Characterization of the stress degradation products of tolvaptan by UPLC-Q-TOF-MS/MS

Abstract

Tolvaptan (TVT) is a selective, competitive vasopressin receptor 2 antagonist used to treat hyponatremia. TVT was subjected to forced degradation under hydrolysis, oxidation, dry heat and photolysis conditions, in accordance with the ICH guideline Q1A (R2). The degradation products (DPs) formed have been characterized through UPLC-PDA and UPLC-Q-TOF-MS/MS studies. The chromatographic separation was achieved on an Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.7 μm) with a mobile phase containing a gradient mixture of solvents A (0.1% formic acid) and B (acetonitrile) at a flow rate of 0.3 ml min⁻¹ at 30 °C. The detection wavelength was set at 266 nm. The drug degraded under acid hydrolysis, base hydrolysis and oxidative conditions to form a total of 7 DPs. When methanol was used as the co-solvent during stress degradation, four additional DPs were formed which were absent when acetonitrile was used as the co-solvent. Comparison of the fragmentation pattern of the DPs with that of the drug helped in the elucidation of the structures of all the degradation products. The degradation pathway of the drug was established, which was duly justified by the mechanistic explanation. The developed UPLC method was validated as per ICH guidelines.

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1. Introduction

Tolvaptan (TVT) is chemically known as (±)-4′-[(7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-toluidide, and is an oral non-peptidic selective vasopressin V₂-receptor antagonist.^1–4 It is indicated for the treatment of clinically relevant hypervolemic or euvolemic hyponatremia associated with heart failure, cirrhosis, or syndrome of inappropriate antidiuresis.^1,2 TVT selectively inhibits arginine vasopressin (AVP) induced water reabsorption in the kidney by competitively blocking the binding of AVP to V₂-receptors in the distal nephron, thereby preventing the antidiuresis caused by circulating AVP.^3–5

The characterization of degradation products is an important task in the drug discovery and development process. The international conference on harmonization (ICH) guidelines emphasizes the identification of the likely degradation products, degradation pathways and intrinsic stability of a molecule. Hence, it is necessary to carry out stability studies of a drug under various forced degradation conditions of hydrolysis, oxidation, thermal and photolysis.

Analytical methods for estimation of TVT include estimation in biological samples^6–10 and in formulation.¹¹ A liquid chromatography-tandem mass spectrometry method for determining tolvaptan and its nine metabolites in rat serum has also been reported, which involves gradient elution using 0.3% acetic acid and acetonitrile with 0.3% acetic acid on a C18 column employing ESI positive ionization mode.¹² Stability methods by UPLC¹³ and HPLC¹⁴ for the impurity profiling of TVT are reported, by which the identification of degradation products have not been attempted. Govind Reddy et al. have reported a RP-HPLC method for the estimation of process impurities in the presence of degradation products with only three known DPs, whereas others were not characterized.¹⁵ In the present work, to better understand the mechanism of degradation of TVT and characterize the degradation products, a chromatographic method involving a diode array detector (DAD) and tandem mass spectrometry (MS/MS) is developed. The degradation of TVT according to the ICH guidelines has been performed and the mechanism for the TVT degradation has been established.

2. Experimental

2.1. Chemicals and reagents

Pure tolvaptan was supplied by MSN Laboratories limited (Hyderabad, India). Commercially available 15 mg tolvaptan tablets (TOLVAT, Sun Pharmaceuticals, Mumbai, India) were purchased from a local pharmacy. HPLC grade acetonitrile (ACN), methanol (MeOH) and formic acid were purchased from Merck (Mumbai, India). Ammonium acetate (HPLC buffer grade) and hydrogen peroxide (30% w/v) were purchased from Finar Chemicals Pvt Ltd. (Ahmedabad). Analytical reagent (AR) grade sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Fine Chemicals Limited, Ahmedabad. High purity water was prepared using a Millipore Milli-Q Plus water purification system (Millipore, Milford, MA, USA).

2.2. Instrumentation

The UPLC system used was an Acquity UPLC H Class system from Waters (Waters, Milford, MA, USA) with a quaternary solvent manager, autosampler, column oven and PDA detector. Data acquisition, analysis, and reporting were performed using Empower3 (Waters) chromatography software.

For LC-MS characterization, an Agilent 1290 series LC instrument (Agilent Technologies, USA) attached to a quadrupole-time of flight (Q-TOF) mass spectrometer (Q-TOF-LC/MS, 6540 series, Agilent Technologies, USA) equipped with an electrospray ionization (ESI) source was used. Data analysis was carried out using Mass Hunter workstation software. The typical operating source conditions were optimized as follows: the fragmentor voltage was set at 140 V, the capillary at 3500 V, and the skimmer at 65 V; nitrogen was used as the drying (325 °C; 10 l min⁻¹) and nebulizing (40 psi) gas. All the spectra were recorded under identical experimental conditions, and are an average of 25 scans. MS scans were carried out in positive ESI mode.

A photo stability chamber (Osworld), consisting of both UV and fluorescent lamps, was used for the photo degradation study. A water bath equipped with a temperature controller was used to carry out degradation studies for all the solutions. A controlled temperature dry air oven (Osworld laboratory oven) was used for the solid-state thermal stress study.

2.3. Chromatographic conditions

The chromatographic separations were carried out on an Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.7 μm) with a mobile phase containing a gradient mixture of solvents A (0.1% formic acid) and B (acetonitrile) at a flow rate of 0.3 ml min⁻¹. The gradient programme was set as follows (time in min/% proportion of solvent B): 0–0.5/25, 0.50–2.50/45, 2.50–3.50/55, 3.50–5.50/60, 5.50–6.50/70, 6.50–7.00/25, 7.00–9.00/25. The eluted compounds were monitored at 266 nm. The column oven and auto sampler temperatures were maintained at 30 °C and 5 °C, respectively. An injection volume of 1 μl was used.

2.4. Preparation of stock and standard solutions

A standard stock solution of TVT (500 μg ml⁻¹) was prepared in HPLC grade ACN. Aliquots of the stock solution were transferred and diluted with ACN : water (50 : 50 v/v) to yield concentrations equal to 25, 50, 75, 100, 125 and 150 μg ml⁻¹. For the assay of the tablets, 20 tablets were crushed. After mixing, an amount equivalent to one tablet was weighed and taken in a 100 ml volumetric flask, dissolved in diluent (ACN : water (50 : 50% v/v)), sonicated for 15 min, diluted up to the mark with the same solvent and filtered through a 0.45 mm Whatman filter paper. From this solution, further dilutions were made with diluent to obtain a solution containing 100 μg ml⁻¹. This solution was analyzed for the assay.

2.5. Forced degradation studies

2.5.1. Hydrolysis. For acid hydrolysis, 5 mg drug was dissolved in 1 ml of ACN, then 4 ml of 1 N HCl was added and the solution was maintained at reflux at 80 °C for 24 h. The studies in alkaline conditions were carried out by dissolving 5 mg of drug in 1 ml of ACN, followed by the addition of 4 ml of 2 N NaOH, and then the solution was refluxed at 80 °C for 17 h. For neutral hydrolysis, the drug was initially dissolved in ACN, then diluted with water and the solution was refluxed at 80 °C for 48 h.

After completion of the degradation treatments, the samples were allowed to cool to room temperature, neutralized as needed and injected into the chromatographic system after dilution with the diluent (ACN : water – 50 : 50, v/v) to 250 μg ml⁻¹.

2.5.2. Oxidative degradation. TVT was dissolved in 1 ml of ACN and treated with a solution of 10% (v/v) H₂O₂ at room temperature in the dark for 24 h.

2.5.3. Photolytic degradation. For the solid sample, approximately 100 mg of TVT was spread on a petri-dish in a layer of 1 mm thickness. For the solution, the drug was initially dissolved in ACN, and then diluted with water. The samples were kept in a photo-stability chamber and exposed to 1.2 million lux h of fluorescent light and 200 Wh m⁻² of UV illumination as per ICH conditions⁸ for 3 days. A parallel set of the drug samples were stored in the dark at the same temperature to serve as a control.

2.5.4. Thermal degradation. The dry heat degradation was carried out by exposing the drug to a temperature of 80 °C for 7 days in a hot air oven.

2.6. Effect of co-solvent on degradation

Initially, ACN was used as the co-solvent for all stress degradation studies. As MeOH is a cheaper solvent, it was also tried as a co-solvent for sample preparation for stress degradation studies.

3. Results and discussion

3.1. Method development

In order to achieve the optimum resolution, numerous changes to the chromatographic conditions, such as the pH and composition of the mobile phase, flow rate, column, etc., were carried out. Different mobile phase compositions using buffers at pH 3.0, pH 4.0, and pH 5.5, and 0.1% formic acid with ACN and MeOH were tried. Isocratic methods were not successful in achieving a favorable resolution. Well resolved degradation product peaks, along with a sharp and symmetrical drug peak, were obtained using 0.1% formic acid in water and ACN, using the proposed gradient method with a UPLC HSS T3 column. For LC/MS analysis, electrospray ionization (ESI) and atmospheric chemical ionization were tried in both positive and negative modes. It was observed that the maximum intensity was obtained with ESI in positive mode. Mass spectrometer conditions were optimized to obtain maximum ionization of TVT and all the DPs. Experiments were conducted using different capillary voltages (3000 V, 3500 V and 4000 V) and different fragmentor voltages (100 V, 125 V, 140 V, 170 V) at a constant source temperature of 325 °C. The maximum intensity of the analytes was observed when using a capillary voltage and a fragmentor voltage of 3500 V and 140 V, respectively.

3.2. Degradation behavior of TVT

The optimized LC-MS method was used to identify the degradation products of TVT. The overlay of the chromatograms of the stress degradation samples are given in Fig. 1. A total of eleven DPs were identified and characterized using LC/ESI/MS/MS experiments and accurate mass measurements. The proposed structures of the DPs and their elemental compositions are given in Scheme 1 and Table 1.

Fig. 1 Overlay of chromatograms showing effect of co-solvent: [A] acid hydrolysis, [B] base hydrolysis, [C] oxidative degradation.

Scheme 1 Proposed structures of protonated degradation products of tolvaptan.

Table 1 MS/TOF data of tolvaptan and degradation products along with their elemental composition and major fragments

	Retention time (min)	Molecular formula	Observed m/z	Calculated m/z	Error	MS/MS fragment ions
TVT	6.55	C26H27ClN2O3^+	449.1620	449.1626	1.34	431, 252, 206, 180, 119, 91, 65
DP-1	3.18	C11H14ClNO^+	212.0833	212.0837	1.89	180, 164, 152, 144, 130, 117, 103
DP-2	4.08	C18H20ClN2O2^+	331.1206	331.1208	0.60	313, 206, 180, 134
DP-3	4.40	C19H22ClN2O2^+	345.1365	345.1364	−0.29	238, 206, 180, 134, 106
DP-4	4.98	C16H16NO3^+	270.1121	270.1125	1.48	226, 178, 119, 91, 65
DP-5	5.48	C26H24ClN2O2^+	431.1535	431.1521	−3.25	339, 252, 206, 178, 119
DP-6	5.53	C27H29N2O4^+	445.2140	445.2122	−4.04	427, 252, 202, 176, 119
DP-7	6.22	C25H24ClN2O3^+	435.1485	435.1470	−3.45	417, 238, 180, 105
DP-8	6.24	C18H17ClN2O^+	313.1096	313.1102	1.92	206, 178, 134, 106
DP-9	6.93	C17H18NO3^+	284.1273	284.1281	2.82	252, 192, 164, 136, 119, 105
DP-10	7.41	C26H24ClN2O2^+	447.1481	447.1470	−2.46	252, 222, 119, 91, 65
DP-11	7.45	C10H11ClN^+	180.0585	180.0575	−5.55	164, 152, 144, 130, 117, 103

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3.2.1. Hydrolysis. The drug degraded significantly to form DP-1, DP-2, DP-3, DP-4, DP-8, DP-9 and DP-11 under acid hydrolysis. Under alkaline hydrolysis conditions, TVT degraded to yield DP-1, DP-2, DP-4 and DP-5, whereas it was stable even after refluxing under neutral hydrolytic conditions for 48 h.

3.2.2. Oxidative degradation. The drug was degraded to form minor degradation products DP-6, DP-7, DP-10.

3.2.3. Photolytic degradation. No degradation was observed under photo-degradation conditions.

3.2.4. Thermal degradation. The exposure of the solid drug to a temperature of 80 °C for 7 days did not result in significant decomposition, which indicates that TVT is stable to dry heat.

3.3. Effect of co-solvent on degradation

As shown in the Fig. 1, DP-1, DP-3 & DP-9 under acid hydrolysis, DP-1 under base hydrolysis and DP-6 under oxidative degradation were formed when MeOH was used as the co-solvent. However, they were not formed when exposed to the same stress conditions using ACN as the co-solvent. This suggests that DP-1, DP-3, DP-6 and DP-9 were formed due to the presence of methanol, which is clear from the chemical structures of the aforementioned degradation products.

3.4. UPLC-MS/MS of TVT and its degradation products

3.4.1. MS/MS of TVT. To elucidate the degradation behavior of TVT (retention time (R_t) = 6.55 min), the ESI-MS/MS spectrum (Fig. 2a) of its [M + H]⁺ ion (m/z 449) was examined. The spectrum shows abundant product ions at m/z 431 (loss of H₂O), m/z 252 (loss of C₁₀H₁₂ClNO), m/z 206 (loss of C₁₅H₁₅NO from m/z 431), m/z 180 (loss of CO from m/z 206), m/z 119 ((2-methylbenzylidene)oxonium), and m/z 91 (loss of CO from m/z 119) (Scheme 2). It can be noted that the product ions at m/z 206 and 180 are characteristic of the 7-chloro-5-hydroxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine-1-carbonyl skeleton of TVT, while the fragment ions with m/z 252 and 119 are diagnostic of the 3-methylphenyl-2-methylbenzamide skeleton of TVT. The elemental compositions of all these ions have been confirmed by accurate mass measurements.

Fig. 2 ESI/MS/MS spectra of (a) TVT (m/z 449) at 10 eV, (b) DP-1 (m/z 212) at 10 eV, and (c) DP-2 (m/z 331) at 10 eV.

Scheme 2 Proposed fragmentation pathway of protonated tolvaptan.

3.4.2. MS/MS of degradation products. On-line LC-ESI-MS/MS experiments were performed to characterize all the degradation products (DP-1 to DP-11) formed under the various stress conditions. The most probable structures have been proposed for all the degradation products based on the m/z values of their [M + H]⁺ ions and the MS/MS data in combination with the elemental compositions derived from accurate mass measurements, as discussed below.
DP-1. The ESI-MS/MS spectrum of the [M + H]⁺ ion (m/z 212) of DP-1 (R_t = 3.18 min) is given in Fig. 2b. The MS/MS spectrum shows structure indicative fragment ions as shown in Scheme 3(a). The peak at m/z 180 is diagnostic for 7-chloro-2,3-dihydro-1H-benzo[b]azepine, which further fragments to give m/z 144 (loss of HCl). As shown in Scheme 4(a), the mass difference of 32 between the [M + H]⁺ ion of DP-1 (m/z 212) and DP-11 (m/z 180) indicates the addition of methanol to DP-11. Based on all these data, DP-1 was identified as 7-chloro-4-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine.

Scheme 3 Proposed fragmentation pathway of protonated tolvaptan degradation products: (a) DP-1 and DP-11; (b) DP-2, DP-3, DP-7 and DP-8; (c) DP-5, DP-6 and DP-10; (d) DP-4 and DP-9.

Scheme 4 Probable mechanisms of formation: (a) DP-1–DP-5, DP-8, DP-9 and DP-11;(b) DP-6, DP-7 and DP-10.

DP-2. The ESI-MS/MS spectrum of the [M + H]⁺ ion (m/z 331) of DP-2 (R_t = 4.08 min) (Fig. 2c) with an elemental composition of C₁₈H₂₀ClN₂O₂ shows product ions which are given in Scheme 3(b). The characteristic product ions of DP-2 include m/z 313 (loss of water), m/z 206 (loss of C₇H₉N from m/z 313), and m/z 134 (loss of C₁₀H₁₀ClN). Based on all these data, the 4-amino-(2-methylphenyl)(7-chloro-5-hydroxy-2,3,4,5-tetrahydro-1H-benzo[b]azepin-1-yl)methanone structure can be proposed for DP-2.
DP-3. The ESI-MS/MS spectrum of the [M + H]⁺ ion (m/z 345) of DP-3 (R_t = 4.40 min) is shown in Fig. 3a. The spectrum displays product ions at m/z 238 (loss of C₇H₉N), m/z 206 (loss of CH₃OH from m/z 238, ((7-chloro-2,3-dihydro-1H-benzo[b]azepin-1-yl)-methylidyne)-oxonium), which are compatible with the structure (4-amino-2-methylphenyl)(7-chloro-4-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepin-1-yl)methanone (Scheme 3(b)).The mechanism of formation of DP-3 is shown in Scheme 4(a). It can be noted that the mechanism may involve an addition of methanol to DP-8.

Fig. 3 ESI/MS/MS spectra of (a) DP-3 (m/z 345) at 15 eV, (b) DP-4 (m/z 270) at 15 eV and (c) DP-5 (m/z 431) at 20 eV.

DP-4. The ESI-MS/MS spectrum (Fig. 3b) of the [M + H]⁺ ion of DP-4 (R_t = 4.98 min.) with an elemental composition C₁₆H₁₆NO₃⁺ displays product ions which are compatible with the structure 2-methyl-4-(2-methylbenzamido)benzoic acid. The characteristic fragments for DP-4 include m/z 226 (loss of CO₂) and m/z 178 (loss of C₇H₈). A probable mechanism for the formation of DP-4 under hydrolytic conditions may involve hydrolytic cleavage of the carbonyl attached to the benzazepine moiety of TVT (Scheme 4(a)).
DP-5. The mass difference between the drug (m/z 449) and DP-5 (m/z 431, Fig. 3c) is 18 Da which suggests that it is formed by the loss of water molecule from the drug during base hydrolysis. The elemental compositions of the [M + H]⁺ of DP-5 and its product ions (Scheme 3(c)) have been confirmed by accurate mass measurements (Table 2). All these data are highly compatible with the proposed structure N-(4-(7-chloro-2,3-dihydro-1H-benzo[b]azepine-1-carbonyl)-3-methylphenyl)-2-methylbenzamide. As shown in the Scheme 4(a), DP-5 may be formed by base catalyzed elimination of water from TVT.

Table 2 High resolution mass spectrometry (HRMS) data of product ions of protonated tolvaptan and its degradation products

	Molecular formula	Observed m/z	Calculated m/z	Error
TVT	C26H27ClN2O3^+	449.1611	449.1626	3.34
	C26H25ClN2O2^+	431.1512	431.1521	2.09
	C16H14NO2^+	252.1006	252.1004	−0.79
	C11H9ClNO^+	206.0356	206.0367	5.34
	C10H11ClN^+	180.0567	180.0575	4.44
	C8H7O^+	119.0494	119.0491	−2.52
	C7H7^+	91.0540	91.0542	2.20
DP-1	C11H14ClNO^+	212.0833	212.0837	1.89
	C10H11ClN^+	180.0574	180.0575	0.56
	C9H7ClN^+	164.0266	164.0262	−2.44
	C8H7ClN^+	152.0249	152.0262	8.55
	C10H10N^+	144.0795	144.0808	9.02
	C9H8N^+	130.0649	130.0651	1.54
	C8H7N^+	117.0563	117.0573	8.54
DP-2	C18H20ClN2O2^+	331.1206	331.1208	0.60
	C18H18ClN2O^+	313.1090	313.1102	3.83
	C10H11ClN^+	180.0572	180.0575	1.67
	C8H8NO^+	134.0594	134.0600	4.48
	C7H8N^+	106.0653	106.0651	−1.89
DP-3	C19H22ClN2O2^+	345.1365	345.1364	−0.29
	C12H13ClNO2^+	238.0608	238.0629	8.82
DP-4	C16H16NO3^+	270.1121	270.1125	1.48
	C15H16NO^+	226.1217	226.1226	3.98
	C9H8NO3^+	178.0490	178.0499	5.05
DP-5	C26H24ClN2O2^+	431.1535	431.1521	−3.25
DP-6	C27H29N2O4^+	445.2140	445.2122	−4.04
	C27H27N2O3^+	427.2018	427.2016	−0.47
	C12H12NO2^+	202.0860	202.0863	1.48
	C11H14NO^+	176.1080	176.1070	−5.68
DP-7	C25H24ClN2O3^+	435.1485	435.1470	−3.45
	C25H22ClN2O2^+	417.1360	417.1364	0.96
	C15H12NO2^+	238.0887	238.0863	−10.08
	C7H5O^+	105.0358	105.0335	−21.90
DP-8	C18H17ClN2O^+	313.1096	313.1102	1.92
	C10H9ClN^+	178.0416	178.0418	1.12
DP-9	C17H18NO3^+	284.1273	284.1281	2.82
	C10H10NO3^+	192.0643	192.0655	6.25
	C8H10NO^+	136.0759	136.0757	−1.47
DP-10	C26H24ClN2O3^+	447.1481	447.1470	−2.46
	C11H9ClNO2^+	222.0311	222.0316	2.25
DP-11	C10H11ClN^+	180.0585	180.0575	−5.55
	C9H7ClN^+	164.0269	164.0262	−4.27
	C10H10N^+	144.0818	144.0808	−6.94
	C9H8N^+	130.0662	130.0651	−8.46

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DP-6. Fig. 4a shows the ESI-MS/MS spectrum of the [M + H]⁺ ion (m/z 445) of DP-6 (R_t = 5.52 min) with an elemental composition of C₂₇H₂₉N₂O₄. The absence of the chlorine isotope pattern in the spectrum confirmed that the lone chlorine atom was absent in DP-6. As explained in Scheme 3(c), the spectrum shows characteristic product ions with m/z 427 (loss of H₂O), m/z 202 (loss of C₁₅H₁₅NO from m/z 427), and m/z 176 (loss of CO from m/z 202) consistent with the structure N-(4-(5-hydroxy-7-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine-1-carbonyl)-3-methylphenyl)-2-methylbenzamide. The probable mechanism of formation of DP-6 under oxidative degradation is shown in Scheme 4(b).

Fig. 4 ESI/MS/MS spectra of (a) DP-6 (m/z 445) at 10 eV, (b) DP-7 (m/z 435) at 15 eV and (c) DP-8 (m/z 313) at 10 eV.

DP-7. The ESI-MS/MS spectrum of the [M + H]⁺ ion (m/z 435) of DP-7 is given in Fig. 4b. The spectrum shows structure indicative product ions which are explained in Scheme 3(b) and listed in Table 2. For example, m/z 417 (loss of H₂O) and m/z 105 (benzylidyne oxonium ion) indicated loss of methylene from TVT (Scheme 4(b)) to form DP-7.
DP-8. The degradant DP-8 at m/z 313 ([M + H]⁺) was eluted at 6.24 min. Its MS/MS spectrum (Fig. 4c) displays product ions at m/z 206 (((7-chloro-2,3-dihydro-1H-benzo[b]azepin-1-yl)-methylidyne)-oxonium), m/z 180 (7-chloro-2,3-dihydro-1H-benzo[b]azepine), m/z 134 ((4-amino-2-methylbenzylidene)oxonium) and m/z 107 (loss of CO from m/z 134) (Scheme 3(b)). The absence of the product ion at m/z 119 and presence of m/z 134 suggested that the probable structure of DP-8 would be (4-amino-2-methylphenyl)(7-chloro-2,3-dihydro-1H-benzo[b]azepin-1-yl)methanone.
DP-9. The ESI-MS/MS spectrum of the [M + H]⁺ ion (m/z 284) of DP-9 (R_t = 6.93 min) with elemental composition of C₁₇H₁₈NO₃ is given in Fig. 5a. The characteristic fragments of DP-9 (Scheme 3(d)) include m/z 192 (loss of C₇H₈), m/z 252 (loss of OCH₃) and m/z 164 (loss of CO from m/z 192). These data indicated the structure to be methyl-2-methyl-4-(2-methylbenzamido)benzoate. DP-9 may be formed by an esterification of DP-3 due to the presence of methanol under acid hydrolysis conditions (Scheme 4(a)).

Fig. 5 ESI/MS/MS spectra of (a) DP-9 (m/z 284) at 15 eV, (b) DP-10 (m/z 447) at 15 eV and (c) DP-11 (m/z 180) at 15 eV.

DP-10. DP-10 was formed under oxidative degradation conditions through the oxidation of alcohol to ketone. The ESI-MS/MS spectrum (Fig. 5b) of the [M + H]⁺ ion of DP-10 (m/z 447, C₂₆H₂₄NClN₂O₃, R_t = 7.40 min) displays a product ion at m/z 222 (((7-chloro-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-1-yl)-methylidyne)-oxonium ion) (Scheme 3(c)). The presence of fragments of m/z 252 and m/z 119 in parallel to those of the drug indicated that oxidation occurred at the N-4-(7-chloro-5-hydroxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine) part of the drug. The mechanism of formation of DP-10 is depicted in Scheme 4(b). Accordingly, the structure of DP-10 may be N-(4-(7-chloro-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-1-carbonyl)-3-methylphenyl)-2-methylbenzamide.
DP-11. The ESI-MS/MS spectrum (Fig. 5c) of the [M + H]⁺ ion of DP-11 (m/z 180, R_t = 7.45 min) displays product ions that are compatible with the structure 7-chloro-2,3-dihydro-1H-benzo[b]azepine (Scheme 3(a)). The elemental compositions of DP-11 and its product ions have been confirmed by accurate mass measurements (Table 2). A probable mechanism for the formation of DP-11 under acid hydrolysis conditions may involve hydrolytic cleavage of the amide functionality in DP-8 (Scheme 4(a)).

3.5. Method validation

The stability indicating assay method was validated for specificity, linearity, precision (inter-day, intra-day and intermediate precision) and accuracy according to ICH guideline Q2 (R1). The specificity of the method was established by determining the peak purity for TVT and the DPs in a mixture of stressed samples using a photodiode array (PDA) detector and evaluation of the resolution factor. Peak purity was also demonstrated by subjecting all the degradation samples to LC-MS. The PDA and mass detector showed an excellent purity across all peaks, which unambiguously proves the specificity of the method. To establish linearity and range, a stock solution containing 1 mg ml⁻¹ TVT in the mobile phase was diluted to yield solutions in the concentration range of 25–200 μg ml⁻¹. The solutions were prepared and analyzed in triplicate. The response for the drug was linear in the investigated concentration range (r² = 0.9992). The linearity data are given in Table S1 (see ESI†). Table S2 (see ESI†) shows accuracy data at three different concentrations in triplicate analysis. The recoveries of the added drug were obtained from the difference between the peak areas of the fortified and unfortified degraded samples. The recovery of TVT in the presence of degradation products ranged from 99.89 to 100.07%. The intra- and inter-day precisions were determined at three different concentrations, 50, 100 and 150 μg ml⁻¹, on the same day (n = 3) and consecutive days (n = 3), respectively. Table S2† shows that the %RSD values for intra and inter-day precision were <0.41% and 0.56% respectively, indicating that the method was sufficiently precise. The robustness of the proposed method was determined by purposely changing the flow rate (0.25–0.35 ml min⁻¹), column temperature (30 ± 5 °C) and the % of formic acid in the mobile phase (0.1 ± 0.02%) at three different concentrations (50, 100, 150 μg ml⁻¹). Each sample was injected in triplicate (n = 3), and the peak areas obtained were used to calculate means and %RSD values. The %RSD was <1%. No significant changes in assay value were observed by changing these chromatographic conditions, which confirms the robustness of the method. The proposed UPLC method was applied to the assay of tolvaptan in TOLVAT tablets and results indicated that the amount of tolvaptan was 99.08 ± 0.84 (mean ± S.D.) in the tablets, which corresponds to the requirement of 90–110% according to the label claim.

4. Conclusion

The degradation behavior of tolvaptan under various stress conditions was studied. The drug was found to degrade extensively to form 4 degradation products under acid and base hydrolysis and showed only 2 minor degradation products under oxidative degradation. When methanol was used as a co-solvent in stress studies, 4 degradation products were formed that were absent when using acetonitrile as a co-solvent. This suggests that acetonitrile is a preferred co-solvent for stress degradation studies. All the degradation products were characterized using ESI/MS/MS in combination with accurate mass measurements of the product ions and precursors. The degradation pathway of tolvaptan was established.

5. Conflicts of interest

The authors have no conflicts of interest in this paper.

Acknowledgements

The authors are thankful to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India, for providing the funds for research at NIPER, Hyderabad.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16644b

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