1.0 INTRODUCTION:

As per WHO, there is a tremendous increase in the number of diabetic patients. Worldwide over and above half a billion people are suffering from diabetes. Between the 15 to 80 age group fall into the prediabetic category i.e., early stage of diabetes, are affected in all population groups including children in all countries. It is also expected that the number is projected to be more than 1.3 billion people in the upcoming 30 years forecasting, with diabetes prevalence at the global level expected to increase. Over the years, multiple discoveries of oral hypoglycemic agents as well as management strategies have made it possible to manage and understand diabetes. Still, the management of diabetes mellitus is challenging due to complex physiology and requires multiple medications¹.

Type 2 diabetes mellitus (T2DM) is the most prevalent metabolic disease and people can develop it even at child age. Customized treatment and targeting factors such as nutrition diet, physical activity, and identifying and characterizing the population accurately based on their demographic factors and risk factors to effectively treat and manage diabetes. Heart attack and stroke risk increased two to three-fold in adults with diabetes along with neuropathy due to reduced blood flow in feet further leading to infection, foot ulcer, and even limb amputation. Diabetic retinopathy is one of the main reasons for blindness, the result of long-term accumulated small blood vessel damage in the retina.

Remogliflozin, an oral antidiabetic drug used for the treatment of progressive diabetes and chemically it is 5-methyl-1-(1-methyl ethyl)-4- ({4-[(1-methyl ethyl)oxy]phenyl}methyl) -1H-pyrazole -3-yl- β-D-glucopyranoside). The structure of remogliflozin is given in Fig. 1. Remogliflozin belongs to sodium-glucose cotransporter 2 inhibitors (SGLT-2 inhibitors) which is the novel approach in the treatment and management of diabetes mellitus^2-4. This category of drugs acts by reducing glucose reabsorption in the kidney which ultimately increases glucose excretion. Reported studies have shown that remogliflozin administration helps in the improvement of clinical glycemic control⁵. Remogliflozin is highly selective and found to be a potent SGLT-2 inhibitor and approved by various regulatory agencies to treat patients with type 2 diabetes mellitus⁶.

(a)

(b)

Fig. 1: Structure of a) Remogliflozin and b) Bixafen

Mostly, UHPLC and liquid chromatography with mass spectrometry detection have been used for the determination of various analytes in biological matrix^7-10. Essential development and validation characteristics for analytical and bioanalytical methods have been discussed with the view to improve the quality of analysis of analytes^11-14. Using rat plasma as a biological matrix for bioanalytical method validation offers advantages such as its availability, cost-effectiveness, and similarity to human plasma making it a suitable surrogate for preclinical studies^15-17. To date for remogliflozin, there are few methods published based on UV visible spectrometry and RP-HPLC^18-20. These methods can detect remogliflozin etabonate down to 1 µg/ml level in bulk and dosage formulations. The HPTLC-densiometric method reported for quantification of remogliflozin in tablet formulation²¹. Assessment of drug interaction risk of remogliflozin etabonate in clinical studies performed²². Accurate and precise estimation of plasma remogliflozin is an effective approach that enables the researcher to establish pharmacodynamics and pharmacokinetic relationships in animal models. So, there is a need for the development of a simple and reliable method that can identify and quantify remogliflozin in biological matrices like plasma. In this study, the UHPLC-MS/MS method has been developed and validated. In this method, bixafen is used as an internal standard that concurrently measures the levels of remogliflozin in rat plasma. The structure of bixafen is given in Fig. 1. This validation was performed as per EMA and USFDA guideline^23,24.

2.0 Materials and methods:

2.1 Chemicals, Reagents and Instrumentation:

Remogliflozin (99.97% purity) was gifted by Glenmark Pharmaceuticals, Nasik, India. Bixafen was purchased from Sigma Aldrich (99.3% purity) and used as an internal standard. All the reagents and chemicals used in the presented research work were of HPLC-grade quality and purchased from Biosolve, India. The LC-MS/MS analysis was performed on an API 4000 Triple Quad Mass Spectrometer coupled with Nexera X2 UHPLC system. X select CSH fluorophenyl (150 mm x 4.6mm, 3.5µm) column was employed and the column temperature was maintained at 40°C. 0.1% formic acid in milli-Q water (20%) and acetonitrile (80%), v/v was used as a mobile phase for isocratic elution method. The autosampler was maintained at 15°C and the injection volume was 10μL. The flow rate was maintained at 0.7 mL/min. The run time was 4 min for each injection. The separated compounds were detected by a CEM (channel electron multiplier) detector. Quantification was achieved by multiple reaction monitoring (MRM) modes with transitions of m/z 451.4→289.4 and m/z 451.4→111.2 for remogliflozin and m/z 414.0→394.0 for bixafen. Data acquisition and control of instruments were done by Analyst software 1.6.3.

2.2 Preparation of Calibration Curve Standards and Quality Control Samples:

Acetonitrile was used as a solvent to prepare stock solution of approx., 1 mg/mL of remogliflozin and bixafen, separately. A stock solution of remogliflozin was diluted with mobile phase to prepare a standard working solution. To prepare the calibration curve standard, 5 μL of the working solution was spiked into 45 μL of blank rat plasma. The range of the concentrations in the calibration curve was around 15-2009 ng/mL. Internal standard (IS) stock solution was diluted with mobile phase to prepare the working solution and like the calibration curve standard, spiking of IS was performed at 5% in the rat plasma.

2.3 Sample Extraction Procedure:

Samples were prepared by the protein precipitation method. Concisely 5μL of the internal standard working solution was added to 45μL of the plasma in the Eppendorf tube. This mixture was precipitated by 1.5 mL of mobile phase and the sample was vortexed for 10 min at 2000rpm. This was followed by centrifugation at 14000 rpm at 4±1°C for 10 min to obtain supernatant and 10μL of supernatant was injected for LC-MS/MS for analysis.

2.4 Method Validation:

The analytical method for the determination of remogliflozin was validated as per US FDA and EMA guidelines^12,13. Parameters covered during validation are specificity, selectivity, autosampler carryover test (ASCOT), lower limit of quantification (LLOQ) determination, linear dynamic curve, precision and accuracy, matrix effect, recovery, and stability.

2.4.1 Specificity and Selectivity:

For specificity, six different lots of blank rat plasma were fortified with remogliflozin (upper limit of quantification (ULOQ)) and internal standard (working internal standard (WIS)) separately and processed and analyzed along with selectivity. The interference at analyte retention time was monitored in the presence of samples of internal standard and vice versa. For selectivity six blank rat plasma from the different lots were processed and analyzed along with six blank lots of plasma with analyte (LLOQ level) and internal standard to determine the extent of endogenous interferences from plasma components contributing to the chromatographic area in addition to the analyte or internal standard responses^12,13.

2.4.2 Autosampler Carryover Test:

Standard blank, LLOQ, and ULOQ standard were processed and samples were injected in sequence as standard blank, LLOQ, ULOQ, and again standard blank (reinjection of the first sample).

Standard blank reinjection of the first sample was performed to observe if any carryover was present at the retention time of the analyte and internal standard. The second injection in the sequence (LLOQ standard) was injected to check whether any interference at a retention time of remogliflozin (analyte) in the standard blank and if any interference then it should be less than 20% of the LLOQ standard response and less than 5% of IS response.

2.4.3 Linearity:

Calibration standard curves were prepared using eight calibration standards which included ULOQ and LLOQ. The range of the calibration standard solution was 15- 2009 ng/mL. The curve was constructed by plotting the peak area ratio of the remogliflozin to the internal standard (y) Vs nominal concentrations of remogliflozin. The linearity was prepared using a weighted (1/X²) least square regression.

2.4.4 LLOQ Determination:

To evaluate the method sensitivity, LLOQ determination was performed. LLOQ samples were processed by spiking the analyte remogliflozin into six individual lots of rat plasma at LLOQ concentration with a working internal standard solution. These samples were analyzed with precision and accuracy and under a calibration curve along with batch quality control (QC) samples. Three batches of LLOQ determination were evaluated.

2.4.5 Precision and Accuracy:

The precision and accuracy batch comprised six replicates of QC’s (LOQQC, LQC, MQC, and HQC) representing the entire standard curve range with concentrations at LOQQC was slightly higher than LLOQ concentration, LQC was approximately 3 times the LLOQ concentration, MQC was at approximately 40-60% of ULOQ and HQC was at approximately 75-85 % of ULOQ. The samples were processed and analyzed under freshly prepared calibration standards. A total of three precision and accuracy batches were performed.

2.4.6 Reinjection Reproducibility:

Reinjection reproducibility was evaluated to establish the validity of the processed samples and to support sample storage before injection. The samples of accepted precision and accuracy batch were stored for a period of 18 h in an autosampler at 2 to 8°C and the entire batch was re-injected for analysis.

2.4.7 Matrix effect:

The matrix effect for the remogliflozin was investigated at LQC and HQC concentrations. Six different lots of blank rat plasma were extracted, and the extract was spiked at LQC and HQC separately. Analyte peak area in the samples was compared with neat standard solutions at the same theoretical concentration (LQC and HQC). IS normalized factor and matrix factor were calculated.

2.4.8 Recovery:

Recovery of remogliflozin was evaluated by analyzing 6 replicates of extracted LQC, MQC, and HQC samples and comparing the same with mean analytes response from post-extracted samples at LQC, MQC, and HQC along with internal standard at its working concentrations. The samples were prepared in rat plasma.

2.4.9 Stability:

The stability of the analyte in rat plasma was established at various storage conditions viz., room temperature (bench top stability), under autosampler condition (autosampler stability), freeze-thaw condition (freeze-thaw stability), and long-term stability for 19 days. All stability determination was performed by using freshly prepared standards of the calibration curve and batch QCs.

3.0 Results and discussion:

3.1 Method Design and Optimization:

In this study, a simple, reliable LC-MS/MS method was developed for the detection of remogliflozin in rat plasma. Both APCI (atmospheric pressure chemical ionization) and ESI (electrospray ionization) techniques were tried but good ionization and sensitivity were obtained with APCI. Bixafen was used as an internal standard. The selection of bixafen as an internal standard is based on the presence of pyrazole linkage and moiety in bixafen and remogliflozin and to minimize the cost involved in the usage of deuterated compounds. The main objective of the usage of the internal standard is to reduce the error (i.e., experimental and processing) and to ensure the ruggedness and accuracy of the developed method.

Method optimization was performed by selecting various bonded stationary phases like C18 and C8, and by changing the composition of the mobile phase for enhancement of sensitivity detection and to improve the shape of the peak. Amongst methanol, acetonitrile, and other available organic solvents, acetonitrile was selected as an organic solvent as it provided lower background noise and high analytical response as compared to methanol. After numerous attempts, 0.1% formic acid in milli-Q water (20): acetonitrile (80) was selected as a mobile phase and X select CSH fluorophenyl (150mm x 4.6mm, 3.5µm) column as a stationary phase for this bioanalytical method.

Liquid-liquid extraction (LLE), solid phase extraction (SPE), and protein precipitation (PPT) are the most commonly used techniques for the extraction of analyte from biological matrix. Protein precipitation is the most commonly used sample preparation method because of its ability to remove unwanted plasma proteins from samples before analysis with minimal method development requirements and low cost. A simple and rapid PPT technique was employed in this experiment. Protein precipitation was performed by using 0.1% formic acid in milli-Q water (20): acetonitrile (80) which is a mobile phase of the analytical method. The selection of the mobile phase as a precipitating agent makes the method simple and effective and offers a high recovery for remogliflozin and bixafen.

3.2 Method Validation:

3.2.1 Specificity and Selectivity:

The method is considered as specific as no interference was observed when analyte remogliflozin was monitored for cross contamination at the retention time of the analyte. Typical retention time for remogliflozin and internal standard were 2.60 and 2.70min, respectively (Fig. 2). The detection of remogliflozin and internal standard by MRM mode was highly selective with no interference from the endogenous substances. LLOQ concentration was found to be within 20% of nominal concentration.

Fig. 2: a) Optimized MS Chromatogram of Remogliflozin and Bixafen extracted from spiked rat plasma at LQC. b) Chromatogram for Calibration Standard Solution

3.2.2 Autosampler Carryover Test:

The results obtained from the autosampler carryover test is depicted in Table 1. No interference or carryover was observed in standard blank and reinjected standard blank at the retention time of remogliflozin and internal standard.

Table 1: Autosampler Carryover Test

Standard Blank	Analyte Peak Area	% Carryover (Analyte)	IS Peak Area	% Carryover (IS)
Standard Blank	0	0	0	0
LLOQ	7620	Nil	318345	Nil
ULOQ	973209	Nil	331584	Nil
Standard Blank (Reinjection)	0	0	0	0

3.2.3 Linearity:

Linearity of the method was carried out by injecting eight concentrations of remogliflozin prepared in the plasma matrix in the range 15 to 2009 ng/mL into the UHPLC-MS/MS system. All samples were analyzed using optimized LC-MS/MS conditions and data of the remogliflozin presented in Table 2. Linearity chromatogram and curves are presented in Fig. 2 (b) and Fig. 3, respectively. At all the selected concentration levels of the calibration curves, back calculated amounts of the calibration standards were always less than ±15% of nominal values.

Table 2: Linearity data of Remogliflozin

Nominal Concentrations (ng/mL)	Recovered Concentrations (ng/mL)	Accuracy (%)	Regression Equation
15	15.1	96.3	Y=0.00144x + 0.00153 (r²=0.997)
31	31.6	100.7
62	59.7	95.1
125	119.6	95.3
251	273.8	109.0
502	518.5	103.2
1004	972.6	96.8
2009	2053.4	102.2

3.2.4 LLOQ Determination:

The LLOQ for remogliflozin in rat plasma was 18.2 ng/mL. S/N ration was found to be greater than 5, recovered concentration of the samples was found within ± 20% of nominal concentrations and percentage of coefficient of variance (% CV) of the samples were found to be less than 20%.

3.2.5 Precision and Accuracy:

Results of the precision and accuracy of remogliflozin are summarized in Table 3. The data is listed for each day individually. The results indicate % CV were below 5.75. The accuracy ranged between 96.3 and 109.5%, which is acceptable. The results illustrated that the developed UHPLC-MS/MS method is precise and accurate.

Fig: 3 UHPLC-MS/MS Calibration curve for Remogliflozin

3.2.6 Reinjection Reproducibility:

The reinjection reproducibility was established for 18 h with the accuracy ranging between 96.2 and 108.2% and the precision value is found below 3.96.

3.2.7 Matrix effect:

Obtained matrix effects indicated that there were no significant matrix effects observed for the analyte as % CV of the IS normalized factor is below 7.84.

3.5.8 Recovery:

As presented in Table 4, the extraction recovery was in the range of 92.7 to 98.5%, which meets the acceptance criteria. % CV of the recoveries at each level were found to be less than 15%.

Table 3: Precision and Accuracy

	Recovery Level	Fortified Concentrations (ng/mL)	Recovered Concentrations (ng/mL)	% Accuracy	% CV
Day 1	LLOQ	18.2	18.6	102.1	4.14
	LQC	49.2	53.9	109.5	2.54
	MQC	1004.7	1076.1	107.1	2.23
	HQC	1607.5	1642.5	102.2	2.93
Day 2	LLOQ	18.2	18.4	100.8	4.47
	LQC	49.2	52.8	107.2	2.25
	MQC	1004.7	1050.6	104.6	2.93
	HQC	1607.5	1567.9	97.5	2.61
Day 3	LLOQ	18.2	17.6	96.3	5.75
	LQC	49.2	53.3	108.2	2.08
	MQC	1004.7	1042.9	103.8	4.41
	HQC	1607.5	1546.4	96.2	2.43

Table 4: Results of % recovery

Recovery Level	Concentrations (ng/mL)	% Recovery
LQC	49.2	98.5
MQC	1004.7	92.7
HQC	1607.5	95.3

3.5.9 Stability:

Results indicated that remogliflozin had no effect in the auto-sample (12h) at 7⁰C, at room temperature for 6h, repeated five freeze-thaw cycle and under frozen conditions at -70⁰C for 19 days. The data of stability were summarized in Table 5.

4.0 Conclusion:

In this study, new, specific and sensitive method developed and validated method for quantification of remogliflozin in the rat plasma. The developed LC-MS/MS method was found to have high extraction recovery with no matrix effect at the end of the precipitation with mobile phase. Moreover, this method can be used to establish pharmacokinetics of remogliflozin in the rat after oral administration.

5.0 ABBREVIATIONS:

LLOQ=Lower limit of Quantification

WHO=World Health Organization

IS=Internal Standard

MRM= Multiple Reaction Monitoring

CEM=Channel Electron Multiplier

WIS=Working Internal Standard

ULOQ=Upper Limit of Quantification

LQC=Low Quality Control

QC=Quality Control

MQC=Middle Quality Control

HQC=High Quality Control

LOQQC=Lower Limit of Quantification Quality Control

EMA=European Medical Agency

6.0 ACKNOWLEDGEMENT:

The authors gratefully acknowledge Jai Research Foundation, India for providing facility and necessary guidance to do work.

7.0 Conflicts of interest:

The authors declare no conflicts of interest.

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Received on 17.12.2023 Modified on 11.03.2024

Research J. Pharm. and Tech 2024; 17(10):5016-5022.

DOI: 10.52711/0974-360X.2024.00771

Condition	Recovery Level	Fortified Concentrations (ng/mL)	Recovered Concentrations (ng/mL)	% Accuracy	% CV
Room Temperature (6 h)	LQC	49.2	52.3	106.3	1.03
Room Temperature (6 h)	HQC	1607.5	1652.5	102.8	1.00
Autosampler (12 h)	LQC	49.2	50.6	102.9	1.20
Autosampler (12 h)	HQC	1607.5	1579.9	98.3	1.00
Freeze-thaw (5 cycle)	LQC	49.2	50.8	103.2	1.52
Freeze-thaw (5 cycle)	HQC	1607.5	1592.9	99.1	1.10
Frozen 70 ⁰C (19 days)	LQC	49.2	51.2	104.0	2.15
Frozen 70 ⁰C (19 days)	HQC	1607.5	1568.5	97.8	1.25