HPLC – Quality by Design Approach for Simultaneous Detection of Torsemide, Spironolactone and Their Degradant Impurities

Unnati Bihola; Prajesh Prajapati; Y. K. Agrawal

doi:10.1515/pthp-2018-0011

Article Publicly Available

HPLC – Quality by Design Approach for Simultaneous Detection of Torsemide, Spironolactone and Their Degradant Impurities

Unnati Bihola , Prajesh Prajapati and Y. K. Agrawal

Published/Copyright: May 15, 2018

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From the journal Pharmaceutical Technology in Hospital Pharmacy Volume 3 Issue 3

Abstract

A simple, highly robust (quality by design (QbD) approach), precise and accurate method using high performance liquid chromatography coupled to mass spectrometry has been established for the simultaneous separation, identification and quantitation of a Torsemide (TOR), spironolactone (SPI) and their degradant impurities. The chromatographic separations of drugs and impurities were achieved on a inertsil ODS-3 µm C18, 150 mm × 4.6 mm, while the isocratic elution using a ternary mobile phase mixture of methanol, acetonitrile and water (5:3:2 v/v/v) at a flow rate of 0.2 mL/min was adopted for achieving optimum separations. The quantitation of torsemide and spironolactone was accomplished by UV detection at 254 nm and identification of the degradants were done by comparing identical mass in mass spectrometer. The recoveries of the torsemide and spironolactone were obtained higher than 98 % with good validation parameters; linearity (r2>0.994), LOD and LOQ was 10 and 33 ng for TOR and 75 and 248 ng for SPI respectively. The quality by design (QbD) approach has been successfully utilized to prove the method is robust even deliberate changes in critical parameters.

Keywords: Quality by Design; HPLC; Torsemide; Spironolactone; Degradant Impurity

Introduction

Torsemide (TOR) and Spironolactone (SPI) (Figure 1) are most widely used drug for treatment of diurectics [1]. A safe, reliable and accurate method has to be incorporated to find out the concentration of these drugs simultaneously as they are available in combined as well as single dosage.

Figure 1:

Molecular structure of (a) Torsemide (TOR) and (b) Spironolactone (SPI).

Extended review reveals that various analytical methods based on spectrophotometric [2, 3, 4, 5, 6, 7, 8], HPLC [9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20], HPTLC [21], LC-ESI-MS [22] have been developed for determination of TOR in pharmaceutical dosage forms and biological fluids or in combination with other drugs [5, 6, 7, 8, 18, 19, 20]. Extended review reveals that various analytical methods based on spectrophotometric [23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36], HPLC [22, 23, 24, 37, 38, 39, 41, 42], HPTLC [9, 43, 44, 44, 45, 46], HPLC-APCI-MS [27], UPLC [47], LC [48] have been developed for determination of SPI in pharmaceutical dosage forms and biological fluids or in combination with other drugs [28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] and also Reported Method for TOR and SPI Combination [49, 50, 51, 52, 53, 54, 55]. The design space covering the influences of various factors was still to be generated for HPLC method. So it was thought proper to use this combination for the present study for development of QbD (Quality by Design) based HPLC method. Several methods viz. HPLC and MS are reported for the stability of the TOR and SPI and acid degradation products [56, 57]. Although, there were lots of method available, we have developed simple, accurate and precise method using LC/MS for identification and detection of degradants of the TOR and SPI. The main advantage of this paper is to analyse simultaneously API and its degradants.

Materials and methods

Instruments

HPLC chromatograph

A GL Science (USA) HPLC chromatograph was used for the separations in this study. It was equipped with binary pump, which were capable to adjust the flow rate (0.01 to 5 mL/min.). It is also equipped with an Autosampler with a capacity of accurately injecting the sample volume of 50 nL to 100 μL into the analytical column. The analytes were detected by using fibre optics based UV detector. The effluent coming from the HPLC was injected in the MS/MS for detection of any impurity if present.

Mass spectrometer

An AB Sciex (Canada) QTRAP-4500 series mass spectrometer was used in the present investigation. It was equipped with linear trap technology and contain TurboV™ source which provides high level of sensitivity with low volume flow rates. The TurboV chamber has two turbo heaters which will improved gas dynamics and helps in ionization. The analyst software was used to perform data acquisition and integration.

LC/MS conditions

Silica column (Inertsil ODS-3 µm C18, 150mm×4.6 mm) protected by precolumn filter cartridges, was used to analyze the samples. After optimization, mobile phase consisting of Acetonitrile: Methanol: Water (5:3:2 v/v/v) was used at flow rate of 0.2 mL/min. The optimized value for MS/MS analyses were as follows: ESI positive ion mode; capillary voltage, 5500 eV; cone voltage, 40 V; Gas 1 (nebulizing gas) and Gas 2 (drying gas) were set to 40 units and 20 units respectively and the source temperature was set at 650°. Mechanically dried air was used as Nebulizing and drying gas. The injection volume and column temperature were set at 10 µL and 40 °C, respectively. Full scan LC–MS spectra were obtained by scanning from m/z 50 to 500. The LC was connected to the mass spectrometer and effluent was injected for detection of impurities if present.

Materials

TOR and SPI standard were obtained as gift specimen from Macleods Pharma Pvt. Ltd (Mumbai, India) and tablet containing TOR and SPI were procured from local market. Acetonitrile (HPLC Grade), methanol (HPLC Grade), water (distilled) – and whatmann filter paper no. 42 (0.45 µm) were used.

Method

Selection of analytical wavelength

The spectra taken at λ_max 254 nm of TOR and SPI in the mobile phase was found to be linear and degraded products were well separated. So 254 nm of TOR and SPI were chosen as detection wavelength in HPLC.

Preparation of mobile phase

The mixture of 30 mL methanol, 50 mL acetonitrile, 20 mL water was filtered through 0.45 µm filter paper and the blend was sonicated for 10 min to degas the mixture and used as mobile phase.

Forced degradation studies

The stress degradation studies were carried out as reported [58, 59]. Standard drug samples have been dissolved in 0.5N HCl and 0.5N NaOH in such a manner that it has given a drug concentration of 1 mg/mL, to study the effect of acid and alkali medium. Similarly, drug concentration of 1 mg/mL in 3 % H₂O₂ solution was used to study effect of peroxide on drug. All the prepared solutions were stored at room temperature for 24 hr. Set of samples also stored under dark and faced similar stress condition as stored under normal condition.

Method validation

Preparation of standard stock solutions and dilution scheme

25 mg of TOR was accurately weighed and transferred into 25 mL volumetric flask and made up with mobile phase. From this take 5 mL above solution and added 25 mg of SPI was accurately weighed than transferred into 25 mL volumetric flask and made up with mobile phase (200 μg/mL TOR and 1000 μg/mL SPI). From this take 5 mL of above solution and transferred into 25 mL of volumetric flask and made up with mobile phase (40 μg/mL TOR and 200 μg/mL SPI) For that pipette out 0.5, 1,1.5, 2,2.5,3 mL of solution and transferred in a same series of 10 mL volumetric flasks and diluted up to mark with mobile phase (2–12 μg/mL TOR and 10–60 μg/mL SPI)

Procedure for estimation of TOR and SPI in tablet

25 mg of Dytor plus tablet was taken into 25 mL volumetric flask and dilute up to the mark with buffer/ACN to get a concentration of 100 µg/mL TOR and SPI. From this 0.6 mL was taken and diluted to 10 mL to get a concentration of 6 μg/mL and 30 μg/mL of TOR and SPI respectively.

Validation of the proposed method

International Conference on Harmonization (ICH) guidelines Q2R1 [60] were used to validate the proposed method.

Linearity

Linearity was observed in a concentration range of 2–12 μg/mL for TOR & 10–60 μg/mL for SPI. The linearity graph of peak area was plotted against concentrations.

Precision (repeatability)

The precision of the method was checked by repeating six solutions TOR (4 μg/mL) for SPI (20 μg/mL) and measured the peak area without changing the parameter of the proposed method. The precision of method was reported in terms of mean, standard deviation and relative standard deviation (% RSD).

Intermediate precision

The intra-day and inter-day precision of the proposed method was done by analyzing the corresponding responses three times on the same day and on three different days for three different concentrations over the calibration range of TOR (2,4 and 8 μg/mL) & SPI (10, 20 and 30 μg/mL). The results were reported in terms of relative standard deviation (%RSD).

Accuracy (recovery study)

The known quantity of standard TOR & SPI were added to the sample and the recovery of the standard from the same were calculated as % recovery. Known amounts of standard solutions of TOR & SPI were added at 80, 100 and 120 % level to pre-quantified sample solutions of TOR & SPI (4 μg/mL and 20 μg/mL).

Limit of detection and limit of quantification

LOD and LOQ were calculated by using following equations.

LOD=3.3×σ/S

LOQ=10×σ/S

Where, σ=the standard deviation of the response

S=slope of the calibration curve

Robustness

Robustness has to studied by analyzing the samples of TOR & SPI by deliberate variation in the method parameters. For that QbD approach was applied to determine the robustness (discuss in following section).

Development and validation of torsemide and spironolactone by HPLC method using QbD approach [61]

Preparation of stock solution of TOR and SPI

25 mg of TOR and SPI was accurately weighed and transferred into different 25 mL volumetric flasks and volume was made up with mobile phase (1000 µg/mL). From this 1 mL of resulting solutions diluted to 10 mL in volumetric flasks using mobile phase (100 µg/mL).

Factorial design

Two-level design with 3 factor i. e., pH, % Composition and Flow rate at 2 different levels was studied as shown in Table 1 (1 and −1)

Table 1:

2 level 3 factorial design.

Level	Factor
	pH	Composition of mobile phase (%v/v)	Flow rate (mL/min)
−1	4	5:3:2 (ACN:MeOH:Water)	0.8
1	5	4:3:3 (ACN:MeOH:Water)	1.0

Experimental runs

As per the design, we have determined the best possibility for experiments that required 8 runs as mentioned in Table 2:

Table 2:

Experimental runs.

Sr No.	pH	Composition of mobile phase (%v/v)	Flow rate (mL/min)
1	4	5:3:2	0.8
2	4	5:3:2	1.0
3	4	4:3:3	0.8
4	4	4:3:3	1.0
5	5	5:3:2	0.8
6	5	5:3:2	1.0
7	5	4:3:3	0.8
8	5	4:3:3	1.0

Characterization of degradation product(s)

LC-MS studies were carried out to determine m/z values of the major degradation products formed under various stress test conditions. The obtained values were compared with the mass spectrum of known degradation products of TOR and SPI.

Results and discussion

The goal of this work was to provide an accurate selective simultaneous estimation method for identification and determination of the TOR and SPI and related substance by LC-MS Acetonitrile: Methanol: Water (5:3:2 v/v/v) as a mobile phase in the LC/MS work.

Selection of mobile phase

For the selection of mobile phase, we have varied the concentration of mobile phase Methanol and Acetonitrile with the addition of water ranging from 10 % to 90 % at a flow rates ranging from 0.1–1.0 mL/min chromatograms were recorded.

Amongst the all result obtained, the optimized system containing Acetonitrile: Methanol: Water (5:3:2 v/v/v) at 0.2 mL/min, was found to be satisfactory and gave well separate peak for TOR and SPI (Figure 2).

Figure 2:

Chromatogram of standard TOR and SPI.

Degradation studies

Under the different condition of stress degradation, three degradant of TOR and one degradant of SPI were separated and identified (Figure 3). The retention time and relative retention time (RRT) of the drug and degraded products are shown in Table 3.

Figure 3:

Chromatogram for (a) Acid degradation (b) Alkali degradation (c) Oxidative degradation and (d) Water degradation of TOR and SPI.

Table 3:

Result of Stress degradation study for TOR & SPI.

Drug	Condition	Time (Hour)	% Degradation	Degradants peaks	RRT
LOR	0.5 N HCL	24	32.9	3 Peak (T1, T2, T3)	2.25, 3.1, 3.2
	0.5 N NaOH	24	27.5	2 Peak (T1, T2)	1.35, 2.25
	Oxidative degradation	24	20.7	1 Peak (T1)	1.35
	Water degradation	24	1.8	No Peak	–
SPI	0.5 N HCL	24	23.7	1 Peak (S1)	1.55
	0.5 N NaOH	24	21.7	No Peak	–
	Oxidative degradation	24	21.0	No Peak	–
	Water degradation	24	1.6	No Peak	–

Stress degradation by hydrolysis under acidic condition and basic conditions

The TOR degraded with given acidic condition (0.5 N HCl, 24 hrs) with time and gave three degradation peak at RRT (w.r.t. TOR) 2.25, 3.1 and 3.2 respectively. While SPI under the same acidic condition gave one degradation peak at RRT (w.r.t. SPI) 1.55.

Similarly, under the imparted alkali condition (0.5 N NaOH, 24 hrs.), TOR degraded with time and gave two peaks at RRT 1.35 and 2.25. There was degradation of SPI (21.7 %) found in the alkali condition but no degradation peak observed in chromatogram. This may be due to the non UV absorbent fragment was removed from the structure.

The results show that both TOR and SPI are sensitive to the acidic as well as basic conditions.

Oxidative degradation and hydrolysis

Exposing the TOR and SPI to the oxidative conditions (3 % H₂O₂, 24 hrs.), It was observed that both the drug degrade in oxidative condition and one major degradant of TOR was observed at RRT 1.35 (w.r.t. TOR).

Similarly, when TOR and SPI bring out in contact with water (24 hrs.), they remain stable and not degraded. Hence, results reveal that both drugs are sensitive to oxidation while quite stable under hydrolysis condition.

Validation of the developed method

Linearity

The linearity study was carried out for both drugs at six different concentration levels. For the linearity study of TOR and SPI, concentration range of 2–12 μg/mL & 10–60 μg/mL was selected.

The data of the peak areas obtained with the respective concentrations in μg/mL are shown in Table 4 for TOR & SPI. The calibration curves for TOR & SPI are shown in Figure 4.

Figure 4:

Calibration curve of (a) LOR (b) SPI in proposed mobile phase.

Table 4:

Linearity data of TOR.

Sr No.	Conc. of TOR (µg/mL)	Conc. of SPI (µg/mL)	Area TOR (Mean ± SD)	CV	Area SPI (Mean ± SD*)	CV
1	2	10	12,539 ± 103	0.82	86,875 ± 154	0.18
2	4	20	24,884 ± 196	0.79	158,292 ± 832	0.53
3	6	30	37,106 ± 145	0.39	269,568 ± 906	0.34
4	8	40	50,131 ± 455	0.91	345,164 ± 1252	0.36
5	10	50	64,572 ± 574	0.89	439,528 ± 1367	0.31
6	12	60	80,264 ± 614	0.76	532,539 ± 2264	0.43

Accuracy (recovery)

Accuracy of method was determined by standard addition at three different concentrations levels i. e. 80 %, 100 % and 120 % to the pre-analyzed sample of the drugs 4 μg/mL and 20 μg/mL and each results was average of three determinations. The results of recovery study for TOR and SPI are shown in Table 5.

Table 5:

Result of Recovery study.

Drug	Conc. level (%)	Amount taken (μg/mL)	Amount added (μg/mL)	Total amount (µg/mL)	%Recovered Mean ± SD*	CV
TOR	80 %	4	2	6	98.55 0.67	0.68
	100 %	4	4	8	99.67 ± 0.52	0.52
	120 %	4	8	10	99.05 ± 0.15	0.14
SPI	80 %	20	10	30	98.82 ± 1.36	1.37
	100 %	20	20	40	98.09 ± 0.27	0.28
	120 %	20	30	50	100.01 ± 0.60	0.60

Table 6:

Result of precision study.

Drug	Parameters	Intraday precision concentration (µg/mL)			Inter day precision concentration (µg/mL)
		2	4	8	2	4	8
TOR	Mean area	12,404	25,008	50,319	12,303	24,996	50,643
	S.D*	98	108	260	84	97	446
	CV	0.79	0.43	0.52	0.68	0.38	0.88
SPI	Mean	87,402	158,501	268,847	86,803	158,612	269,211
	S.D	470	501	883	530	642	914
	CV	0.53	0.32	0.33	0.61	0.41	0.34

Precision

The value of % RSD for TOR for intra-day precision and inter-day were found to be in the range 0.43 to 0.64 of % and 0.38 to 0.88 % respectively which indicated that the method was precise.

The value of % RSD for SPI for intra-day precision and inter-day were found tobe in the range of 0.03 to 0.53 % and 0.20 to 1.07 % respectively as shown in Table 6 which indicated that the method was precise.

Robustness

The robustness of the method checked by the QbD approach and experimentation done as per Table 2 and found that when changed in essential parameter, results remains unaffected (Table 7).

Table 7:

Result of robustness study by QbD.

Sr No.	pH	Composition of mobile phase (%v/v)	Flow rate (mL/min)	%Recovered (TOR) Mean ± SD*	CV	%Recovered (SPI) Mean ± SD*	CV
1	4	5:3:2	0.8	99.14 ± 0.26	0.26	98.89 ± 0.74	0.74
2	4	5:3:2	1.0	100.37 ± 0.52	0.51	99.67 ± 0.52	0.52
3	4	4:3:3	0.8	99.05 ± 0.15	0.14	99.05 ± 0.15	0.14
4	4	4:3:3	1.0	98.82 ± 1.36	1.37	98.82 ± 1.36	1.37
5	5	5:3:2	0.8	98.09 ± 0.27	0.28	98.09 ± 0.27	0.28
6	5	5:3:2	1.0	100.01 ± 0.60	0.60	100.01 ± 0.60	0.60
7	5	4:3:3	0.8	98.55 ± 0.67	0.68	98.55 ± 0.67	0.68
8	5	4:3:3	1.0	99.67 ± 0.52	0.52	99.67 ± 0.52	0.52

Limit of detection and limit of quantification

The limit of detection and limit of quantification for TOR & SPI were calculated theoretically and found to be 10 and 33 ng for TOR and 75 and 248 ng for SPI respectively.

Statistical comparison of the developed method

Comparison of RP- HPLC method for TOR and SPI by pair t- test

The Assay result for TOR and SPI in tablet dosage form obtained using RP-HPLC method was compared statistically by applying two tail pair t-Test. The calculated t-Value for TOR (0.78) and SPI (0.03) was less than tabulated t-Value at 95 % confidence interval (Table 8).

Table 8:

Comparison of Methods for TOR and SPI.

Parameter	TOR		SPI
	Reported	Experimental	Reported	Experimental
% Assay	99.96	101.00	99.90	103.00
	99.94	99.30	99.95	101.66
	99.90	99.96	99.87	102.02
Tabulated t-value	4.30		4.30
Calculated t-value	0.78		0.03

There for no significant difference were found in the content of TOR and SPI determined by proposed RP-HPLC method.

Characterization of degradation products

Mass chromatograms in the positive electron spray ionization (ESI) mode for the drug and degradation products are shown in Figure 5. The probable structures and observed mass of impurities identified in mass spectrometer are tabulated in Table 9.

Figure 5:

Mass Spectra of (a) Torsemide (b) Spironolactone (c) Impurity of a Torsemide at RRT 3.2 (1-ethyl-3-{[4-[(3-methylphenyl)amino]pyridine-3-yl]sulphonyl}urea (d) Impurity of a Torsemide at RRT 3.1 (4-(3-Methylphenyl)-2H-pyrido[4,3-e]-1, 2, 4-thiadiazin-3(4H)-one 1,1-Dioxide) (e) Impurity of a Torsemide at RRT 2.25 (4-[(3-methylphenyl)amino]pyridine-3-sulphonamide) (f) Impurity of a Spironolactone at RRT 1.55 (Canrenone).

Table 9:

Probable structure and molecular mass of observed Impurities.

Chemical formula	Exact molecular mass	Observed molecular mass	RRT
C₂₂H₂₈O₃	340.456	341.1	1.55 (w.r.t SPI)
C₁₂H₁₃N₃O₂S	263.32	264	2.25 (w.r.t TOR)
C₁₃H₁₁N₃O₃S	289.31	289.9	3.1 (w.r.t TOR)
C₁₅H₁₈N₄O₃S	334.40	335.9	3.2 (w.r.t TOR)

Evidently, the molecular weight of Torsemide (348.42) was obtained at m/z 349.0 (M+H) and molecular weight of Spironolactone (416.57) was seen at m/z 417.0 (M+H), thus validating the output of the mass spectrometer. The m/z values obtained for the degradation products resolving at RRT 2.25, 3.1, 3.2 and 1.55 in the same run were 264, 289.9, 335.9 and 341.1 respectively. These were compared to the mass spectrum of known degradation products of TOR and SPI.

The m/z 264 at RRT 2.25 was observed might be due to the cleavage of NH-CO bond next to the SO₂ – NH bond. M/z 183.0 was found to be the most prominent fragment ion (Figure 5). Impurity m/z 289.9 was corresponds to RRT 3.1 and detected might due to the ring formation of SO₂ and NH – CO bond and cleavage of the NH – (CH₃)₂. Mass spectra of m/z 335.9 at RRT 3.2 was observed due to the branch cleavage at C – C bond, results in removal of methylene group. The m/z 341.1 at RRT 1.55 was observed due to the cleavage of C – S bond in spironolactone.

Conclusion

To the best of our knowledge this is the first method that utilised for the simultaneous determination of Torsemide and Spironolactone and related compound by stress degradation by QbD with LC-MS approach. Torsemide and Spironolactone are susceptible to the acid, base and oxidative degradation while they are stable when exposed to water. The use of QbD approach confirmed the robustness of the method. The LC-MS method successfully separates and identifies the LOR and SPI in formulations and also separates degradant impurities simultaneously.

Research funding: This study was completed with support of the Institute of Research and Development and without funding by other funding agencies.
Conflict of interest statement: The authors state no conflict of interest. The authors have read the journal’s Publication ethics and publication malpractice statement available at the journal’s website and hereby confirm that they comply with all its parts applicable to the present scientific work.

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Received: 2018-03-13

Revised: 2018-04-23

Accepted: 2018-04-23

Published Online: 2018-05-15

Published in Print: 2018-08-28

Articles in the same Issue

https://doi.org/10.1515/pthp-2018-0011

Keywords for this article

Quality by Design; HPLC; Torsemide; Spironolactone; Degradant Impurity