Separation and Identification of Related Substances in Candesartan Cilexetil Tablets by UHPLC-Q-TOF–MS

Related substances, such as process-related substances and degradation products, may affect the efficacy of drugs and cause adverse reactions. Therefore, identifying and controlling them is of the importance. A rapid ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF–MS) method has been developed for the separation and characterization of related substances in candesartan cilexetil tablets. After optimization, the method validation was followed according to ICH guidelines. The developed UHPLC method showed adequate specificity, sensitivity, accuracy, linearity, precision, stability and robustness for validation of analytical procedures. Commercial candesartan cilexetil tablets was subjected to stress testing (60 ℃, 90% RH and 4500 lx for ten days) and forced degradation studies (acidic, alkaline, oxidative and photolytic degradation conditions). A total of eleven related substances were detected and characterized. Among them, four related substances have not been reported in the literature yet, and one of them (RS7) was confirmed as candesartan cilexetil methoxy analog by reference substance. In addition, plausible mechanisms for the formation of these related substances are discussed. This study provides a useful reference for the quality control of candesartan cilexetil tablets.


Introduction
Candesartan cilexetil (CDC) (Fig. 1) is the prodrug of candesartan (CD), an angiotensin II receptor antagonist. Candesartan binds selectively and non-competitively to the angiotensin II receptor (AT1), thus preventing the actions of angiotensin II. Clinical trials have demonstrated its efficacy in hypertension of all grades, heart failure, in reducing urinary albumin excretion in diabetes mellitus and in coexisting hypertension and renal failure [1]. At present, quality standards of CDC substances and tablets are official in United States Pharmacopoeia (USP) [2], British Pharmacopoeia (BP) [3] and European Pharmacopoeia (EP) [4]. EP lists nine specified impurities, named A-I. Impurity separation and identification in CDC substances and tablets are reported in several articles. Kumar et al. developed an UPLC method for the determination of CDC impurities in tablet formulations [5]. Rao et al. reported a stability-indicating LC method for CDC in the bulk drug and in pharmaceutical dosage forms [6]. Mohan et al. found out the isolation and structural identification of five potential degradation products (EP impurities B, C, D, E and F) in CDC tablets during accelerated testing and thermal degradation study [7]. Mehta  oxidation and thermal stress [8]. Bhanu et al. figured out one process-related impurity (N1-ethyl ethylcandesartan) in CDC substance using LC/ESI-ITMS and NMR methods [9]. Nonetheless, no comprehensive study has been made for identification of related substance present in CDC tablets, which is essential for ensuring drug safety and efficacy. The present manuscript describes (1) degradation study of CDC tablets under ICH Q1 [10] prescribed stress conditions; (2) development and optimization of UHPLC method for resolving CDC and its RSs (including process-related impurities and degradation products); (3) validation of the UHPLC method (4) identification of unknown RSs by Q-TOF/MS study; (4) possible formation mechanisms of RSs.

Sample Preparation
The test solutions were prepared according to the EP method by dissolving an accurately weighed portion of powder (20 tablets crushed with mortar and pestle), equivalent to 8 mg of CDC in 10 mL diluent A (acetonitrile/water = 3:2 v/v). After shaking for around 10 min, the volume was made up to 20 mL, resulting in a concentration of 0.4 mg/mL. Forced degradation of CDC tablets obtained from company A was carried out by hydrolysis, photolysis and oxidation treatments as defined in the ICH guideline Q1A (R2) [10]. The thermal degradation study was performed in the stress testing (60 ℃ for ten days). For hydrolytic degradation, powder equivalent to 8 mg of CDC was mixed with 10 mL of diluent A in 20 mL volumetric flasks. After shaking for 10 min, 0.8 mL of 0.1 M HCl and 0.1 M NaOH were separately added into the flasks. Acid and alkaline hydrolysis samples were kept at room temperature for 15 h and 1 h, respectively. The acid and alkaline hydrolyzed solutions were neutralized followed by diluting to the desired volume. For oxidative degradation, powder equivalent to 8 mg of CDC was dispersed in 10 mL of diluent B (methanol/ Fig. 1 Chemical structure of Candesartan cilexetil water = 3:2 v/v) in 20 mL of volumetric flask. After 10 min of shaking, 0.8 mL of 30% H 2 O 2 was added to the mixture. The oxidative degradants were obtained after heating the mixture on a 80 ℃ water bath for 1.5 h, and finally the volume was made up to volume with diluent B. Photolytic degradation was performed by exposing the test solution to UV light at 254 nm at room temperature for 4 h. During accelerated stability studies, CDC tablets were respectively exposed to dry heat at 60 ℃ in an oven, relative humidity of 90% in a climate chamber and 4500 lx in a light box for 10 days. Then the preparation of stress testing samples was the same as that of the test solution.
As it was found that acetonitrile would react with CDC under oxidative stress, resulting in acetonitrile adducts, diluent B was chose as optimal diluent for the preparation of oxidative degradation sample. Each diluted sample was centrifuged at 11,500 rpm for 10 min to eliminate insoluble excipients. Finally, the supernatant liquid was used for chromatographic analysis.

Method Development
A time-consuming gradient HPLC method (acetonitrile/ water/acetic acid in a ratio of 57:43:1 (v/v/v) and acetonitrile/water/acetic acid in a ratio of 90:10:1 (v/v/v) are used as mobile phase A and mobile phase B, respectively.) for related substances test of CDC is recruited in USP and BP monographs. To save time, an UHPLC method was developed in this study. The UHPLC method was initiated with a gradient program and flow rate obtained by converting EP method with an Agilent HPLC/UHPLC translator. When optimum resolutions were not achieved on the flow rate of 0.2 mL/min obtained by the translator, 0.3 mL/min was used for method optimization. The optimal resolution, elution time, and peak shapes were achieved using the following gradient program 0 min (100%A-0%B) → 0.6 min (100%A-0%B) → 6.6 min (0%A-100%B) → 10.0 min (0%A-100%B) → 10.2 min (100%A-0%B) → 13.0 min (100%A-0%B). In an effort to promote the response of compounds in mass spectrometry, 1% acetic acid involved in the HPLC method was replaced by 0.1% formic acid. ACQUITY UPLC BEH C18 (2.1 × 150 mm, 1.7 μm) and ZORBAX Eclipse XDB-C18 column (2.1 mm × 100 mm, 1.8 μm) were the columns initially selected for RS study. It was found that the interference of gradient to RS9 was considerable and the resolution between RS3 and RS4 (R < 1.5) was poor when using the former one. Thus ZORBAX Eclipse XDB-C18 column (2.1 mm × 100 mm, 1.8 μm) was employed to achieve the resolution of individual related substances from CDC. The optimized chromatographic conditions are shown in "Instruments and conditions".

Specificity
The specificity of the UHPLC method was demonstrated by interference tests and stress tests. An interference test was carried out by spiking the CDC drug substance with appropriate levels of impurities A, B, C, D, E, F, G and I. As shown in Fig. S1, the detection of CDC and the impurities did not interfere with each other. The excipients and solvent blanks did not interfere with the determination of impurities. As shown in Fig. 2, effective separation was found between each degradation impurity and CDC.

Linear and Range
A series of linearity solutions were prepared containing impurities A, B, C, D, E, F, G and I solution at different concentrations, i.e., 2 to 200% of the target limit for each impurity. The measured impurity peak areas were linearly regressed with least squares to verify the linearity of the method. The correlation coefficients for each impurity were greater than 0.999. The linear range and correlation coefficients for all impurities were listed in Table S1.

Precision
Precision was assessed by determining six independence spiked samples at 100% of the target limit for impurities A, B, C, D, E, F, G and I. As shown in Table S1, the results showed that the RSD values of repeatability were less than 4.7% (N = 6).

Limit of Detection (LOD) and Limit of Quantitation (LOQ)
LOQ was calculated with the S/N ratio of 10, and LOD was calculated with the S/N ratio of 3. The LOQs and LODs of impurities A, B, C, D, E, F, G and I were in the range of 0.087-0.194 μg/ml and 0.037-0.97 μg/mL, respectively. The LOQ and LOD results for above impurities were tabulated in Table S1.

Accuracy
Spiked samples were prepared at 50, 100 and 150% of the target limit for each impurity at triplicate. The accuracy of the method was evaluated by determinating the spiked samples. Recoveries of spiked impurities at the analytical concentration level were obtained within the acceptance limits of 94.9-108.3%, which met the requirements of ICH 1 3 guidelines. The %recovery for each of the impurities at all levels were listed in Table S1.

Stability
The stability study was investigated by injecting impurity standard solution, 100% spiked sample and test solution which were placed at room temperature for 0 and 24 h respectively, calculating the peak area of each impurity at 0 and 24 h. As shown in Table S2, the ratios of peak area of each impurity in the above three solutions at 24 h and 0 h were in the range of 95.45-109.89%, indicating that solutions were stable at room temperature for at least 24 h.

Robustness
To examine the degree to which the resolution was affected when the method parameters changed slightly, column oven temperature (± 5 ℃) and flow rate (± 0.02) were investigated. The results showed that the developed method was unaffected upon applying small variations to the chromatographic conditions. The resolution under these changing conditions are summarized in Table S3. Figure 2 shows the chromatograms of CDC test solution and all the degradation samples. Among them, ten forced degradation products (RS1-6, 8-11) and one process-related impurity (RS7) were detected. RS1, 2, 6, 8, 9 and 10 correspond to EP impurities G, B, C, D, E and F, respectively. Although the amount of RS9 did not increase significantly under forced degradation conditions in this study, it was still classified as degradation product according to the study by Mehta et al. [8]. Degradation of CDC was observed under acid hydrolysis (RS2 and 9), alkaline hydrolysis (RS1), oxidative (RS1-6, 8 and 10), photolytic (RS10) degradation conditions and stress testing (60 ℃ for ten days) (RS2 and 11) as described in "Sample preparation". None of degradation product was observed under accelerated stability studies (90% RH and 4500 lx for ten days for ten days) (Fig. S2).

UHPLC-Q-TOF-MS Study on CDC
UHPLC-Q-TOF-MS spectrum of CDC was recorded to outline mass fragmentation pattern for assisting the characterization of related substances (Fig. 3). The protonated molecule of CDC produced a base peak at m/z 611.2613, corresponding to the formula of C 33 H 34 N 6 Fig. 4, and the chromatographic and mass spectrometric data are summarized in Table 1.

UHPLC-Q-TOF-MS Studies on Known RSs
The RS 1, 2, 6, 8, 9 and 11 were identified as EP impurity G, B, C, D, E and F, respectively, by comparing their retention times and fragment ions with those of reference substances used in the analysis of related substances of CDC. The UHPLC chromatogram of system suitability solution (containing EP impurity B, C, D, E, F, G, I and CDC references) is shown in Fig. S2. MS/MS spectrum and possible mass spectral fragmentation pathway of RS1, 2, 6, 8, 9, 11 are shown in Fig. S3 and Fig. S4, respectively.

RS3 and RS5
RS3 and RS5 were the major degradation products under oxidative degradation condition with decreased retention compared to the drug (Fig. 2e). The observed accurate m/z value of RS3 and RS5 were 627.2511 and 627.2553, respectively. Based on the exact mass data, the elemental compositions of RS3 and RS5 are both C 33 H 34 N 6 O 7 . By comparing with the elemental composition of the drug, it was found that RS3 and RS5 had an additional oxygen atom due to oxidation. As shown in Fig. 3 .0923, suggesting the structural difference was attributed to biphenyl-tetrazole structure. The characteristic fragment ions at m/z 180.0824 and 180.0804, which were assigned as methylene-carbazole ion, were separately found in MS/MS spectra of RS3 and RS5, indicating the extra oxygen atom was in tetrazole structure. Considering the weaker retention in comparison with that of CDC, it was reasonable to speculate that RS3 and RS5 were N-hydroxyl derivatives of the drug. Hydroxyl addition could be possible at both N1 and N2 positions, and it was difficult to distinguish between them merely on the basis of mass studies. The calculated cLogP values (Chemdraw software 14.0) of EP impurity E (N1-ethyl CDC) and EP impurity F (N2-ethyl CDC) were 7.86889 and 7.65889, respectively. The calculated cLogP values (Chemdraw software 14.0) of N1-hydroxyl CDC and N2-hydroxyl CDC were 6.399 and 6.031, respectively, which indicated the relative retention time of RS3 and RS5 was similar as that of EP impurities E and F. Moreover, as observed in the stability sample (60 ℃ for ten days), EP impurity F was formed in higher amount than EP impurity E, The rationale for this might be due to the steric hindrances present in EP impurity E [7]. RS3 and RS5 also displayed a  similar kind of the relative extent of formation under oxidative stress. Therefore, RS3 and RS5 were proposed to be N1-hydroxyl CDC and N2-hydroxyl CDC, respectively. The formation of similar N1-hydroxyl product on the oxidative process has been reported in the case of valsartan [11]. The possible mass spectral fragmentation pathways of RS3 and RS5 are shown in Fig. 4.

RS4
RS4 was one of the major degradation products under oxidative degradation condition with reduced retention. Similar to RS3 and RS5, the observed accurate m/z value of RS4 was 627.2562. Elemental composition calculator suggested the same molecular formula for it as RS3 and RS5, indicating an additional of hydroxyl group compared  As m/z 377.1711 was not found in the MS/MS spectrum of CDC, it is reasonable to speculate that the additional hydroxyl group in RS4 made 14-position C-N bond easier to break. The fragment ion at m/z 289.1543 was explained by the cleavage of ester linkage from m/z 377.1711 followed with cyclohexyloxy transfer. According to exact mass data, the fragment of m/z 207.0767 was assigned as protonated 2-ethoxy-1H-benzimidazole-4-carboxylic acid, which was different from equivalent fragment of drug with 207.0923. Based on the above information, RS4 was identified as 14-hydroxyl CDC. Similar oxidation mechanism in benzyl position through a free radical mediated mechanism in the case of morphine has been discussed in the literature [12][13][14]. The possible mass spectral fragmentation pathway of RS4 is shown in Fig. 4.

RS7
RS7 was a process-related impurity with decreased retention compared to the drug in the test solution a (Fig. 2a) (Fig. S5), RS7 was confirmed to be CDC methoxy analog. It is not reported as a related substance in CDC substances or preparations in the literature. According to the synthesis routine of CDC, the detritylation step was carried out in aqueous methanol containing hydrochloric acid. RS7 is possible to be the overreacting product during this step in methanol/HCl system [15]. The mass possible spectral fragmentation pathway of RS7 is shown in Fig. 4.

RS10
RS10 was the major degradation product under photolytic condition.  [8]. The possible mass spectral fragmentation pathway of RS10 is shown in Fig. 4.

Discussion
The schematic representation of the degradation pathway in tested degradation conditions is shown in Fig. 5. The ester linkages make CDC susceptible to alkaline and oxidative degradation conditions, yielding RS1. RS2 was formed by the hydrolysis of the ether bond between the benzimidazole and ethyl moieties under acid, oxidative degradation conditions and stress testing (60 ℃ for 10 days), subsequently underwent keto-enol tautomerism and stabilized as stable keto form. The leaving ethyl moiety was attacked by the N1 and N2 of tetrazole ring of RS2 to yield RS6 and RS8 under oxidative condition, respectively, which are regioisomers. Similarly, the leaving ethyl moiety also reacted with the CDC molecules leading to the formation of another set of regioisomers (RS9 and RS11) under acid degradation Fig. 5 The degradation pathways of CDC condition and stress testing (60 ℃ for 10 days). When the drug was subjected to oxidative condition, tetrazole group on CDC was susceptible to hydroxylation by HO· action to produce RS3 and RS5, which are isomeric to each other. The molecule of CDC contains a benzylic (C14) position which is quiet susceptible to H abstraction by a free radical generated by H 2 O 2 under the same oxidative condition to produce RS4. Dehydrogenation was found in the case of RS10 when the drug was subjected to photolytic degradation conditions. In general, the detritylation reaction in the synthesis step of the CDC was conducted in methanol aqueous containing hydrochloric acid, which may accompany with the formation of RS7 as a major impurity in the final product. RS7 has similar properties to CDC so that it is difficult to purify the final product.

Conclusion
A UHPLC-Q-TOF-MS method was developed and optimized to separate and characterize eleven related substances in CDC tablets. The specificity, sensitivity, accuracy, linearity, precision, stability and robustness of the method were confirmed by methodological validation results. Degradation studies and accelerated stability studies on CDC tablets were carried out under various degradation conditions such as hydrolysis (acidic and alkaline), oxidation, photolysis conditions and three stress testing conditions (60 ℃, 90% relative humidity and 4500 lx for ten days). The elemental compositions and fragmentation pathways were confirmed by UHPLC-Q-TOF-MS studies. One process-related impurity and ten degradation products were identified. Among them, RS3-5, 7 were not reported before. The structure of RS7 was identified as CDC methoxy analog by comparing the retention time with that of the reference substance.
Author Contributions Material preparation, data collection, analysis and original draft preparation: LY, XG and CY. Conceptualization, writing-review and editing: QT and XZ. Project administration: LH and JZ.
Funding The project was supported by Zhejiang Province Public Welfare Technology Application Research Project > (NO. LGC21H300001).
Data availability Data is available from the authors upon request.