Simultaneous estimation of finerenone and canagliflozin using HPLC: application in metabolic stability studies

ABSTRACT

Aim

A robust, sensitive, and reliable method was developed and validated on HPLC for the simultaneous estimation of Finerenone (FNR) and Canagliflozin (CFZ).

Method

The resolution was performed by using mobile-phase acetonitrile (ACN): Water (51:49) at a flow rate of 0.75 ml/min with the C18 analytical column Phenomenex Luna (250 mm, 4.6 mm, 5 µm). FNR and CFZ were estimated at a retention time of 6.33 and 8.26 min with 10.0 min analysis run time and detected by PDA detector at a λ_max 249 and 290 nm, respectively. The calibration curve was linear over the concentration range of 0.05–10 µg/ml with R² 0.998 and 0.999 for FNR and CFZ, respectively. For FNR and CFZ, the limit of detection (LOD) was 0.53 and 0.36 μg/ml & limit of quantitation (LOQ) was 1.62 and 1.10 μg/ml, respectively. The method was validated using specificity, linearity, accuracy, precision, LOD, LOQ, robustness, and stability.

Conclusion

According to the International Council for Harmonisation (ICH) guideline, the validation studies confirmed that the optimization method is specific, simple, highly sensitive, reliable, robust, and reproducible. The developed method was successfully applied for simultaneous estimation with applications in in-vitro metabolic stability studies of pooled microsomes of humans, monkeys, dogs, rabbits, rats and mice.

1. Introduction

Finerenone (FNR) is chemically called as (S)-4-(3-cyano 5-methoxyphenyl)-5-ethoxy-2, 8-dimethyl-1, 4-dihydro-1, 6-naphthyridine-3-carboxamide: C₂₁H₂₂N₄O₃; MW 378.43 g/mol; see Figure 1(A) (for chemical structure). FNR is a novel compound, selective and nonsteroidal mineralocorticoid receptor antagonist (MRA) approved by USFDA for the treatment of chronic kidney disease (CKD) associated with type 2 diabetes [1–3]. Compared between all MRAs, spironolactone is potent but nonselective, eplerenone is more selective but less potent and FNR is potent and selective in both properties [4–6]. FNR was reducing kidney failure, disease progression in diabetic kidney disease and also reduced the urine albumin-to-creatinine ratio (UACR) in patients with CKD associated with type 2 diabetes [7–11]. FNR was also protects CKD associated with diabetes which induced cardiac dysfunction (systolic and diastolic), fibrosis despite similar renal dysfunction and mild or moderate hepatic impairment [12–17]. MR was enhanced by cortisol that causes increased expression of inflammation, reactive oxygen species, and fibrosis [18,19].

Figure 1.

Chemical structure of compound (A) FNR and (B) CFZ.

Canagliflozin (CFZ) is chemically called as (2S,3 R,4 R,5S,6 R)-2-(3-{[5-(4-fluorophenyl)thiophen2-yl]methyl}x-4-methylphenyl)-6-(hydroxymethyl)oxane3,4,5-triol: C₂₄H₂₅FO₅S; MW 444.52 g/mol; see Figure 1(B). CFZ is a novel oral hypoglycemic agent which treats the type 2 diabetes mellitus (T2DM) by inhibiting the sodium glucose co-transporter 2 (SGLT2) [20]. CFZ inhibits glucose reabsorption, which is expressed in renal proximal tubular cells and it decreases the filtrated glucose level. CFZ has multiple therapeutic effects, such as diminish reabsorption of glucose, increase urinary glucose excretion thus reduce plasma glucose level and promote weight loss [21]. CFZ can be used as a single or combined to any other group of glucose-lowering agents [22]. CFZ exhibited protective effects against inflammation, oxidative stress, extracellular matrix, fibrosis and treated the cardiovascular-related deaths, heart failure, cardiac arrest, myocardial infarction, atrial fibrillation, risk of adverse cardiovascular events and arterial and venous thrombosis. CFZ was inhibited the inflammatory pathological mechanisms of cardiovascular complications indirectly affect the AMPK/SIRT1 pathway and suppress the TNF-α, IL-1 and IL-6 signaling pathways [23–26]. SGLT2 inhibitor was decreased the circulating leptin, while elevated circulating adiponectin suggesting it may improve insulin resistance. SGLT2 inhibitors might provide the beneficial effects on metabolic homeostasis, reduced cardiovascular risk, and reduced leptin level [27]. SGLT2 inhibitors (SGLT2i) were treated for diabetes as well as cardioprotective effects and enhanced the treatment of ischemic heart disease (IHD) in patients with T2DM [28].

CKD is a major global health issue, it is a slowly developing disorder which causes end-stage renal disease (ESRD), abnormal renal function, and progressive decrease in glomerular filtration rate (GFR) [29].

Metabolic stability studies are important techniques to determine the metabolic behavior of drugs in different species. Metabolism understands the problems of drug – drug interactions, drug alone differences and bioavailability; if a drug is fairly metabolized, it may be good bioavailability in-vivo [30]. Metabolic stability determines how much time a drug can be stable in the body's system, toxicity, elimination of drugs from the human body and their pharmacological activity. Development and design of the new drug a metabolic study is an important technique to make a decision about drug’s acceptance or rejection. Different species microsomes were used to determine the species differences in the metabolism process and recognize the species closest to humans. Microsomal enzymes of different species were determined for the various degradation rates in both phase I and II reactions and presystemic clearance. The metabolic stability studies will not only suggest biotransformation data but it also provide the selecting and designing a compound with pharmacokinetic properties. The current study will also analyze the species closest to humans to accomplish the pharmacokinetic, metabolism, toxicity, safety, and efficacy studies [31–34]. Metabolic stability study of FNR and CFZ is determined the combination of that drugs may give mutual effect of metabolism. Since both FNR and CFZ are metabolizing by liver so may be chances of both drugs affecting each other’s metabolic stability.

Survey of different literature, the HPLC method of FNR and CFZ was individually reported in publishes paper. For example such as, analytical method of FNR was reported by Imran et al., the separation was achieved by column Hemochrom C18 (250 mm, 4.6 mm, 5μ) using mobile phase ACN and 0.1% trifleuroacetic acid 30:70 ratio at 1 ml flow rate. The FNR was detected at 252 nm wavelength and elute the 4.17 min [35]. Arulselvan et al., developed a method of FNR by using X-bridge C18 Column (150 mm, 4.6 mm, 3.5 µm) and mobile phase ACN and orthophosphoric acid (70:30% v/v) at a flow rate of 1 ml/min [36]. Similarly, another method was developed by Sumalatha et al., separation achieve by using column Inertsil ODS 3 V C18 column (250 mm, 4.6 mm, 5 μm) and mobile-phase ammonium dihydrogen phosphate buffer (pH 4.5) and ACN (60:40) ratio at flow rate of 1 ml/min [37]. Arsalan et al., analytical method was developed by using column Inertsil ODS 3 V C18 (250 mm, 4.6 mm, 5μ) column, and mobile phase 0.03 M potassium dihydrogen orthophosphate in water pH 3.2 with orthophosphoric acid:ACN (30:70 v/v) at a flow rate of 1 ml/min [38]. Jyothi et al., separation achieve by using column a Symmetry ODS C18 (4.6, 250 mm, 5 μm), and mobile phase in combination of 80:20% v/v acetonitrile: methanol at a flow rate of 1.0 ml/min [39]. Gabirel et al., LC-MS/MS method was developed for the quantification of FNR in gradient method. The separation was achieved by using column Luna C18 Mercury (20 mm, 2.0 mm, 3 µm) analytical column, and mobile phase ACN: 10 mM ammonium acetate buffer (pH 3) at 1 ml/min flow rate [1].

CFZ method development and validation was reported in a previous paper. Ishpreet et al., the separation was achieved by column C18 Column (250 mm, 4.6 mm, 5 μm) using mobile phase in a ratio of 55:45 v/v ACN: orthophosphoric acid at flow rate of 1 ml/min [40]. Ajitha et al., developed a method of CFZ by using Supelcosil C18 column (250 mm, 4.6 mm, 5 μm), and mobile phase using trifluoroacetic acid in water:ACN (80:20% v/v) at a flow rate of 1.0 ml/min [41]. Darshan et al., develop a method by using column nonpolar inertsil ODS-3 (250 mm, 4.6 mm, 5 μm) and mobile-phase ratio of 30:70% v/v ammonium acetate buffer (pH-4.5) and acetonitrile at a flow rate 1 ml/min [42]. Somarouthu et al., LC-MS/MS method was developed and drug estimation by SPE method, sample analysis by using column Zodiac C18 (100 mm, 4.6 mm, 5 μm), and 2 mM ammonium acetate and methanol use as a mobile phase with ratio of 15:85 v/v at a flow rate of 1.0 ml/min [43]. Shinji et al., LC-MS/MS method was developed and using column Quicksorb ODS C18 (100 mm, 2.1 mm, 5 μm) and acetonitrile : 0.1% formic acid (90:10 v/v) use as a mobile phase at a flow rate of 0.2 ml/min [44].

The simultaneous method has create challenging in designing a single method for multiple analyte require multiple testing with additional development time. Some factor has required additional time like as optimization of mobile phase, flow rate, mobile-phase ratio, and type of column. Chromatographic conditions might cause overlapping of analyte, sometime one analyte high sensitive to other analyte. Accuracy and precision was difficult to achieve when both analytes have significantly different properties. The sample preparation can be challenging, if analyte has different physical and chemical properties.

The simultaneous analyte has analyzing in biological matrices facing many challenges. Most of the interference, matrix effect is important which changes in analyte response signal because some other substance present in the sample. Analyte concentration in biological sample is present at low, facing problem in detection and quantification. The extracting of the analyte from biological sample is difficulty without adding co-extraction substances. The interfering substance in biological sample has facing problem in reduce sensitivity, signal overlap and selectivity. The liquid-liquid extraction (LLE) and solid-phase extraction techniques were used to overcome matrix interferences.

The aims of current research were to develop a new simultaneous method for the quantification of FNR and CFZ; it’s more sensitive and robust than previously reported methods. To the best of my knowledge, no HPLC and LC–MS/MS method was reported yet for the simultaneously quantification of FNR and CFZ. For better resolution of FNR and CFZ, Phenomenex Luna column C18 (250 mm, 4.6 mm, and 5 μm) was used. The mobile phase was used ACN as an organic phase and water as an aqueous phase with ratio of 51:49% v/v at 0.75 ml/min flow rate. This method can illustrate the principle applicable and reliable for simultaneous estimation of both drugs within the same mobile phase. After method development, this will be applied for stability study at room temperature and cool temperature. The forced degradation (FD) stability was also performed in different conditions such as base, acid, oxidation, thermal, and photo degradation. This method will be further utilizing for stability study of different species liver microsomes for FNR and CFZ. The aim of the metabolic studies, to determine the metabolism of drug in different animal can significantly varying rate due to species to specific differences in metabolizing enzyme. This minimizes risk associated with unexpected metabolic variation. We want to examine if there is any drug–drug interaction or mutualistic effect between FNR and CFZ during metabolic stability studies. We are performing a metabolic stability study in different species microsomes. This simultaneous method may be helpful in establishing newer drug delivery system in-vitro preclinical pharmacokinetic and metabolic stability studies.

2. Methods

2.1. Chemicals & reagents

FNR was purchased from Molnova Chemical (USA), and CFZ was purchased from SNB Innovation (USA). Testosterone, Alamethicin, Nicotinamide adenine dinucleotide phosphate (NADPH), uridine 5 -diphosphoglucuronic acid (UDPGA), phenyl methyl sulfonyl fluoride, and ethylendiaminetetraacetic acid (EDTA) were purchased from Sigma Aldrich (MO, USA). Magnesium chloride and tris-buffer were purchased from Sisco Research Laboratories (SRL). Potassium chloride was purchased from Thermo Fisher Scientific. Human, monkey, dog, and rabbit liver microsomes were procured from Corning Gentest™ (USA). Mouse and rat liver microsomes were prepared in in-house facilities of CSIR-CDRI. ACN (chromatographic grade) was purchased from J T Baker (Mumbai, India). MilliQ water purification system (EMD Millipore, USA) was acquired to prepare Triple distilled water (TDW).

2.2. Instrumentation and chromatographic specification

The samples were injected using a Shimadzu HPLC system from Kyoto, Japan, which consist of degasser (DGU-20A3), binary pump (LC-20AD), a controller auto sampler (SIL-HTc), Column oven (CTO-20AC) and attach with PDA detector (SPD-M20A). The chromatographic conditions were optimized by changing in different organic and aqueous mobile phase. The primary trial of mobile phase was optimized with different ratios of methanol (MeOH) or ACN with aqueous buffer. We try to another solvent-like MeOH in mobile phase resulting in broad and peak tailing of CFZ. Another observation is that, if increasing the ratio of aqueous content to RT of CF, the FNR peak was changed approx 11 min and peak broad, at the same time FNR peak around 4 min. Therefore, finding this result, ACN was selected instead of MeOH.

Composition of mobile phase was selected on the basis of a good peak parameter such as separation between both drugs, tailing factor, capacity factor, number of theoretical plates and resolution. FNR and CFZ were separated on Phenomenex Luna (250 mm, 4.6 mm, 5 µm) column with isocratic mobile phase consists of ACN: TDW, 51:49 v/v at 0.75 ml/min flow rate. The oven temperature was 30°C, injection volume 30 μl and total analysis run time was 10.0 min. Both FNR and CFZ were checked the peak parameter such as peak areas, retention time (RT), the number of theoretical plates, capacity factor, and resolution.

In bioanalytical sample, Extraction of FNR and CFZ from plasma matrix were process by different method to optimize the higher recovery. Firstly, the simple protein precipitation techniques (PPT) procedure was performed because it’s simple, lower cost and less time-consuming. A number of organic solvents, such as ACN, methanol, ethanol, 0.1% formic acid in MeOH, 0.1% acetic acids in ACN were evaluate as precipitation solvents in protein precipitation technique (PPT). PPT procedure was shown to have low sensitivity and low extraction recovery for FNR and CFZ. After that PPT followed by liquid–liquid (LLE) was performed, matrix mixtures with PPT solvents like ACN and MeOH after that extracting solvent add like ethyl acetate, diethyl ether, n-hexane and TBME. PPT solvent ACN and extracting solvent TBME were observed higher extraction recovery and reduced matrix effect for both FNR and CFZ. In running stock solutions, DMSO concentration was very less and did not cause any interaction with biological matrices.

2.3. Stock sample preparation

The primary stock solutions of FNR and CFZ were prepared in dimethylsulfoxide separately by dissolving 1 mg/ml concentration. The running stock solutions were prepared in ACN. For calibration range sample were prepare 10 time higher concentration in stepwise dilutions in ACN to reach suitable concentration. All stock solutions were kept in the refrigerator at 4°C until use.

3. Method validation

The analytical method was developed and validated according to the ICH guidelines Q2 (R1) [45–47].

3.1. System suitability

The system suitability was performed by preparing a mixture of FNR and CFZ at 5 μg/ml concentration in blank ACN solvent. The sample was injected by six replicates of the mixture of FNR and CFZ. The system suitability was evaluated by calculating the mean area, standard deviation, and %RSD.

3.2. Preparation of calibration curve

For FNR and CFZ, the analytical standards were prepared by adding a 10 μl of working stock solution with 90 μl of blank ACN, its reaches to actual concentration. The calibration ranges of FNR and CFZ were from 0.05 to 10 μg/ml. Other analytical standard samples were prepared at low, mid, and high (50%, 100%, 150%) levels in analytical solvent (ACN). The final concentrations of FNR and CFZ were 2.5, 5, and 7.5 μg/ml in ACN. Samples were stored in the refrigerator at 4°C until analysis.

3.3. Accuracy

The accuracy was defined the similarity of the results acquired by this method and it is close to the true value. Accuracy was performed at three known concentrations level in six replicates for both drug of FNR and CFZ. The analytes were 50%, 100%, and 150% as low, middle, and higher levels, respectively, injected into HPLC. The data were analyzed the % RSD and % recovery by calculating the pre-analyzed drug concentrations and comparing them with spiked drug concentrations.

3.4. Precision

The precision was defined as the degree of that similarity to the true value. The precision and reproducibility of both FNR and CFZ was analyzing six replicates over three levels (2.5, 5, and 7.5 µg/ml) at the day 1, 2 and 3 day (intra- and interday). The results were interpreted for both FNR and CFZ as percent relative standard deviation (%RSD) and % recovery.

3.5. Limit of detection and limit of quantification

In the sample, a calculated amount of drug that can be recognized called LOD. For LOQ, the minimum amount of compound in the sample was determined accurately and adequately.

The LOD and LOQ for FNR and CFZ were interpretate by calculating the standard deviations of the response and slope which is finding in the calibration curve.

3.6. Robustness

The robustness was performed by minimum changes in different circumstances, like as a change in flow rate, composition of mobile phase and oven temperature in the developed method. It was evaluated the precision results for FNR and CFZ. The robustness was evaluated and compared to developed method and also calculate the recovery of FNR and CFZ.

3.7. Solution stability

The solution stability was determined by sample solutions spiked in the ria vial and capped tightly to prevent moisture. The samples were kept at room temperature (24 ± 2°C) for 4 h and other sample placed refrigerator at 4°C. The solution stability was calculated and checks the difference in the peak areas of FNR and CFZ compared to the optimized sample.

4. Forced degradation studies

Forced degradation studies were performed to check the stability of FNR and CFZ at different environmental conditions. Analytes were performed at 5 μg/ml (mid level) concentration of stock solution. Both FNR and CFZ were applied the stress conditions, such as basic hydrolysis (0.1 N NaOH), acid hydrolysis (0.1 N HCl), oxidation (3% H2O2), thermal, and photolytic degradation (UV).

4.1. Basic degradation

Forced degradation in basic media was performed using FNR and CFZ from the stock solution and mixing with 0.1 N NaOH in ria vial. The ria vial was placed in an incubator at 60°C ±2°C at 4 h for control environment. The samples were injected (N = 6) under optimized chromatographic conditions.

4.2. Acid degradation

Forced degradation in acid media was performed by taking FNR and CFZ from the stock solution and in addition with 0.1 N HCl in ria vial. The ria vial was placed in an incubator at 60°C ±2°C at 4 h for control environment. The samples were injected (N = 6) under optimized chromatographic conditions.

4.3. Oxidative degradation

Forced degradation in oxidative conditions was performed using FNR and CFZ from the stock solution and mixing with 3% v/v of hydrogen peroxide in ria vial. The ria vial was placed in an incubator at 60°C ±2°C at 4 h for control environment. The samples were injected (N = 6) under optimized chromatographic conditions.

4.4. Thermal degradation

For thermal stress, the sample of FNR and CFZ was transferred in ria vial and then kept in an oven and heated at 80°C at 4 h. The samples were injected (N = 6) under optimized chromatographic conditions.

4.5. Photolytic degradation

Photolytic degradation was performed by aliquot of FNR and CFZ into a ria vial and direct exposure to UV light for a period of 4 h. After exposure, the samples were injected (N = 6) under optimized chromatographic conditions.

5. Preparation of microsomes in different species

Mouse liver microsome (MLM) and Rat liver microsome (RLM) was prepared in animal house facility as per approved protocol IAEC/2022/102/Renew-0/Dated-09/09/2022. Before the experiment, the animal was fasted previous night only free access water. First, the young rats were anesthetized and sacrificed by cervical dislocation. Upper Abdomen cut 3–4 cm and perfuse the liver by perfusion buffer via a hepatic portal vein. The blood was eliminating after perfusion the liver with ice-cold saline. The liver were collected and rinsed with saline, weighed, and homogenize by using homogenizing buffer which contain 0.1 M tris-buffer (pH 7.4), 0.125 MKCL) and 1 mM sodium EDTA. Centrifuge the homogenate at 9000 g for 30 min at 4°C. After centrifuge, supernatant was produced and it was centrifuge at 100,000 rpm for 60 min at 4°C. The pallet was resuspended in resuspension buffer which contains glycerol (20%), sodium EDTA, phenyl methyl sulfonyl fluoride, and 0.1 M tris-buffer (pH 7.4). Protein concentration in microsome was calculated by Bradford assay method. The protein content was adjusted to 20 mg/ml and aliquots of microsome in different microcentrifuge tube and kept in a refrigerator at −80°C until analysis [48].

5.1. Determination of microsomal stability

In-vitro microsomal stability study was determined the effect of liver microsomes on different species [30,49–54]. The in-vitro phase I metabolic stability of FNR and CFZ was individually conduct with different species microsomes such as human liver microsome (HLM), monkey liver microsome (MnLM), dog liver microsome (DLM), rabbit liver microsome (RbLM), rat liver microsome (RLM), and mouse liver microsome (MLM) respectively. The different species microsomes were used and FNR and CFZ at 10 μM concentration alone and its combination. To evaluate the microsomal activity, testosterone used as a positive control at 10 µM concentrations.

For phase I reaction, in glass test tubes, 50 mM tris-HCl buffer (pH 7.4), 40 mM MgCl₂, microsomes (0.5 mg/ml), and FNR and CFZ (10 μM) were pre-incubated in a shaking water bath at 37°C for 10 min. the reaction was start by adding Nicotinamide adenine dinucleotide phosphate (NADPH), and samples were collected at 0, 5, 15, 30, 45, and 60 min. For phase II reactions, uridine 5-diphosphoglucuronic acid (UDPGA) use instead of NADPH and addition of alamethacin for increase the pore size of the microsome. The negative control was also performed by excluded of NADPH in phase I and UDPGA in the phase II reaction. The sample was withdrawn 100 µL as per given above time point and transferred into a 1.5 ml centrifuge tube, already containing 200 µl of ACN. Samples were centrifuged for 10 min at a speed of 10,000 g, and the supernatant was collected and used for analysis. To check the metabolism of different species was calculated by comparing 0 min sample peak area to peak area of different time and calculating the percent remaining of drug at 1 h.

6. Results

6.1. Method development

The present scenario was indicating the separation of FNR and CFZ by reversed-phase column procedure because both compounds are hydrophobic or low water solubility. FNR and CFZ were separated by C18, Phenomenex Luna column by using ACN and water. FNR and CFZ were separated simply with a clear resolution and analyzing run time 10 min. First, we injected the blank solvent and analyze the no peak observed at the RT of both drugs, Figure 2(A). The chromatogram results of FNR and CFZ were RT of 6.33 min and 8.26 min, respectively Figure 2(B,C). For FNR and CFZ, the peak parameters were evaluated such as less tailing factor (1.2% and 1.11% respectively), theoretical plates (3926 and 3797 respectively), capacity factor (1.14 and 1.81 respectively), and resolution (9.54 and 4.11). The peaks of FNR and CFZ were observed in symmetric shape, and peak parameters were acceptable the limit range shown in Table 1. The bioanalytical method was performed in matrix by using fresh blank Sprague-Dawley rat plasma spike with known concentrations of FNR and CFZ at 5 µg/ml and observes the no peak at the RT of both drugs, as represented in Figure 3(A and D).

Figure 2.

High pressures LC for (B) FNR and (C) CFZ resolution. In chromatograms no interference observed in blank sample (A) at RT of FNR (6.33 min) and CFZ (8.26 min) were detected at 249 (B) and 290 nm (C), respectively.

Table 1.

Parameters of FNR and CFZ.

S.No.	Parameter	FNR	CFZ	Acceptance criteria
1	Retention time (RT)	6.33	8.26	–
2	λmax (nm)	249	290	–
3	Theoretical plates (N)	3926	3797	>2000
4	Tailing factor (T)	1.2	1.11	<2
5	Capacity factor	1.14	1.81	<2
6	Resolution	9.54	4.11	>2
7	Linearity range(µg/mL)	0.05–10	0.05–10	–
8	Detection limit(µg/mL)	0.53	0.36	–
9	Quantification limit (µg/mL)	1.62	1.10	–

Abbreviations: FNR: Finerenone; CFZ: Canagliflozin; nm: Nano meter.

Figure 3.

In chromatogram, blank plasma matrix detected at 249 (A) and 290 nm (B), respectively, FNR and CFZ spike with plasma detected at 249 (C) and 290 nm (D) at RT of FNR (6.08 min) and CFZ (8.37 min), respectively.

The samples were processing as follows: 45 µL of blank plasma sample and 5 µL stock of low, mid, and high-level sample, mixture was precipitated with 200 µL of ACN. Later, extraction solvent was adding 2 mL of TBME and vortexed for 10 min at 2000 rpm on Benchmixer. The mixed samples were followed by centrifugation for 5 min at 8,000 rpm (Eppendorf, Germany). The separated supernatant (1.6 mL) was transferred to other vials and dried with TurboVap nitrogen drier at 40°C. Dried residue remaining in ria vial was reconstituted with 100 µL of ACN, and injected into UPLC was 30 µL. Matrix effects were determined with the help of postextraction spike technique for both FNR and CFZ. The average matrix effect of FNR and CFZ was found 1.01 ± 0.29% and 1.04 ± 0.16%, respectively. The percentage recovery of FNR and CFZ during LLE was calculated by peak areas of analyte extracted plasma sample (pre-spiked) and post extracted plasma samples (post-spiked). The average percentage recovery of FNR and CFZ was found 97.58 ± 5.88% and 93.15 ± 4.43%, respectively.

6.2. HPLC method validation

The developed analytical method was validated by evaluating different parameters such as system suitability, linearity, precision, accuracy, LOD, LOQ, robustness, and stability. All the analytical samples were analyzed and found to be the %RSD within the acceptable limits according to the ICH guidelines.

6.2.1. System suitability

The system suitability was determined and calculated using the mean area, standard deviation and %RSD. The sample was analyzed and found to be the %RSD (< 2) within the acceptable limits shown in supplementary Table S1.

6.2.2. Linearity

Linearity was performed and calculated both FNR and CFZ at eight different concentrations. The peak area response was obtained linearity curves over the concentration range of 0.05–10 μg/ml for FNR and CFZ. The linearity was determined correlation coefficient value and slope 0.998 and 96,612 for FNR and 0.999 and 74,483 for CFZ, respectively, shown in supplementary Table S2.

6.2.3. Accuracy

The accuracy was performed by measuring the recovery at three levels (50%, 100%, and 150%) for FNR and CFZ. To determine the accuracy by known concentration of each drug was added, processed, and evaluate the percentage recovery. The percentage recovery was found to be 98.72–101.91% for FNR and 98.30–101.75% for CFZ. The results of accuracy shown the method was highly accurate for both drugs. The results of recovery studies are shown in Table 2.

Table 2.

The percentage recovery and relative standard deviation to determination of accuracy and precision for FNR and CFZ.

FNR
		Accuracy (N = 6)				Precision (N = 6)
		Intraday		Interday		Intraday	Interday
Level	Concentration (µg/mL)	Obs. conc	Accuracy	Obs. conc	Accuracy	%RSD	%RSD
50%	2.5	0.02 ± 2.46	1.18 ± 98.72	0.03 ± 2.50	1.41 ± 100.19	1.20	1.03
100%	5	0.03 ± 5.04	0.74 ± 100.86	0.02 ± 5.07	0.50 ± 101.44	0.73	0.77
150%	7.5	0.06 ± 7.64	0.90 ± 101.91	0.06 ± 7.58	0.80 ± 101.15	0.88	1.40
CFZ
50%	2.5	0.02 ± 2.45	1.18 ± 98.30	0.01 ± 2.46	0.59 ± 98.71	1.20	1.19
100%	5	0.03 ± 5.08	0.75 ± 101.75	0.01 ± 5.07	0.24 ± 101.59	0.74	0.94
150%	7.5	0.09 ± 7.57	1.32 ± 101.00	0.03 ± 7.61	0.46 ± 101.53	1.31	1.14

Abbreviations: FNR: Finerenone; CFZ: Canagliflozin; RSD: Relative standard deviation.

6.2.4. Precision

Precision was evaluated by performing six replicate injections at three levels (50%, 100%, and 150%) for FNR and CFZ. Results of precision were analyzed in terms of %RSD, for FNR 0.73–1.40 and for CFZ 0.74–1.31 (Table 2). Both drugs had % RSD < 2 within the acceptable limit, according to ICH guidelines.

6.2.5. Determination of LOD & LOQ

According to the ICH guidelines Q2 (R1), the LOQ and LOD were calculated using the given equations:

$$\mathit{LOD}=\frac{3.3\ast \mathit{standard}\mathit{deviation}\mathit{of}\mathit{the}\mathit{intercept}\mathit{of}\mathit{regression}\mathit{line}}{\mathit{Slope}\mathit{of}\mathit{calibration}\mathit{curve}}$$

For FNR and CFZ, LOD values were determined as 0.53 µg/ml and 0.36 µg/ml, respectively. Similarly, LOQ values for FNR and CFZ were determined as 1.62 µg/ml and 1.10 µg/ml, respectively. The observation of the lowest value of LOD and LOD indicates that the newly developed method is more sensitive to quantification of FNR and CFZ.

$$\mathit{LOQ}=\frac{10\ast \mathit{standard}\mathit{deviation}\mathit{of}\mathit{the}\mathit{intercept}\mathit{of}\mathit{regression}\mathit{line}}{\mathit{Slope}\mathit{of}\mathit{calibration}\mathit{curve}}$$

6.2.6. Robustness

Robustness was determined by any variation among the chromatographic resolution in the optimized method for FNR and CFZ. The sample was prepared at 5 μg/ml (mid level) for FNR and CFZ and evaluated with significant variation to optimized parameters, such as mobile-phase ratio, flow rate, and column temperature (°C). Data were calculated the % recovery of both drugs were 97.11–102.42% for FNR and 97.46–102.85% CFZ. Slight modifications in the method such as flow rate, composition of mobile phase and oven temperature caused deviations in the RT of FNR and CFZ. The results of robustness were calculated and % RSD found to be within the acceptable limits according to ICH guidelines. The result shows in Table 3, which the method is highly robust.

Table 3.

Determination of method robustness for FNR and CFZ.

Parameter	RT	Recovery	% RSD
FNR
Mobile phase (51:49) ACN: TDW	6.33	0.43 ± 101.1	0.42
Mobile phase (53:51) ACN: TDW	6.65	0.47 ± 97.86	0.48
Mobile phase (49:47) ACN: TDW	8.25	0.76 ± 102.42	0.75
Flow rate (0.75 mL/min)	6.33	0.43 ± 101.03	0.43
Flow rate (0.85 mL/min)	5.64	0.42 ± 97.11	0.44
Flow rate (0.65 mL/min)	7.38	1.39 ± 102.36	1.36
Column temperature (30◦C)	6.33	0.43 ± 101.02	0.43
Column temperature (35◦C)	6.35	0.58 ± 99.78	0.58
Column temperature (25◦C)	6.33	0.22 ± 98.71	0.22
CFZ
Mobile phase (51:49) ACN: TDW	8.26	0.53 ± 100.2	0.52
Mobile phase (53:51) ACN: TDW	8.53	0.47 ± 97.46	0.48
Mobile phase (49:47) ACN: TDW	11.11	1.14 ± 102.36	1.12
Flow rate (0.75 mL/min)	8.25	0.63 ± 101.85	0.62
Flow rate (0.85 mL/min)	7.41	1.33 ± 97.66	1.36
Flow rate (0.65 mL/min)	9.69	1.31 ± 102.85	1.28
Column temperature (30◦C)	8.26	0.63 ± 101.85	0.62
Column temperature (35◦C)	8.30	0.31 ± 99.75	0.31
Column temperature (25◦C)	8.10	0.15 ± 99.94	0.15

Abbreviations: FNR: Finerenone; CFZ: Canagliflozin; ACN: Acetonitrile; TDW: Triple distilled water; RT: Retention time; RSD: Relative standard deviation.

6.2.7. Solution stability

The stability of the analytical sample was checked by kept the FNR and CFZ at refrigerator condition (4°C) and room temperature (24 ± 2°C). The samples were processed and evaluate by compare the standard sample and also perform at different time intervals. The %RSD of FNR and CFZ for the duration of solution stability was found to be acceptable within the limit. The chromatograms of standard solution and the sample solution demonstrated that no significant difference observed at different time intervals. The observation in the peak, which is no degradation found in peak hence proved that the sample is stable in investigation condition, result shown in supplementary Table S3.

6.3. Forced degradation studies

Forced degradation studies were performed on FNR and CFZ to apply various stress-related conditions and check the RT of active compound and RT of degradation compound at different time. In some circumstances, both compounds were observed a decrease in peak area and peak height rather than separate peaks of the actives compound. The degradation compound peaks were observed and compared with the standard solution, shown in supplementary Figure S1. Forced degradation studies for FNR and CFZ were performed by basic (0.1N NaOH), acid (0.1N HCL), oxidation (3% H2O2), thermal degradation (80°C), and photolytic degradation (UV rays). The degradation studies were observed the FNR more stable against photolytic, basic, thermal studies, oxidation, and acid degradation. For CFZ more stable against photolytic, basic, oxidation, thermal studies, and acid degradation. In comparison to acid and base conditions, FNR is more prone to acid condition rather than basic, while CFZ less degraded in basic condition. The results are described in Table 4.

Table 4.

Force degradation under different conditions for FNR and CFZ.

S.No.	Degradation Type	RT of Degradation products	% Residual drug	Drug decomposed (%)
FNR
1.	Standard drug	6.33	100	–
2.	Acid degradation	5.41	75.39	24.61
3.	Basic degradation	6.49	97.45	2.55
4.	Peroxide degradation	6.47	85.33	14.67
5.	Thermal degradation	6.43	94.86	5.14
6.	Photolytic degradation	6.21	98.24	1.76
CFZ
1.	Standard drug	8.26	100	0
2.	Acid degradation	8.46	89.61	10.39
3.	Basic degradation	8.46	98.30	1.70
4.	Peroxide degradation	8.53	94.14	5.86
5.	Thermal degradation	8.39	92.79	7.21
6.	Photolytic degradation	8.30	98 .45	1.55

Abbreviations: FNR: Finerenone; CFZ: Canagliflozin; RT: Retention time.

6.3.1. Basic degradation

The basic degradations of FNR and CFZ were observed approximately 2.55% and 1.70%, respectively.

6.3.2. Acid degradation studies

Acid degradation studies were observed the small change in peak area, decrease in peak height, and additional peaks at 7.8 RT in case of CFZ. Acid degradation for FNR and CFZ was observed at approximately 24.61% and 10.39%, respectively.

6.3.3. Photo degradation

Photo degradation was observed the degradation of 1.76% and 1.55% for FNR and CFZ, respectively.

6.3.4. Peroxide degradation

FNR and CFZ were observed to have degradations of 14.67% and 5.86%, respectively.

6.3.5. Thermal degradation

Thermal degradation was observed the degradation of FNR and CFZ showed 5.14% and 7.21%, respectively.

6.4. Application in microsomal stability

For FNR and CFZ, different microsome (HLM, MnLM, DLM, RbLM, RLM, and MLM) were used to determine the metabolism behavior in different species. The metabolic stability of FNR and CFZ was performed alone and combinations at 10 μM concentration for phase I and II reactions. The incubation times for reaction was performed for 1 h, and each sample was withdrawn at 0, 5, 15, 30, 45, and 60 min. The incubation reactions of FNR and CFZ were stopped as per given time point. The metabolic stability was performed in different species with the help of co-factors NADPH and UDPGA for phase I and II reactions, respectively. The negative control reaction was also performed in the absence of both cofactors NADPH and UDPGA for phase I and II reactions, respectively. The % remaining of FNR and CFZ in absence of cofactor was found 99.50% and 98%, respectively. The positive control reaction was also performed by using testosterone and confirmed the activity of microsomal protein. FNR and CFZ was metabolized by different microsomal enzyme species and found to be metabolizing different rate in phase I and II reactions. The metabolic stability was calculated by % remaining of FNR and CFZ value alone and combination at different times with NADPH and UDPGA for phase I and II reactions shown in Table 5. The concentrations were calculated in triplicate at different time points and error bars indicate the deviation of a single value from the mean value. The graph of FNR and CFZ was plotted between the percent drugs remaining vs time for phase I alone and combination shown in Figure 4(A–D) and phase II alone and combination shown in figure 5(A–D). For FNR, human species are more similar to MLM, RLM, and DLM, and significant differences in MnLM and RbLM species and for CFZ more similar to all species except DLM, in phase I reaction. FNR was more similar to MnLM, DLM, and RbLM species and CFZ was found non-significant difference in all species, at phase II reactions.

Table 5.

Percentage of drug remaining versus time profile for microsome stability in different species in phase 1 and phase 2 consisting of FNR and CFZ alone and combination at 10 μM concentrations.

	HLM		MnLM		DLM		RbLM		RLM		MLM
Drug concentration at 10 μM	Alone	Combination	Alone	Combination	Alone	Combination	Alone	Combination	Alone	Combination	Alone	Combination
Phase 1
FNR	54.93 ± 3.78	49.18 ± 3.75	91.43 ± 2.27	95.14 ± 2.68	73.58 ± 1.85	68.08 ± 6.55	89.13 ± 2.69	91.68 ± 4.84	84.00 ± 3.35	82.04 ± 4.04	70.47 ± 3.82	69.76 ± 2.75
CFZ	89.39 ± 2.88	92.31 ± 2.53	81.75 ± 1.43	78.0 ± 2.24	77.27 ± 4.00	74.03 ± 5.80	84.94 ± 2.49	81.79 ± 5.20	78.52 ± 2.41	81.18 ± 2.92	90.04 ± 3.76	92.66 ± 0.35
Phase 2
FNR	77.89 ± 3.25	75.65 ± 5.77	81.78 ± 5.21	83.90 ± 1.67	81.81 ± 4.62	74.86 ± 1.33	73.58 ± 4.39	71.29 ± 4.12	95.55 ± 3.89	95.22 ± 4.62	95.87 ± 2.98	97.97 ± 4.08
CFZ	78.39 ± 5.13	76.13 ± 6.18	73.99 ± 6.06	79.96 ± 5.34	71.91 ± 1.03	61.02 ± 2.07	79.96 ± 5.34	75.30 ± 4.04	74.57 ± 5.08	84.63 ± 6.63	85.10 ± 3.05	87.12 ± 4.46

Abbreviations: FNR: Finerenone; CFZ: Canagliflozin; HLM: Human liver microsome; MnLM: Monkey liver microsome; DLM: Dog liver microsome; RbLM: Rabbit liver microsome; RLM: Rat liver microsome; MLM: Mouse liver microsome; μM: Micro molar.

All data were performed in triplicate (mean ± standard deviation).

Figure 4.

(A–D) liver microsomal stability were performed in different species (human, monkey, dog, rabbit, rat and mouse) in phase I reaction consisting of FNR and CFZ alone and combination at 10 μM concentrations. All data were performed in triplicate (mean ± standard deviation).

Figure 5.

(A–D) liver microsomal stability were performed in different species (human, monkey, dog, rabbit, rat and mouse) in phase II reaction consisting of FNR and CFZ alone and combination at 10 μM concentrations. All data were performed in triplicate (mean ± standard deviation).

7. Discussion

FNR are mineralocorticoid antagonist for the treatment of chronic kidney disease associated with diabetes. FNR has strong binding to Mineralocorticoid Receptor (MR) compared to other drug like as spironolactone and eplerenone. FNR was inhibited inflammation, fibrosis, reducing kidney failure, diabetic kidney disease (DKD), and urine albumin-to-creatinine ratio. CFZ are inhibiting the sodium‐glucose co‐transporter 2 (SGLT2) to control the type 2 diabetes mellitus. SGLT2 is expressed in the proximal renal tubules and is responsible for the reabsorption of glucose from lumen.

We survey the published literature and a number of papers to determine the combination of chronic kidney disease associated with diabetes. In the current scenario, many number of paper published to quantification of FNR and CFZ separately, but no one published the simultaneously. To the best of my knowledge, a new HPLC method was developed for the simultaneous quantification of FNR and CFZ.

We prepare the solution of FNR and CFZ in DMSO because both drugs are insoluble in aqueous medium. Secondary working stocks were prepared in ACN. FNR and CFZ were separated on the basis of polarity at RT of 6.33 and 8.22 min and evaluated by HPLC PDA at wavelengths 249 and 290 nm, respectively. To optimize the mobile-phase composition (ACN: TDW), a ratio of (51:49) was chosen based on better separation, resolution, peak shape, and intensity of both drugs. The peak parameters, such as fronting and tailing was decreased by change the increasing or decreasing the mobile-phase concentration. Column temperature was determined the 30°C to prevent peak overlap and noise variation. In brief, the method was validated successfully for the simultaneous quantification of FNR and CFZ according to ICH guidelines. All validation parameter was determined such as system suitability, accuracy, precision, LOD, LOQ, robustness, and stability, all are acceptable within the range. This method will initially help in the method development and validations of other SGLT2i such as empagliflozin and dapagliflozin with slight modification. Because every drug has unique structure property attached with different functional groups and different molecular weight, so might be require same or different mobile phase and column. The validated method was evaluated for greenness by using Modified Green Analytical Procedure Index (MoGAPI) software. MoGAPI has been generated score 79 for environmental friendliness of analytical methods. Several recent publications have implemented these tools to evaluate the greenness of their methods. Several renowned research papers now use this software to demonstrate the greenness of their method [55].

The force degradation studies were performed and optimize the how much concentration of drug was degraded. In acid and base degradation study was optimized the more amount of drug degraded in acid compared to basic buffer.

In-vitro microsomal stability assay were evaluated in different species of liver microsomes (HLM, MnLM, DLM, RbLM, RLM, and MLM). Microsomal stability was performed alone and a combination of phase I and II reactions to find the metabolism variations in different species and metabolic behavior most similar to humans. The reaction was incubated for 1 h at a concentration of 10 μM for FNR and CFZ alone and its combination and finds the metabolism of drug in different species. The drug concentration was calculated the percentage of drug remaining versus time profile of FNR and CFZ alone and its combination. FNR were observed the metabolized in phase I due to different groups of enzymes (hydrolysis, oxidation, and reduction) capable of metabolism but less metabolism in phase II reactions. CFZ were found the metabolism in phase II reaction due to UDPGA enhance the glucuronidation or conjugation pathways. FNR was metabolized by mainly CYP3A4 (90%) and CYP2C8 (10%) to make clearance from the system. CYP3A4 expressed in the gut wall contributes to the first-pass metabolism of finerenone to clearance of the drug. CFZ is less metabolized by CYP3A4 approx 7% but it mainly metabolized by the UGT1A9 and UGT2B4 enzyme.

8. Conclusion

In the current scenario, many numbers of papers have been published on HPLC methods to quantify FNR and CFZ separately, but simultaneously has no one reported. We have developed a new simultaneous method for the quantification of FNR and CFZ to diminish overcome financial issues. The HPLC method was validated successfully according to ICH guidelines, and all validation parameters were acceptable limits. Accuracy and precision were calculated and %RSD less than 2 for intra- and inter-day. The method was specific by evaluating the blank sample and both drug samples and no interference found in blank sample at the RT of both drugs. The method has proved to be precise, accurate, linear, robust, and stable. The force degradation studies were evaluated the how much concentration of the drug degraded. This method is applicable to quantify the metabolic activity of different microsomal species.

Funding Statement

This paper was not funded.

Article highlights

• Current research has illustrated the importance of FNR and CFZ in the treatment of chronic kidney disease associated with diabetes.

• To the best of my knowledge, currently no analytical method has been developed that can quantify both drugs simultaneously.

• This work has illustrated the development and validation on HPLC – PDA method for the simultaneous quantification of FNR and CFZ.

• The separations were achieved by C18 Phenomenex Luna HPLC column by using mobile-phase acetonitrile (ACN): Water (51:49) at a flow rate of 0.75 ml/min.

• According to International Council for Harmonisation (ICH) guidelines, the method was validated by performing different parameters such as linearity, accuracy, precision, limit of detection, limit of quantification, robustness, and solution stability.

• The developed method was linear over the linearity range of 0.05–10 µg/ml for FNR and CFZ.

• The force degradation approach was also used to evaluate the degradation of FNR and CFZ under different environmental conditions.

• The developed method was applicability in simultaneous quantification of FNR and CFZ and also helps in the microsomal stability.

• The developed method was performed in in-vitro studies and microsomal stability in different species such as human, monkey, dog, rabbit, rat and mouse liver microsomes.

Author contributions

Shivam Rathaur. Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosures

Peer reviewers in this manuscript have no relevant financial or other relationships to disclose.

Ethical Declaration

The authors declare that they have taken approval from of Institutional Animal Ethics Committee of CDRI and follow the principle declaration of Helsinki of human and animal experimental. According to guidelines of Institutional Animal Ethics Committee of CDRI, India, approval number IAEC/2022/102/Renew-0/Dated-09/09/2022.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17576180.2025.2565142