INTRODUCTION:

Empagliflozin (EMPA) is an orally administered antidiabetic drug in patients with type II diabetes, used alone or with other medications such as Linagliptin or Metformin. It is suitable for patients with type II diabetes and cardiac problems as it reduces the risk of heart attack, stroke, or death¹.

It blocks glucose reabsorption in the kidney as it is one of the sodium-glucose co-transporter 2 (SGLT2) inhibitors The IUPAC name of EMPA is 2S,3R,4R,5S,6R)-2-[4-chloro-3-({4-[(3S)-oxolan-3-yloxy]phenyl}methyl)phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol².

Linagliptin is another antidiabetic drug for type II diabetes used either alone or with other medications. It is a reversible dipeptidyl peptidase (DPP)-4 enzyme inhibitor. The IUPAC name of Linagliptin (LINA) is (R)-8-(3-aminopiperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methylquinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione.³ The chemical structures of EMPA and LINA are given in figure 1. EMPA and LINA are available as fixed-dose combinations with two different strengths for empagliflozin (5:10 and 5:25 of LINA and EMPA respectively with the ratios in milligrams)

(a) (b)

Figure 1. Chemical structures of EMPA (a) and LINA (b)

An exhaustive survey on literature has shown the presence of some analytical methods for Empagliflozin and Linagliptin either alone or in fixed dose combinations. Empagliflozin is analysed by UV spectrophotometry,^4,5 and LC methods^6-8 and Linagliptin is studied by UV spectrophotometry,⁹ and LC methods¹⁰. The fixed-dose combination of Empagliflozin and Linagliptin is studied by UV spectrophotometry^11,12LC^13–16 and HPTLC methods.¹⁷Metformin is analysed with Empagliflozin by UV spectrophotometry,¹⁸and with Linagliptin by UV method.¹⁹The fixed-dose combination of Empagliflozin Linagliptin and Metformin is studied by LC method.²⁰Since the UV spectrophotometric methods are minor, we have developed two new strategies based on chemometrics for the simultaneous estimation of the fixed-dose combination.

Two Chemometrics methods are developed; namely, the difference between the adjacent data point (DBADP) method and the ratio mean centering (RMC) method. In chemometrics methods, helpful information can be extracted using statistical or mathematical tools to facilitate the QC analysis.

Afkhami and Bahram^21,22 reported analytical works using the mean centering method for the simultaneous analysis of binary and ternary samples. The analytical parameters obtained by the mean centering strategy are much better than the derivative method. The data points collected as the differences between adjacent values in the ratio spectra are used for the DBADP method.²³

Compared to other analytical methods, spectrophotometric methods are preferable due to cost consideration and ease of handling of the instrument. As binary or ternary mixtures cannot be analysed by simple spectrophotometric methods, chemometrics methods are a good option. In the developed methods, the use of an organic solvent is maximum avoided, and hence the methods are termed the green method.

MATERIALS AND METHODS:

Methods:

Instrumentation:

A Double beam UV-Visible spectrophotometer that functions with UV probe software was used for primary data acquisition, and Python and MATPLOTLIB were used to write the software program for data processing.

Materials:

Reference standards and marketed formulation:

Gratis samples of Empagliflozin and Linagliptin were obtained from Hetero drugs Ltd, Hyderabad, India. Commercially available tablets (Glyxambi) containing Empagliflozin (25/10mg) and Linagliptin (5mg) manufactured by Hetero drugs Ltd. were procured from a local pharmacy.

Chemicals:

Analytical reagent grade Urea (sd fine-Chem Limited Mumbai, India) was used without further purification for solvent preparation.

Analytical method development and validation:

For both methods, EMPA and LINA solutions were scanned in the UV range of 220nm to 370nm at a wavelength interval of 1nm using 0.1M urea as the blank solution at a medium scan speed.

The Ratio Mean Centering Method (RMC):

For RMC, the zero-order spectrum of each drug are divided by the zero-order spectrum of the suitable drug, and the concentration of the second drug, and the resulting ratio spectra were subjected to the mean centering process. A calibration graph for each drug was prepared with the amplitude values obtained for the RMC method concerning the wavelength.

Difference Between Adjacent Data Point Method (DBADP):

The calibration graph for EMPA was obtained from the ratio spectra for which the zero-order spectra of standard solutions with varying concentrations of EMPA were collected and were divided by the zero-order spectrum of LINA of a specific concentration. Then, the difference between the adjacent data points of the ratio values concerning wavelength was obtained. R’afat Mahmoud N EJEM et al¹³ explained the mathematics of the method using a triple combination. The minimum or maximum amplitude values obtained in the DBADP method was plotted against the concentration to get the calibration graph of each drug. The zero-order spectrum of the mixture was divided by the zero-order spectrum of the standard solution of LINA and EMPA, respectively, to estimate the concentration of EMPA and LINA in the marketed formulations.

Method validation:

We extended the method validation study for accuracy, precision, linearity, LOD, and LOQ according to ICH guidelines.²⁴

Linearity:

Ten mg of each drug was separately dissolved in 1ml of methanol and then diluted up to 10mL using 0.1M urea solution for the stock solutions of 1000µg/mL of EMPA and LINA. Aliquots were diluted with 0.1M urea to get end concentrations in the range of 2-10µg/mL of EMPA and LINA.

The method linearity is the capability to obtain test results directly proportional to the analyte concentration in the sample. We prepared the calibration graphs for EMPA and LINA with five calibrators (2-10µg/mL for both drugs). The data of amplitude versus concentration were obtained by ratio mean centering and the difference between adjacent data points methods by a simple software program written using matplotlib. Empagliflozin and Linagliptin were showing the linear relationship between concentration and amplitude. Both the drugs were linear in the range of 2-10µg/mL, and the linear regression analysis, showed the coefficient of determination, R² for both the drugs, as 0.999.

Accuracy:

The standard addition process did the accuracy, assessing the degree of closeness between the actual and the value found. Standards were spiked at three different levels (80%, 100% and 120%) to commercially available tablets in triplicate. The method's accuracy and reproducibility are proved by calculating the amount of drug recovered and the values of % relative standard deviation (RSD) that should be <2.0 by both methods.

Limit of detection and limit of quantitation:

We used samples containing low concentrations of the analyte to determine the detection limit (LOD) and quantitation limit(LOQ). Formulae 1 and 2 are used for calculating LOD and LOQ.

LOD = 3.3 σ/S Formula 1

LOQ = 10 σ/S Formula 2

S = slope of the calibration curve and σ = standard deviation of the response

Precision:

The precision assessment is a measure of the closeness of test results upon multiple sampling of the homogenous sample. The method repeatability (intra-day precision) was determined by the triplicate analysis of three standard solutions of EMPA and LINA at 2, 6 and 10µg/mL of concentrations for both the drugs. Inter-day (n=3) analysis of three standard solutions of EMPA and LINA at the concentration of 2, 6 and 10µg/mL for both the drugs was used for Intermediate precision. The statistical analysis on precision data proved good precision of the methods as the % RSD was <2.0 for both drugs in interday and intraday precision studies.

Analysis of the pharmaceutical dosage form:

The marketed fixed-dose formulation of EMPA and LINA, Glyxambi tablets are available in two strengths: Empagliflozin (10mg) and Linagliptin (5mg) and Empagliflozin (25mg) and Linagliptin (5mg). Twenty Glyxambi tablets of each strength were powdered. A quantity equivalent to 10mg of EMPA was transferred to a 10ml volumetric flask, added 1mL of methanol and dissolved and further diluted with 0.1N urea with the help of sonication for 15 min. The solutions were filtered. An aliquot was diluted to get a sample solution of 10µg/ml of EMPA and 5µg/ml of LINA for the tablets with a fixed-dose combination of Empagliflozin (10mg) and Linagliptin (5mg). For the tablets with higher strength of Empagliflozin (25mg) and Linagliptin (5 mg), dilution was done to get a sample solution of 10 µg/ml of EMPA and 2µg/ml of LINA. For the preparation of ratio spectra, the formulation's spectra were divided by the spectra of each drug one by one. The amplitude values corresponding to 260 and 300nm were considered for the ratio mean centering method. For the DBADP method, the amplitudes were measured at 290 and 280nm for EMPA and LINA, respectively, to assess drug content. The substitution of the amplitudes into the straight-line equation yields the content of EMPA and LINA by both methods.

RESULT:

Method optimization:

The method depends on the spectral manipulation of the ratio spectra of each drug for which the selection of divisor concentration was critical in getting minimum or maximum signal in the RMC graph or DBADP graph of each drug. Hence, for EMPA, the divisor was selected as 6µg/mL of LINA; and for LINA, it was 10µg/mL of EMPA. Δλ was chosen as 1.0nm and did not affect the signal.

Method validation:

Calibration plot for EMPA and LINA:

Linearity data have shown a linear relationship between concentration (µg/ml) and the corresponding amplitudes for EMPA and LINA by RMC and DBADP methods in the range of 2-10µg/ml. By linear regression analysis, coefficient of determination, R² for EMPA and LINA were calculated by both methods. Figure 2 represents the RMC spectra and the DBADP spectra for EMPA, which indicated the linearity between the signals and the drug concentration. Similarly, figure 3 indicated the linear relationship between the drug concentration and the corresponding signals for LINA. The spectral characteristics for EMPA and LINA, linearity range and regression equation by the developed methods are depicted in Table 1. For the ratio mean centering method, the amplitude values corresponding to 260 and 300nm were measured for the calibration graph for EMPA and LINA, respectively. For the DBADP method, the amplitudes were measured at 290 and 280nm for EMPA and LINA, respectively.

Table 1. Spectral characteristics for EMPA and LINA by RMC and DBADP methods

Method	Drug	λ (nm)	Linearity range (µg/mL)	Regression equation, R²
RMC	EMPA	260	2-10	y = 0.847x-0.539,0.9991
RMC	LINA	300	2-10	y = 1.113x+0.227,0.9992
DBADP	EMPA	290	2-10	y = 0.851x-0.551,0.9992
DBADP	LINA	280	2-10	y = 0.755x+0.214,0.9991

Figure 2. Overlay ratio spectra of EMPA (2-10 µg/mL) by RMC (top) and DBADP methods (bottom)

Figure 3. Overlay ratio spectra of LINA (2-10 µg/ml) by RMC (top) and DBADP methods (bottom)

Accuracy:

The drugs' percentage recoveries were computed from the corresponding regression equations obtained in the linearity studies in the RMC and DBADP methods. Drug concentrations of EMPA and LINA present in the spiked samples were calculated by the RMC method and tabulated in table 2. The % recoveries of EMPA and LINA were obtained in the range of 99.59 – 100.69 and 100.22 - 102.26, respectively, by the RMC method. The percentage recoveries of the drugs by the DBADP method were calculated and tabulated in table 3. The % recoveries of EMPA and LINA were obtained in the range of 99.21 – 99.88 and 99.72 - 102.51, respectively, by the DBADP method. The %RSD values were found to be <2.0 in both ways.

Table 2. Recovery data by RMC method

Spiking Level (%)	Drug	Conc. of Std (µg/mL)	Conc. of sample (µg/mL)	Actual con. (µg/ml)	*Con. recovered (µg/ml) AM±SD (n=3)**	Recovery (%)	% RSD
80	EMPA	4	3.2	7.2	7.25 ± 0.15	100.69	0.45
80	LINA	2	1.6	3.6	3.61 ± 0.06	100.27	1.39
100	EMPA	4	4	8	7.99 ± 0.64	99.87	1.58
100	LINA	2	2	4	4.21 ± 0.05	102.26	1.01
120	EMPA	4	4.8	8.8	8.62 ± 0.51	99.59	1.1
120	LINA	2	2.4	4.4	4.41 ± 0.04	100.22	0.77

*Acceptance Criteria: % RSD should not be more than 2

Table 3. Recovery data by DBADP method

Spiking Level (%)	Drug	Conc. Of Std (µg/mL)	Conc. of sample (µg/mL)	Actual con. (µg/ml)	*Con. recovered (µg/ml) AM±SD (n=3)**	Recovery (%)	% RSD
80	EMPA	4	3.2	7.2	7.11 ± 0.21	99.21	0.61
80	LINA	2	1.6	3.6	3.59 ± 0.06	99.72	1.38
100	EMPA	4	4	8	7.99 ± 0.51	99.87	1.27
100	LINA	2	2	4	4.12 ± 0.05	102.51	0.98
120	EMPA	4	4.8	8.8	8.79 ± 0.45	99.88	1.04
120	LINA	2	2.4	4.4	4.41 ± 0.67	100.22	1.21

*Acceptance Criteria: % RSD should not be more than 2

Limit of detection (LOD) and limit of quantitation (LOQ)

LOD and LOQ of EMPA and LINA were calculated by both RMC and DBADP methods, and the values are presented in table 4.

Table 4. LOD and LOQ values

Method	EMPA (µg/ml)		LINA (µg/ml)
Method	LOD	LOQ	LOD	LOQ
RMC	0.04	0.12	0.03	0.09
DBADP	0.06	0.18	0.02	0.06

Precision:

The method repeatability (intra-day precision) was determined by inter-day (n=3) analysis of three of the selected standard solutions of EMPA and LINA at the concentration of 2, 6 and 10µg/mL. The intra-day precision data is represented in Table 5. The inter-day precision analysis was also performed at the same concentration levels but on different days, and data are reported in Table 6. The statistical analysis on precision data proved good precision of the methods as the % RSD was <2.0 for both drugs in interday and intraday precision studies.

Table 5. Precision data by RMC methodology

Drug name	Actual Conc. (µg/mL)	Intra-day precision		Inter-day precision
		Conc. Found (µg/mL) (AM ± SD) (n=3)	% RSD	Conc. Found (µg/mL) (AM ± SD) (n=3)	% RSD
		Conc. Found (µg/mL) (AM ± SD) (n=3)	% RSD	Conc. Found (µg/mL) (AM ± SD) (n=3)	% RSD
EMPA	2	2.101 ± 0.08	0.81	1.981 ± 0.14	1.53
	6	6.012 ± 0.08	0.90	5.991 ± 0.03	0.14
	10	10.101 ± 0.06	0.21	10.022 ± 0.17	0.57
LINA	2	2.102 ± 0.07	0.81	1.983 ± 0.07	1.40
	6	5.923 ± 0.06	0.90	6.121 ± 0.20	1.92
	10	10.021 ± 0.11	0.21	10.112 ± 0.07	0.48

*Acceptance Criteria: % RSD should not be more than 2

Table 6. Precision data by DBADP methodology

Drug name	Actual Conc. (µg/mL)	Intra-day precision		Inter-day precision
		Conc. Found (µg/mL) (AM ± SD) (n=3)	% RSD	Conc. Found (µg/mL) (AM ± SD) (n=3)	% RSD
		Conc. Found (µg/mL) (AM ± SD) (n=3)	% RSD	Conc. Found (µg/mL) (AM ± SD) (n=3)	% RSD
EMPA	2	1.912 ± 0.06	0.66	1.931 ± 0.06	0.67
	6	5.971 ± 0.30	1.53	5.899 ± 0.08	0.42
	10	10.121 ± 0.10	0.36	10.103 ± 0.04	0.15
LINA	2	2.103 ± 0.05	1.01	1.991 ± 0.08	1.63
	6	6.121 ± 0.18	1.81	6.011 ± 0.13	1.29
	10	9.822 ± 0.12	0.83	10.112 ± 0.07	0.46

*Acceptance Criteria: % RSD should not be more than 2

Analysis of commercial tablets (assay):

The utility of the methods was done by applying them to analyse the drug content in marketed formulations. Assay of Glyxambi tablets containing two different doses of Empagliflozin was performed (10mg and 25mg of EMPA) and Linagliptin (5 mg). The results obtained for Empagliflozin and Linagliptin were compared with the respective labelled amounts and reported in Table 7. The % RSD for assay results of the formulation was <2, which indicated the developed methods' accuracy.

Table 7: Assay results of commercial tablets

Formulation with label claim	Amount found in mg (AM) ± SD; %RSD (n=3)
Glyxambi, EMPA=10 mg, LINA=5 mg	RMC		DBADP
	EMPA	LINA	EMPA	LINA
	9.92±0.081; 0.806	4.99 ±0.048;0.96	9.99±0.088;0.819	4.96 ± 0.051; 0.92
% Assay	99.25	99.86	99.90	99.12
Glyxambi, EMPA=25 mg, LINA=5 mg	24.52±0.14 0.57	4.92 ±0.051 1.03;	25.23±0.25;0.99	5.12 ± 0.038; 0.742
% Assay	98.08	98.40	100.92	102.40

DISCUSSION:

The developed methods are based on chemometrics that depends on ratio spectral manipulations using a software program written with PYTHON and MATPLOTLIB for which the ratio spectra are obtained by division of zero order spectra of the drug by a suitable concentration of the second drug and its concentration is critical in getting minimum or maximum signal in the RMC graph or DBADP graph of each drug. For EMPA, the divisor was selected as 6µg/mL of LINA; and for LINA, it was 10 µg/mL of EMPA. The wavelength interval was fixed as 1.0nm. The methods were validated for linearity, accuracy, precision, Detection limit and quantitation limit in tune with International Conference on Harmonization. Linear response was obtained in the concentration range of 2-10µg/mL for both LINA and EMPA by the two methods. Method repeatability and accuracy was proved by the low RSD (%). The methods utility was proved by extension of the method in the analysis of marketed formulation. The assay values and the %RSD values were within limits.

CONCLUSIONS:

The major objective was to develop economical and eco-friendly analytical methods based on chemometrics that uses UV spectroscopy for the concurrent analysis of EMPA and LINA in the marketed tablet. The developed methods do not require any sophisticated instruments nor costly reagents or solvents as that of HPLC or HPTLC. Since two chemometrics methods are used without any derivative steps, maximum information is available without loss of signal-noise ratio.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are thankful to Hetero drugs Ltd, Hyderabad, India. for providing the drugs as gift samples. The authors are thankful to the management of Gokaraju Rangaraju college of Pharmacy for the lab facilities and to Dr. Sudheer Joseph, Scientist-F, INCOIS for the software assistance in using python and MATPLOTLIB.

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Received on 10.08.2021 Modified on 06.12.2021

Asian J. Pharm. Ana. 2022; 12(1):43-48.

DOI: 10.52711/2231-5675.2022.00008