, M. (2012). Chapter 3. Candesartan Cilexetil. In: Profiles of Drug Substances, Excipients and Related Methodology, 1st ed. Volume 37: Academic Press, pp.79-112.
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18. Schug, K. and Taylor, T. (n.d.). The Essential CHROMacademy Guide: Mobile Phase Optimization Strategies for Reversed Phase HPLC.
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23. Godefroid, M. (2009). Kwantitatieve bepaling van adrenaline en noradrenaline in plasmastalen van patiënten onder anesthesie via HILIC-MS/MS. Eerste master in de Geneesmiddelenontwikkeling. Universiteit Gent.
24. Iowa State University. (2015). Mass Spectrometry Tutorial. [online] Available at: http://www.cif.iastate.edu/mass-spec/ms-tutorial [Accessed 15 Mar. 2016].
25. Huber, L. (n.d.). Validation of Analytical Methods. 1st ed. [ebook] Agilent Technologies, pp.2-28. Available at: https://webcache.googleusercontent.com/search?q=cache:X1AKz1A-YfcJ:https://www.agilent.com/cs/library/primers/Public/5990-5140EN.pdf+&cd=1&hl=nl&ct=clnk&gl=us [Accessed 14 Mar. 2016].
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27. Armbruster, D., Tillman, M. and Hubbs, L. (1994). Limit of Detection (LOD)/Limit of Quantitation (LOQ): Comparison of the Empirical and the Statistical Methods Exemplified with GC-MS Assays of Abused Drugs. Clinical Chemistry, 40(7), pp.1233-1238.