INTRODUCTION:

MK-8835, another name for ertugliflozin 5, is chemically (1S,2R,3S,4R,5S)-5- {4-chloro-3- [(4-ethoxy phenyl) methyl] phenyl}-1-(hydroxymethyl) octane-2,3,4-triol [3.2.1]-6,8-dioxabicyclo. Ertugliflozin was originally approved globally in the USA on December 19, 2017, as a dietary and exercise supplement to help adults with type-II diabetes improve their glycaemic control. It received a favourable opinion from the EU Committee for Medicinal Products for Human Use (CHMP) in January 2018 and is presently pending EU clearance. To enhance glycaemic control in adults with type-II diabetes, Pfizer, Merck Sharp, and Dohme Corp. produce and sell it under the names Steglatro, Segluromet, and Steglujan, respectively.

Ertugliflozin has a T-max of 1.0hours and is quickly absorbed. It is well absorbed by humans and is primarily eliminated by glucuronidation. A slight increase in LDL and HDL cholesterol levels has been reported, but it also lowers triglyceride levels. It is well tolerated, and most side effects are mild to moderate in intensity. Because vaginal mycotic and urinary tract infections are the most prevalent side effects, ertugliflozin medication is not recommended for patients with moderate renal impairment, renal disorders, or dialysis patients¹.

An inhibitor of the sodium glucose co-transporter (SGLT2) is ertugliflozin. By increasing urine glucose excretion and decreasing renal glucose reabsorption, SGLT2 inhibitors reduce blood glucose levels during fasting and after meals. It is a white powder that dissolves in acetone and ethanol but very slightly dissolves in water, acetonitrile, and ethyl acetate. It is available commercially as a stand-alone drug or in combination with sitagliptin and metformin hydrochloride.²

The recommended initial dosage is 5 mg of ertugliflozin given once daily in the morning, with or without meals. If greater glycaemic control is needed and tolerated, the 5 mg dose can be increased to a maximum of 15 mg once daily³. In addition to diet and exercise, ertugliflozin and metformin (SeglurometTM) and ertugliflozin and sitagliptin (SteglujanTM)⁵ have been approved as fixed-dose combinations (FDCs) for the treatment of type 2 diabetes in the United States^3,
4.

CHEMISTRY:

Chemical Structure of Ertugliflozin

· Category: SGLT2 Inhibitor

· Molecular Formula: C23H28Cl2O6S

· Molecular Weight: 500.44 g/mol

· Melting Point: 160-162°C

· Solubility: Slightly soluble in water; soluble in organic solvents like DMSO, methanol, and ethanol

· Appearance (Ertugliflozin L-pyroglutamic acid): White, odorless, and slightly amorphous powder.

MATERIALS AND METHODS:

Table No. 1. List of Reagents Used

Sr. No.	Chemicals/ Reagents/ Solvents	Supplier	Grade
1	Methanol	Merck	HPLC grade
2	Acetonitrile	Merck	HPLC grade
3	Water	Siddhi Lab	HPLC grade

HPLC method development:

Preparation of Standard Solution:

In order to prepare stock solution, weighed accurately 25.91mg Ertugliflozin L-pyroglutamic acid (Equivalent to 20mg of Ertugliflozin) and transferred into 20ml volumetric flask, added 15ml of methanol and sonicated to dissolve the standard completely and diluted up to the mark with methanol (1000PPM). Further diluted 0.5mL to 25mL with methanol. (20PPM)

Preparation of standard stock solution for Chromatographic development:

Ertugliflozin Standard stock solution was prepared by dissolving 25.91mg Ertugliflozin L-pyroglutamic acid (Equivalent to 20mg of Ertugliflozin) into a 20mL clean and dried volumetric flask added about 15mL of methanol to dissolve it completely and made volume up to the mark with methanol (1000PPM). Further diluted 1 ml of stock solution to 10mL with mobile phase (100 PPM).

Preparation of System suitability test (Ertugliflozin standard solution):

Weighed about 32.38mg of Ertugliflozin L-pyroglutamic acid (Equivalent to 25mg of Ertugliflozin) and transferred in 50mL volumetric flask, added 35mL of methanol, sonicate to dissolve it, made volume up to the mark with methanol. Pipette out 0.5ml from standard stock solution and transferred into 25ml volumetric flask and made volume up to the mark with mobile phase (Approx 10µ/mL= working concentration), chromatograms were recorded.

System suitability is a Pharmacopoeial requirement and is used to verify, whether the chromatographic system is adequate for analysis to be done. The tests were performed by collecting data from Five replicate injection of standard drug solution and the results are recorded.

Analysis of marketed Test sample:

Marketed tablet formulation was not available, hence physically lab mixture of tablet prepared at lab level and used for analysis and for doing validation.

Tablet Preparation:

Physical lab mixture of Tablet prepared at lab level by following formula:

150mg as average weight considered for preparing tablet mixture.

Sr. No.	Ingredients	Role	Qty (mg)
1	Ertugliflozin L-pyroglutamic acid API	Active ingredient	19.43
2	Placebo	NA	130.57
Total			150.00

Table no. 2. Placebo prepared at lab level by using formula as follows:

Sr. No.	Ingredients	Role	Qty (mg)
1	Lactose	Filler	80
2	Starch	Binder	5
3	Magnesium stearate	Lubricant	5
4	Talc	Glidant	5
5	crospovidone	Disintegrants	5
Total			100 mg

Total 10 gm of placebo prepared.

Sample preparation of Marketed test sample:

Weighed the powder material equivalent to 20mg of Ertugliflozin (200.0mg of powder material) from physical lab mixture. Transferred to clean and dried 50 mL of volumetric flask. Added 35mL of methanol, sonicated for 10minutes with intermittent shaking. After 10minutes allow to cool the solution to room temperature and made volume up to the mark with methanol. Filtered the solution through suitable 0.45µ syringe filter discarding 3-5mL of initial filtrate. Further diluted 0.5ml of filtered stock solution to 20ml with mobile phase. (10mcg of Ertugliflozin), injected the resultant solution and chromatograms were recorded and results are recorded.

Table no.3. Sample Prepared in duplicate. Summary of sample preparation as follows:

Sample	Sample (mg)	Diluted to (mL)	Volume taken	Diluted to (mL)
Sample 1	200.9	50	0.5	20
Sample 2	200.3	50	0.5	20

Formula for % Assay calculation:

VALIDATION OF RP-HPLC METHOD:

1) Filtration Study:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample.

This study was conducted with Ertugliflozin Test sample (Physical lab mixture).

Filtration study carried out with unfiltered and filtered test solution. During filtration activity 0.45µm PVDF and 0.45µm Nylon syringe filters used by discarding 5 mL of aliquot sample.

2) Stability of Analytical Solution:

Stability study was conducted for standard and test sample solution. Stability study was performed at normal laboratory conditions.

The solution was stored at normal illuminated laboratory conditions and analyzed after 12hours and 24hours.

Standard and Test solution stability study was performed by calculating the difference between results of test solution at each stability time point to that of initial.

3) Specificity:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present.

Following solution shall be prepared and injected to prove the specificity nature of the method.

1. Blank (Mobile phase as a diluent)

2. Placebo

Placebo Sample solution preparation:

Weighed 174.09mg of placebo material (Which is equivalent to 20mg of Ertugliflozin) and transferred to clean and dried 50mL of volumetric flask. Added 35mL of methanol, sonicate for 10minutes with intermittent shaking. After 10minutes allow to cool the solution to room temperature and made volume up to the mark with methanol. Filtered the solution through suitable 0.45µ syringe filter discarding 3-5mL of initial filtrate. Further dilute 0.5ml of filtered stock solution to 20ml with mobile phase, injected the resultant solution and chromatograms were recorded.

4) LINEARITY AND RANGE

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Linearity Ertugliflozin stock solution:

Weighed 32.38mg of Ertugliflozin L-pyroglutamic acid (Equivalent to 25mg of Ertugliflozin) and dissolved in 50mL with methanol. Further diluted 5.0ml of stock solution to 50mL with Methanol. (50PPM).

5) Limit of Detection (Lod) And Limit of Quantitation (Loq):

Detection limit:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

Quantitation limit:

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

As per ICH Q2R1 guidelines LOD and LOQ was determined by using the approach Based on the Calibration Curve in which residual standard deviation of a regression line was calculated and determined the LOD and LOQ by using following formula:

LOD = 3.3 σ / S

LOQ = 10 σ / S

Where,

σ = residual standard deviation of a regression line

S = Slope of regression line

6) Accuracy (% Recovery):

The accuracy of the analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value of the value found, Accuracy will be conducted in the range from 50 % to 150% of working concentration. Solution of each accuracy level was prepared in triplicate. Calculated % Recovery for each sample, Mean % recovery for each level and overall recovery and also calculated % RSD for each level and %RSD for overall recovery.

7) Precision:

Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous test under the prescribed conditions. Precision is of two types, Repeatability and Intermediate precision. It is performed on tablet test sample.

8) Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

RESULT AND DISCUSSION:

Selection of analytical wavelength:

Fig. No. 1 UV spectrum of Methanol as a blank

Optimized Chromatographic Conditions:

Table no 4. Optimized Chromatographic Conditions

Parameter	Description
Mode	Isocratic
Column Name	Phenomenox C18, 250 mm X 4.6 mm, 5 µm
Detector	UV Detector
Injection Volume	20µl
Wavelength	223nm
Column Oven temp	35ºC
Mobile Phase	Acetonitrile: Water (70:30%V/V)
Flow Rate	1.0ml/min
Run time	08Minutes

Fig. No. 2 Typical chromatogram of Trial 3

VALIDATION OF RP-HPLC METHOD:

1) Filtration Study:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. Performed on tablet test sample.

Table no.5.Results of Filter study

Sample description	Area	% Absolute difference
Unfiltered	15216923	NA
0.45 µ PVDF filter	15146100	0.47
0.45 µ Nylon filter	15059172	1.04

2) Solution Stability:

Stability study was conducted for Standard as well as Test Sample. Stability study was performed at normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analyzed at initial, after 12 hours and 24 hours.

Table no.6. Results of Solution stability.

Sample solution
Time point	Area	% Absolute difference
Initial	15196323	NA
12 Hours	15109463	0.57
24 Hours	15059041	0.90
Standard solution
Time point	Area	% Absolute difference
Initial	15439721	NA
12 Hours	15364020	0.49
24 Hours	15324976	0.74

3) Specificity:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present. Blank, standard solution prepared and injected to check peak purity.

Table no.7. Results of Specificity.

Description	Observation
Blank	No interference at R.T. of Ertugliflozin due to blank
Placebo	No interference at R.T. of Ertugliflozin due to placebo

4) Linearity and Range:

Linearity of an analytical method is its ability to elicit test results that are proportional to the concentration of analyte in samples within a given range.

Fig. No. 3 Calibration curve of Ertugliflozin

Table No.8. Data of linearity of Ertugliflozin:

Sr No	Parameter	Result value	Acceptance criteria
1	Beer's linearity range	1.0-15.0µg/mL	NA
2	Correlation coefficient (R²)	0.99997	NLT 0.98
3	Intercept	-36696.784	To be report
4	Slope	1544105.561	To be report
5	% RSD for area at each level	NA	NMT 2.0

The respective linear equation for Ertugliflozin was

Y = M X + C

Y = 1544105.561X + -36696.784

Where,

X = concentration of Analyte in µg/mL

Y = is area of peak.

M = Slope

C = Intercept

Conclusion:

From the calibration curve it was concluded that the Ertugliflozin shows linear response in the range of 1.0-15.0μg/ml. The Regression value was found well within the limit.

5) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

σ = 70214.775 (Residual standard deviation of a regression line)

s = 1544105.561 (Slope)

Detection limit (LOD):

LOD = 3.3 σ / S

LOD = 3.3 x 70214.775 / 1544105.561

LOD = 0.150µg/mL

Quantitation limit (LOQ):

LOQ = 10 σ / S

LOQ = 10 x 70214.775 / 1544105.561

LOQ = 0.455 µg/Ml

6) Accuracy (Recovery):

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method is determined by applying the method to analyzed samples to which known amounts of analyte have been added.

Table no.9.Result and statistical data of Accuracy of Ertugliflozin

Level (%)	Area	Recovered conc (µg/mL)	Added conc (µg/mL)	% Recovery	Mean Recovery	% RSD
50	7760258	5.04	5.10	98.82	99.54	0.802
	7819174	5.08	5.06	100.40
	7803682	5.07	5.10	99.41
100	15568075	10.11	10.15	99.61	100.00	0.957
	15402140	10.00	10.07	99.30
	15737562	10.22	10.11	101.09
150	23090752	15.00	15.09	99.40	99.29	0.508
	22935805	14.90	15.09	98.74
	23064234	14.98	15.02	99.73

Overall Recovery: 99.61 %

% RSD for Overall Recovery: 0.744

Table no.10. Result of Intra- day and Inter- Day Precision for Ertugliflozin test sample assay

Sr. No	Parameters	Intraday Precision	Interday Precision
1.	Mean	98.56	99.13
2.	STD	1.564422	1.567743
3.	% RSD	1.587	1.582

Table no.11. Result of Robustness study of Ertugliflozin

Change in Parameter	R.T.	Standard area	Asymmetry	Theoretical plates
Wavelength by +3 NM (226 NM)	3.78	14892036	1.25	7719
Wavelength by -3 NM (220 NM)	3.77	15169253	1.27	7870
Flow rate by +10% (1.1mL/min)	3.43	13964802	1.26	7086
Flow rate by -10% (0.9mL/min)	4.19	18622536	1.31	6824
Column oven temp by +2ºC (37 ºC)	3.77	15169206	1.25	7614
Column oven temp by -2ºC (33 ºC)	3.77	15215517	1.27	7796